Electron tomographic analysis of post-meiotic cytokinesis during pollen development in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.Abbreviations AGP Arabinogalactan protein - MT Microtubule     

Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron tomography

The three-dimensional architecture of syncytial-type cell plates in the endosperm of Arabidopsis has been analyzed at approximately 6-nm resolution by means of dual-axis high-voltage electron tomography of high-pressure frozen/freeze-substituted samples. Mini-phragmoplasts consisting of microtubule clusters assemble between sister and nonsister nuclei. Most Golgi-derived vesicles appear connected to these microtubules by two molecules that resemble kinesin-like motor proteins. These vesicles fuse with each other to form hourglass-shaped intermediates, which become wide (approximately 45 nm in diameter) tubules, the building blocks of wide tubular networks. New mini-phragmoplasts also are generated de novo around the margins of expanding wide tubular networks, giving rise to new foci of cell plate growth, which later become integrated into the main cell plate. Spiral-shaped rings of the dynamin-like protein ADL1A constrict but do not fission the wide tubules at irregular intervals. These rings appear to maintain the tubular geometry of the network. The wide tubular network matures into a convoluted fenestrated sheet in a process that involves increases of 45 and 130% in relative membrane surface area and volume, respectively. The proportionally larger increase in volume appears to reflect callose synthesis. Upon fusion with the parental plasma membrane, the convoluted fenestrated sheet is transformed into a planar fenestrated sheet. This transformation involves clathrin-coated vesicles that reduce the relative membrane surface area and volume by approximately 70%. A ribosome-excluding matrix encompasses the cell plate membranes from the fusion of the first vesicles until the onset of the planar fenestrated sheet formation. We postulate that this matrix contains the molecules that mediate cell plate assembly.     

Early Endosperm Development in Ovules of Soybean, Glycine max (L) Merr. (Fabaceae)

Endosperm development was studied in normally setting flowersand pods of soybean from anthesis to a pod length of 10–20mm. The free-nuclear stage following double fertilization istypified by loss of starch and increasing vacuolation. The cytoplasmprovides evidence of extensive metabolic activity. Wall ingrowths,already present at the micropylar end of the embryo sac wallprior to fertilization, develop along the lateral wall of thecentral cell as well as at the chalazal endosperm haustorium.Endosperm cellularization begins when the embryo has developeda distinct globular embryo proper and suspensor. Cellularizationstarts at the micropylar end of the embryo sac as a series ofantidinal walls projecting into the endosperm cytoplasm fromthe wall of the central cell. The free, growing ends of thesewalls are associated with vesicles, microtubules, and endoplasrnicreticulum. Pendinal walls that complete the compartmentalizalionof portions of the endosperm cytoplasm are initiated as cellplates formed during continued mitosis of the endosperm nuclei.Endosperm cell walls are traversed by plasmodesmata. This studywill provide a basis for comparison with endosperin from soybeanflowers programmed to abscise. Glycine max, soybean, endosperm, ovules     

花生胚乳细胞化的超微结构观察

花生（ＡｒａｃｈｉｓｈｙｐｏｇｅａｅＬ．）心形胚期的胚乳游离核多瓣裂,或具长尾状结构。胚乳细胞质内有大量线粒体、质体、高尔基体、小泡及少量内质网。中央细胞壁有壁内突。球胚及心形胚期常见胚乳瘤。心形胚晚期,胚乳开始细胞化,胚乳细胞壁形成有３种方式,分别存在于不同的胚珠中：（１）从胚囊壁产生自由生长壁形成初始垂周壁,具有明显的电子密度深的中层,其生长主要靠末端的高尔基体小泡及内质网囊泡的融合。两相邻的自由生长壁末端或其分枝末端相连形成胚乳细胞。（２）核有丝分裂后产生细胞板,细胞板向外扩展并可分枝。间期的非姊妹核间也观察到形成了细胞板。小泡与微管参与细胞板的扩展,高尔基体和内质网是小泡的主要来源。细胞板的扩展末端相互连接,形成胚乳细胞的前身。小泡继续加入细胞板的组成,以后形成胚乳细胞壁。（３）胚乳细胞质中,出现一些比较大的不规则形的片段性泡状结构,它们可能来源于高尔基体小泡,这些片段性泡状结构随机相连形成细胞壁,未见微管参与。胚乳细胞外切向壁及经向壁上有壁内突。     

