Cotton embryogenesis: The pollen cytoplasm

Summary The ultrastructure and composition of cotton (Gossypium hirsutum) pollen, exclusive of the wall, was examined immediately before and after germination. The pollen grain before germination consists of two parts: the outer layer and a central core. The outer layer contains large numbers of mitochondria and dictyosomes as well as endoplasmic reticulum (ER). The core contains units made of spherical pockets of ER which are lined with lipid droplets and filled with small vesicles; the ER is rich in protein and may contain carbohydrate while the vesicles are filled with carbohydrate. Starch-containing plastids are also present in the core as are small vacuoles. The cytoplasm of the pore regions contains many 0.5 spherical bodies containing carbohydrate. After germination the ER pockets open and the lipid droplets and small vesicles mix with the other portions of the cytoplasm. With germination the pore region becomes filled with mitochondria and small vesicles. The vegetative nucleus is large, extremely dense and contains invaginations filled with coils of ER. A greatly reduced nucleolus is present in the generative cell which is surrounded by a carbohydrate wall. The cytoplasm of the generative cell is dense and contains many ribosomes, a few dictyosomes and mitochondria, many vesicles of several sizes, and some ER. No plastids were identified. The generative nucleus is also dense with masses of DNA clumped near the nuclear membrane. An unusual tubular structure of unknown origin or function was observed in the generative cell.     

棉花花粉水合和萌发时超微结构的变化：着重论述内质网和被膜小泡

棉花(Gossypium hirsutum L.)花粉在授粉后水合至萌发时期的营养细胞中贮藏的大量淀粉粒和脂体被动用。超微结构的观察表明,首先是造粉质体中的淀粉粒降解,尔后是脂体。在花粉水合至萌发时期,营养细胞中内质网和高尔基体十分活跃,并含丰富的被膜小泡。内质网的构型发生明显的变化:花粉刚水合时内质网潴泡高度扩张,不同程度扩张的内质网潴泡连续成网状并折迭形成许多囊袋状结构单位,其中包含造粉质体、脂体和被膜小泡群;其后,内质网潴泡形成的囊袋状结构消失,变为分支互通的网状结构;至萌发时,内质网潴泡略为扩张,有些连续成简单的网状,有些呈游离的囊泡状。被膜小泡始终是成群地分布,并与脂体联结,当脂体降解时一些被膜小泡与之融合。根据棉花花粉在水合至萌发时期,营养细胞质中存在独特形态的内质网系统和含丰富的被膜小泡,它们的动态行为及与淀粉和脂体的转化和降解之间的密切关系,讨论了这两种细胞器可能的功能。     

朱顶红花粉萌发过程中营养细胞质的超微结构变化

本文应用透射电镜对朱顶红成熟花粉水合、活化和萌发的动态过程中营养细胞质的结构和组成变化进行了观察。成熟花粉具质体、线粒体、内质网、高尔基体。微丝束以聚集体的形式存在。花粉活化后,细胞器的数目和结构发生显著变化：质体和线粒体的片层明显增加,内质网片层狭窄,高尔基体活跃产生小泡,脂体降解及微丝聚集体散开。花粉萌发后,细胞质中出现周质微管和被刺小泡,此期细胞器的变化不明显。微丝以纤丝状遍布整个花粉管中。     

Histochemical studies of pollen: storage pockets in the endoplasmic reticulum (ER)

Summary The pollen grain of cotton (Gossypium hirsutum) was examined histochemically at the light and electron microscope level. The cytoplasm of the pollen contains an unusual storage unit which consists of a pocket of endoplasmic reticulum (ER) containing lipid droplets and dictyosome vesicles. The ER pockets are large enough to be seen with the light microscope if thin enough sections are used (0.3–1.5). The results of the histochemical analyses show that the dictysome vesicles are rich in carbohydrate and contain protein and lipid as well. The ER contains large amounts of protein which may be arginine rich. Some carbohydrates may also be present in the ER. The ER is covered with ribosomes so that the pockets are unusually rich storage units containing abundant protein, carbohydrate, lipid and RNA. The light microscope localization of carbohydrates was confirmed by the periodic acid-silver method. Other storage units in the cytoplasm were also studied. A new method for the embedding of plant tissue for thin sectioning for light microscopy is presented.This work was supported by a Public Health Service fellowship 5-F2-GM-22, 031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-RO1-CA 0356-10 and by the Miller Institute for Basic Science.     

