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 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.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

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.     

RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.     

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。     

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。     

Absence of Callose and Tetrad in the Microsporogenesis of Pandanus odoratissimus with Well-formed Pollen Exine

In the microsporocytes of Pandanus odoratissimus, cytokinesisis successive with centrifugal cleavage in both the meioticdivisions. The dyads move apart from each other after the firstdivision, and the microspores likewise after the second division,so that only monads are formed at the end of meiosis. Althoughno trace of callose wall is found at any stage around the microsporocyteor microspore, fertile, monocolpate pollen with well-developed,spinescent exine develops, and is shed at the two-celled stage. Pandanus odoratissimus, microsporogenesis, centrifugal cleavage, absence of callose, monad formation     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

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。     

DEVELOPMENT OF OMNIAPERTURATE POLLEN IN TRILLIUM KAMTSCHATICUM (LILIACEAE)

Ultrastructural changes during omniaperturate pollen development in Trillium kamtschaticum Pall, was examined using transmission electron microscopy. The pollen mother cells are not enveloped within a thick callosic wall. The microspores resulting from successive meiosis are divided by scanty deposition of callosic wall in the tetrad. A primexine/exine template is not recognizable within the tetrad during formation of exinous components. Preexinous globules, originating from vesicles in the callosic wall, accumulate electron-dense materials and develop into exinous globules. The preexinous globules have ca 10 nm wide contacts with tilted and invaginated plasma membrane of the microspore within the callosic wall. After dissolution of the callosic wall, the microspores separate and mitosis subsequently leads to the formation of a generative cell and vegetative cell encased in a loose aggregation of developing exinous globules. When the generative cell is at the pollen grain surface, the channeled zone is initiated at the opposite side of the microspore on the surface of the vegetative cell. Just before pollen maturity, a new layer develops under the channeled zone. Thus, development of the omniaperturate pollen grains of T. kamtschaticum involves some processes that are distinct from those of Canna and Heliconia and some that are similar.     

Anther ontogeny in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Brachypodium distachyon has emerged as a model plant for the improvement of grain crops such as wheat, barley and oats and for understanding basic biological processes to facilitate the development of grasses as superior energy crops. Brachypodium is also the first species of the grass subfamily Pooideae with a sequenced genome. For obtaining a better understanding of the mechanisms controlling male gametophyte development in B. distachyon, here we report the cellular changes during the stages of anther development, with special reference to the development of the anther wall. Brachypodium anthers are tetrasporangiate and follow the typical monocotyledonous-type anther wall formation pattern. Anther differentiation starts with the appearance of archesporial cells, which divide to generate primary parietal and primary sporogenous cells. The primary parietal cells form two secondary parietal layers. Later, the outer secondary parietal layer directly develops into the endothecium and the inner secondary parietal layer forms an outer middle layer and inner tapetum by periclinal division. The anther wall comprises an epidermis, endothecium, middle layer and the secretory-type tapetum. Major documented events of anther development include the degradation of a secretory-type tapetum and middle layer during the course of development and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The tapetum undergoes degeneration at the tetrad stage and disintegrates completely at the bicellular stage of pollen development. The distribution of insoluble polysaccharides in the anther layers and connective tissue through progressive developmental stages suggests their role in the development of male gametophytes. Until sporogenous cell stage, the amount of insoluble polysaccharides in the anther wall was negligible. However, abundant levels of insoluble polysaccharides were observed during microspore mother cell and tetrad stages and gradually declined during the free microspore and vacuolated microspore stages to undetectable level at the mature stage. Thus, the cellular features in the development of anthers in B. distachyon share similarities with anther and pollen development of other members of Poaceae.     

POLLEN PORE DEVELOPMENT AND ITS SPATIAL ORIENTATION DURING MICROSPOROGENESIS IN THE GRASS SORGHUM BICOLOR

The spatial relationships observed during microsporogenesis and pollen development in Sorghum bicolor indicate that a strong polarization exists in the anther locule and within individual microspores and pollen grains. During all developmental stages, each sporogenous cell and its derivatives lie continuously adjacent to the tapetum. The microspores and pollen grains form depressions in the tapetal orbicular wall. When the single pore of each microspore is initiated, as a gap in the primexine, it too lies adjacent to the tapetum and remains tightly appressed there until pollen maturity. A sequence of polar phenomena in microspores and pollen grains centers on an axis through the pore and perpendicular to the tapetal surface. These events include migrations of the microspore and vegetative nuclei, initial placement of the generative cell opposite the pore and its later migration, and a polar engorgement process whereby the pore end of the pollen grain (adjacent to the tapetum) fills with starch grains first. The tapetal cytoplasm completely degenerates at precisely the time of pollen engorgement, and its degradation products are believed to be available for pollen uptake at this time. The continuous association of the sporogenous cells or their cellular derivatives and their pores with the tapetum is thought to play an indispensible role in pollen development in sorghum and probably in all other grasses as well. The consistent position of the pore adjacent to the tapetum should be considered another common feature of microsporogenesis in the Gramineae. The characteristic exine pattern forms over the operculum and annulus of the pore, but the lamellae, which underlie the annulus, form a highly modified multilayered nexine. Membrane-like cores are observed in these lamellae and are believed to be involved in the initiation of sporopollenin deposition, but they are obliterated by pollen maturity. Neither the cores nor the lamellae are found in other parts of the pore or in the nonapertured wall.     

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。     

Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, <Emphasis Type="Italic">Pancratium maritimum</Emphasis>

Dynamic changes in the distribution of lipid and insoluble polysaccharide reserves of Pancratium maritimum L. (Amaryllidaceae) anthers were investigated throughout the successive stages of pollen development, using cytochemical methods, to determine whether the synthesis, transformation, and mobilization of reserve materials in developing anthers follow the regular pathway in angiosperms and support the physiological activities in developing pollen. Polysaccharides and lipid reserves exhibited a variable pattern of distribution from the sporogenous cell stage to the anthesis. Starch granules and lipid bodies were scarce in the cytoplasm of sporogenous cells, but their number increased significantly at the premeiotic stage. Conversely, starch and lipid reserves of meiocytes reduced at the early prophase of the first meiotic division, and then their amount showed fluctuations during the microsporogenesis. The cytoplasm of free and vacuolated microspores was poor regarding the polysaccharide and lipid reserves. However, at the late vacuolated microspore stage, small insoluble polysaccharides began to appear in the microspore cytoplasm, and their number increased remarkably in the cytoplasm of the bicellular pollen grain. During the maturation of pollen grains, polysaccharide reserves were replaced with lipids. The starch and lipid reserves of the staminal envelope also showed variations at different stages of the anther development. The dynamic changes in the polysaccharide and lipid reserves of P. maritimum anthers were consistent with the physiological activities such as differentiation, cell division and material synthesis that occur in the anther tissue at different stages of the male gametophyte development, and supported the normal pollen development.     

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.