Studies on the Development of the Embryo and Endosperm of Nitraria sibirica Pall

The pollen tube enters the embryo sac through the crassinucellus at the micropylar end, and brings about the porogamy. The embryogeny corresponds to the Solanad type. The defference of the suspensor structure is notable by comparing it with the other genera of Zygophyllaceae that have been studied. The endosperm is of the Nuclear type. Mitosis is the main form of the free endosperm nuclei proliferation. No cell plates develop in the early free nuclear division, however, they appear in late development, without developing into the cell wall and disappear ultimately. At the late globular embryo stage, cell formation in endosperm starts first from the micropylar end. The first anticlinal walls develop from the cell plate that is initiated from tile phragmoplast as a result of normal cytokinesis. Follwing this a wall begins to grow from the base of the cell plates, the outer growing margin soon fuses with the wall of the central cell, and the inner growing margin continues to grow towards the central vacuole. The growing walls branch and eventually fuse on the side nearest the central vacuole. Thus, the first periclinal walls are initiated, and a complete endosperm cell is formed. Along with the development of embryo, cell is gradually formed in the endosperm from the micropylar end towards the chalazal end, but the chalazal endosperm is still coenocytic until the endosperm disintegrate completely. The mature seed has no endosperm.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Polarization predicts the pattern of cellularization in cereal endosperm

Summary The endosperm of cereal grains develops as a multinucleate mass of wall-less cytoplasm (syncytium) that lines the periphery of the central cell before becoming cellular. The pattern of initial wall formation is precisely oriented and is followed by a round of precisely oriented formative cell division that gives rise to initials for the two tissues of endosperm. The initial anticlinal walls form at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubules emanating from nuclei in the syncytium. Polarized growth of the NCDs in axes perpendicular to the embryo sac wall and centripetal elongation of the anticlinal walls results in a single layer of open ended alveoli overtopped by the remaining syncytial cytoplasm. This arboreal stage, so named because the elongate nucleate columns of cytoplasm resemble an orchard of trees, predicts the division polarity of the imminent formative division. Mitosis occurs as a wave which, like polarization, moves in both directions from ventral to dorsal. Spindles are oriented parallel to the long axis of the alveoli and cell plates give rise to periclinal walls. The outer daughter nuclei (aleurone initials) are thus completely enclosed by walls and the inner nuclei (starchy endosperm initials) are in alveoli adjacent to the central vacuole.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

The osmoregulatory tissue around the afferent blood vessels of the coxal gills in the estuarine amphipods, Grandidierella japonica and Melita setiflagella

By electron microscopy of the coxal gills in two species of estuarine amphipod crustaceans, Grandidierella japonica and Melita satifragella, we found a patch-like, specialized tissue area which consisted of unique cells closely resembling the salt-excreting cells in the gill of the brine shrimp and so-called chloride cells in teleost gills. These cells were characterized by an abundance of mitochondria, two kinds of extensive networks of cytoplasmic tubules, well-developed lamellar infoldings of the basal cell membrane, sparse microvillous projections of the apical border, and numerous large vacuoles with several incomplete partitions. The large (60 nm in diameter) and the small (30 nm) cytoplasmic tubular networks were found in the basal and the apical portions of the cell, respectively. The large networks, which were both directly and indirectly (through the lamellar system) continuous with the basal cell membrane, were regarded as extensions of the cell membrane. Both the outer walls and the partition walls of the vacuoles were reinforced with a parallel array of microtubules. The results suggest that this unique tissue plays an important role in the active transport of electrolytes to maintain a constant osmotic pressure of the hemolymph under widely fluctuating salinities of the estuarine environments.     

Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.     

Changes in cell wall polysaccharides of germinating barley grains

Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.     