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae)

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Interaction of lipid bodies with other cell organelles in the maturing pollen of Magnolia × soulangeana (Magnoliaceae)

The pollen grain maturation in Magnolia × soulangeana was studied ultrastructurally and cytochemically using both the light and transmission electron microscope. Emphasis was given on the storage lipid bodies of the vegetative cell (VC) and their interaction with other cell organelles. Stereological analysis of electron micrographs was performed to evaluate the variation in volume density (V_V), surface density, and surface-to-volume ratio (S/V) of various cell organelles during pollen maturation. The size and numerical density of the lipid bodies, and their frequency of association with other cell organelles, were also determined. It was noted that during pollen ontogeny and maturation, the lipid bodies changed their pattern of distribution in the VC cytoplasm, which may be a good marker for the succeeding stages of pollen development. Also, the size, osmiophily, and V_V of the lipid bodies were progressively reduced during pollen maturation whereas the S/V was significantly increased. This seems to indicate that the lipid bodies are mobilized in part during this period of pollen maturation. In particular, the intermediate and mature pollen showed a high percentage of lipid bodies establishing a physical contact with either glyoxysomes, either protein storage vacuoles, or small vesicles presumably originated from dictyosomes. This physical contact was found in both the chemically fixed and rapid freeze-fixed pollen indicating that it is neither artifactual nor casual. On the basis of this intimate association with other cell organelles and the morphometric analysis performed, we suggest that the mobilization of lipid bodies is likely mediated not only by glyoxysomes but also by other catabolic pathways involving the interaction of lipid bodies with either protein storage vacuoles or small Golgi vesicles.     

Investigation on the Development of Male Cametophyte in Anemarrhena Asphodeloides

Anemarrhena asphodeloides is a monotypic genus of Liliaceae, endemic to China and Korea. This genus is characterized by possessing three stamens. From development of male gametophyte, three features of the species are noteworthy. (1) During meiosis of the micros- pore mother cells, the Golgi vesicles are immediately incorporated into the formation of the material of callose wall; The latter lying at the outer tangential is about 4 gm in thickness dining formation of the tetrad. In the outer tangential callose wall there are certain cytoplasmic canals, which are about 0.6 to 1 μm in diameter. During the development of pollen grains, there are a number of other vesicles dispersing in the cytoplasm of the microspores. The activity of these vesicles seems to be involved in accumulation and formation of lipid bodies. But the above vesicles, which were derivxed from Golgi or endoplasmic reticulum, have not been known in this genus. (2) By two-celled stage of pollen grains, the unequal distribution of lipid bodies is very prominent, and they are singular in being placed on the boundary between the plasmalemma of vegetative and generative cells. While the generative cell is delached from the intine of pollen grain, the generative cell is surrounded by the lipid bodies which had been called the corona of them. By the observation of TEM, these lipid bodies come from the cytoplasm of vegetative cell and did not remain a constant surrounding layer. Towards the stage of pollen maturation, the lipid bodies lying oppositely to the nucleus of vegetative cell were gradually dispersed in the cytoplasm. Their function is unknown but the observation shows that some of them move to the plasmalemma of the pollen grain. (3) An important feature of the mature pollen grain in Anemarrhena is that the generative cell does not contain plastids during polle development. On the basis of cytological mechanisms of the plastid inheritance, Hagemann (1983) has classified the angiosperms into four groups of species, of which the Lycopersicum type, Solanum type, and Triticum type belong to the mode of a uniparental maternal inheritance of plastids; while the Pelargonium type represents the mode of biparental inheritance of plastids. Our studies have confirmed that the mode of plastid inheritance in Anemarrhena asphodeloides is similar to Gasteria verrucosa, both show the same mode of plastid inheritance of Lycopersicum type.     

Cotton embryogenesis: The pollen tube in the stigma and style

Summary The ultrastructure and composition of the pollen tube of cotton (Gossypium hirsutum) growing in the tissues of the stigma and style of the flower were examined. The distal portion of the tube is densely cytoplasmic and contains the vegetative nucleus and the two sperms. The vegetative nucleus is highly convoluted and the membrane contains many pores and connections with the ER. No organized nucleolus is present but 4–6 membrane-bound, protein containing bodies are found in the nucleus. Mitochondria containing numerous cristae are abundant in the cytoplasm. Dictyosomes are also plentiful and are engaged in the production of many large vesicles. Rough ER is conspicuous and polysomes are found in the cytoplasm. Plastids are few in number, poorly developed, and contain little starch. Many uniform, small vesicles are found throughout the cytoplasm. Lipid bodies frequently with small vesicles associated with them are found in the tube. In the proximal region vacuoles form and the cytoplasm becomes pressed against the wall. In the transition zone the ER frequently becomes distended and filled with protein. The wall has two distinct layers: one strongly PAS positive, the other faintly PAS positive. The inner wall is apparently formed by the deposition of large dictyosome vesicles. Plug structure and development were studied.     

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.     