Development of the endosperm in rice (Oryza sativa L.): Cellularization

The syncytial endosperm of rice undergoes cellularization according to a regular morphogenetic plan. At 3 days after pollination (dap) mitosis in the peripheral synctium ceases. Radial systems of microtubules emanating from interphase nuclei define nuclear-cytoplasmic domains (NCDs) which develop axes perpendicular, to the embryo sac wall. Free-growing anticlinal walls between adjacent NCDs compart-mentalize the cytoplasm into open-ended alveoli which are overtopped by syncytial cytoplasm adjacent to the central vacuole. At 4 dap, mitosis resumes as a wave originating adjacent to the vascular bundle. The spindles are oriented parallel to the alveolar walls and cell plates formed in association with interzonal phragmoplasts result in periclinal walls that cut off a peripheral layer of cells and an inner layer of alveoli displaced toward the center. Polarized growth of the newly formed alveoli and elongation of the anticlinal walls occurs during interphase. The next wave of cell division in the alveoli proceeds as the first and a second cylinder of cells is cut off inside the peripheral layer. The periods of polarized growth/anticlinal wall elongation alternating with periclinal cell division are repeated 3–4 times until the grain is filled by 5 dap.     

Development of endosperm in Arabidopsis thaliana

 The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms. Received: 21 September 1998 / Revision accepted: 17 November 1998     

Intracisternal tubules and intramitochondrial filaments in cells of a snail, Limnaea

Epithelial and peritubular cells associated with the reproductive tract of the snail, Limnaea stagnalis, contain an extensive system of endoplasmic reticulum that is often dilated with many closely packed intracisternal tubules. The intracisternal tubules are approximately 24-28 nm in diameter and they are often hexagonally packed. They have a two-layered wall, possess fine interconnections, and extend linearly for considerable distances, but angular bends in the tubules also occur. Mitochondria in the peritubular cells contain solid, filamentous structures 9-12 nm in diameter and triangular-shaped structures when sectioned in the mitochondrial matrix.     

Cytoskeletal organization of the micropylar endosperm in Coronopus didymus L. (Brassicaceae)

Summary. The micropylar chamber of the mustard Coronopus didymus is a developmental domain distinct from the contiguous central chamber and the more extreme chalazal chamber. Early in syncytial development the micropylar endosperm surrounding the embryo becomes populated with unusual fusiform to multilobed nuclei. These nuclei are sheathed by unique parallel arrays of microtubules that focus at tips of the nuclei and flare to connect with a reticulate network in the common cytoplasm. F-actin does not closely invest the nuclei but instead forms an extensive but separate cytoplasmic reticulum. When the embryo is in the early heart stage, the cytoskeleton of the endosperm undergoes a remarkable transition in preparation for cellularization. Microtubules become reorganized into radial arrays emanating from the nuclei, which themselves become spherical. Radial microtubule systems (RMSs), which replace both the parallel microtubules and the cytoplasmic reticulum, organize the common cytoplasm into evenly spaced nuclear cytoplasmic domains (NCDs). F-actin gradually becomes coaligned with the RMSs. Phragmoplasts are initiated adventitiously at the interfaces of opposing RMSs in the absence of mitosis. Cell plate deposition, which is initiated at multiple sites, results in a network of walls formed more or less simultaneously around the densely packed NCDs. The walls, which are rich in 1–3-β-glucans, join with one another and with the existing walls of both the central cell and embryo to complete cellularization in the micropylar chamber. In the adjacent central chamber where the syncytium is restricted to a thin peripheral layer by the large central vacuole, basic organization of the syncytium into NCDs is followed by alternating cycles of alveolation and periclinal cell division resulting in cellularization. Received July 19, 2001 Accepted October 16, 2001     

Microtubular configurations during the cellularization of coenocytic endosperm in Ranunculus sceleratus L.

Summary Endosperm cellularization in Ranunculus sceleratus was studied in terms of the initiation of cell-wall formation in the coenocytic endosperm. The first endosperm cell walls were in an anticlinal position relative to the cell wall of the embryo sac and originated from the cell plates and not from wall ingrowths from the embryo-sac wall itself. Alveolar endosperm was formed 3 days after pollination. Microtubules were associated with the freely growing wall ends of the anticlinal walls and were observed in various orientations that generally ranged from angles of 45 ° to 90 ° to the plane of the wall. They were absent in the regions where vesicles had already fused. These microtubules may function in maintaining the growth and the direction of growth of the anticlinal wall until cellularization is completed. At the site where three neighbouring alveoli share their freely growing wall ends, remarkable configurations of microtubules were observed: in each alveolus, microtubules ran predominantly parallel to the bisector of the angle formed by the common walls. These microtubules may form a physically stable framework and maintain the direction of growth of the wall edges. It is concluded that the growing edge of the anticlinal endosperm wall and its associated microtubules are a special continuum of the original phragmoplast that gave rise to the anticlinal wall.     

Capsella embryogenesis: The suspensor and the basal cell

Summary The suspensor and basal cell ofCapsella were examined with the electron microscope and analyzed by histochemical procedures. The suspensor cells are more vacuolate and contain more ER and dictyosomes, but fewer ribosomes and stain less intensely for protein and nucleic acids than the cells of the embryo. The end walls of the suspensor cells contain numerous plasmodesmata but there are no plasmodesmata in the walls separating the suspensor from the embryo sac. The lower suspensor cells fuse with the embryo sac wall and the lateral walls of the lower and middle suspensor cells produce finger-like projections into the endosperm. At the heart stage the suspensor cells begin to degenerate and gradually lose their ability to stain for protein and nucleic acids.The basal cell is highly vacuolate and enlarges to a size of 150 X 70. An extensive network of wall projections develops on the micropylar end wall and adjacent lateral wall. The nucleus becomes deeply lobed and suspended in a strand of cytoplasm traversing the large vacuole. The cytoplasmic matrix darkens at the late globular stage and histochemical staining for protein becomes very intense. The basal cell remains active after the suspensor cytoplasm has degenerated. It is proposed that the suspensor and basal cell function as an embryonic root in the absorption and translocation of nutriments from the integuments to the developing embryo.Research supported by NSF grant GB 3460 and NIH grant 5-RO 1-CA-03656-09.     

Formation of macromolecular lignin in ginkgo xylem cell walls as observed by field emission scanning electron microscopy

Formation of macromolecular lignin in ginkgo cell walls. In the lignifying process of xylem cell walls, macromolecular lignin is formed by polymerization of monolignols on the pectic substances, hemicellulose and cellulose microfibrils that have deposited prior to the start of lignification. Observation of lignifying secondary cell walls of ginkgo tracheids by field emission scanning electron microscopy suggested that lignin-hemicellulose complexes are formed as tubular bead-like modules surrounding the cellulose microfibrils (CMFs), and that the complexes finally fill up the space between CMFs. The size of one tubular bead-like module in the middle layer of the secondary wall (S2) was tentatively estimated to be about 16+/-2 nm in length, about 25+/-1 nm in outer diameter, with a wall thickness of 4+/-2 nm; the size of the modules in the outer layer of the secondary wall (S1) was larger and they were thicker-walled than that in the middle layer (S2). Aggregates of large globular modules were observed in the cell corner and compound middle lamella. It was suggested that the structure of non-cellulosic polysaccharides and mode of their association with CMFs may be important factors controlling the module formation and lignin concentration in the different morphological regions of the cell wall.     

CYTOPLASMIC MICROTUBULES : I. Hydra

Small cytoplasmic tubules are present in the interstitial cells and cnidoblasts of hydra. They are referred to here as "microtubules." These tubular elements have an outside diameter of 180 A and an inside diameter of 80 A. By difference, the membranous wall is estimated to be 50 A thick. The maximum length of the microtubules cannot be determined from thin sections but is known to exceed 1.5 µ. In the interstitial cells the microtubules are found in the intercellular bridges, free in the cytoplasm and in association with the centrioles. In the cnidoblast they form a framework around the developing nematocyst and in late stages are related to the cnidocil forming a tight skein in the basal part of the cell. Especially in this cell, confluence of microtubules with small spherical vesicles of the Golgi complex has been observed. It is proposed that these tubules function in the transport of water, ions, or small molecules.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.