Reproductive timetable for the tropical Vireya rhododendron,R. Macgregoriae

Summary The reproductive development of Rhododendron macgregoriae F.v.M., Section Vireya (Ericaceae) has been followed from 10 days before anthesis to the production of mature germinable seeds about 145 days after anthesis. The species is self-compatible but shows both protandry and physical separation of pollen from the receptive stigma. Pollen is mature and viable from shortly before anthesis until the corolla and attached anthers abscise some 9–12 days after the flowers open. Spontaneous dehiscence, however, occurs mostly in the first few days after opening. The stigma becomes receptive 6–7 days after anthesis, and nectar is produced at this time. Female gametophytes are not mature until about 10 days after anthesis. Fertilization occurs about 6–7 days after pollination, and although the endosperm commences development immediately, development of the embryo proper does not begin until some 40–45 days later. Pollinations made with fresh pollen between 5 and 14 days after anthesis were successful, but those made on dry stigmas in the first 4 days after anthesis, or on senescing pistils 21 days after anthesis, gave little or no seed set. In nature, autogamy is probably uncommon, the majority of pollinations are likely to be geitonogamous, but there is considerable potential for outcrossing.     

The ultrastructure of polymorphic pollen grains of Canna indica L.

Summary Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

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.     

The transmitting tract in Trimezia fosteriana (Iridaceae).

Observations of the transmitting tract cells in Trimezia fosteriana were made from the pre-secretory stage until anthesis. Secretory products appear about 14 days before anthesis in all parts of the pistil. Simultaneously starch disappears from the plastids and the dictyosomes are surrounded by more and larger vesicles than before. In the beginning of the secretory stage multivesicular bodies may be in contact with ER profiles and an ER origin is therefore presumed. Later during the secretory stage the multivesicular bodies are larger and more abundant. Their envelope is often partly fused with the plasma membrane and vesicle swarms are common on the outside of it. Close to flower opening many organelles change in appearence. RER becomes more abundant and the mitochondrial matrix highly electron dense and the cristae expanded. Starch grains reappear and large ones are common in the stigma and ovary plastids. The dictyosomes are numerous and surrounded by the largest vesicles observed during the secretory stage. Osmiophilic granules are common in the dictyo-some vesicles and under the cell walls abutting the transmitting tract. In the ovary they are present one week before anthesis. In the stigma and style corresponding granules of high electron density appears at the day of flower opening. The extracellular secretory product contains fibrillar and granular substances consisting of carbohydrates, including pectic substances, and proteins.     

Ultrastructure of the vegetative cell ofBrassica napus pollen with particular reference to microbodies

Summary The ultrastructure of the vegetative cell ofBrassica napus tricellular pollen grains, just before anthesis with standard chemical fixation, is reported. The vegetative cell may be regarded as a highly differentiated and metabolically active fat-storage cell. It contains many mitochondria with a well developed internal membrane system, starchless plastids, microbodies, lipid bodies, dictyosomes and numerous vesicles thought to originate from the dictysomes. Rough endoplasmic reticulum organized in stacks of cisternae is also spatially associated with certain organelles, mainly lipid bodies, microbodies and plastids. There are also randomly distributed polyribosome areas. The microbodies are mainly polymorphic in shape and are often observed in contact with lipid bodies. The above spatial relationship implies that the microbodies may have a glyoxysomal function. In the late period of vegetative cell maturation, the microbodies are probably involved in the process of glyconeogenesis in which the conversion of lipid reserves to sugar takes place.Abbreviations VC vegetative cell - VN vegetative nucleus - SC sperm cell - M mitochondria - MB microbodies - L lipid body - P plastid - D dictyosomes     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

Hermodactylus tuberosus L. (Iridaceae) pollen organisation before and after anther dehiscence

ABSTRACT

The morphology, cytology and viability of Hermodactylus tuberosus L. (Iridaceae) pollen were examined from the first mitosis until maturation and after anther opening. During maturation, the pollen coat becomes modified, and the vegetative cell cytoplasm accumulates several types of reserve substances. In the vegetative cell cytoplasm, starch is quickly utilised whereas lipid inclusions of different dimensions, shape and composition occur during pollen maturation. Pollen from opened anthers have a thin pollen coat; the cytoplasm has mostly lipid reserves, and many small vesicles and vacuoles. It is similar in size or larger than pollen located inside the anther, and its viability does not decrease until one day after anther dehiscence. Large osmiophilic bodies, different from those of the vegetative cell cytoplasm, are present in the generative cell cytoplasm starting from the first stage of pollen development. The poorly developed pollen coat in pollen from opened anthers suggests that it plays a minor role in attracting insects for pollination. The size and structural and ultrastructural features of mature pollen indicate that it does not undergo dehydration and possesses sufficient vigour for immediate germination.