Zygote formation inCosmarium botrytis studied by scanning and transmission electron microscopy,phase-contrast,and Calcofluor staining

Summary Rapid zygote formation byCosmarium botrytis was induced in a liquid medium by incubation in 5% CO₂. Conjugation and zygote formation were studied by SEM, TEM, phase-contrast, and Calcofluor fluorescence microscopy. It was observed that the cells divided immediately prior to conjugation and formed Calcofluor fluorescent conjugation papillae as soon as the primary wall was shed. The conjugating cells and the resultant zygote were envelopped by a non-fluorescent mucilagenous envelope which was eventually pierced by the zygote spines, but never shed. The very young smooth-walled zygote had a thick Calcofluor fluorescent wall. At that stage the zygote could be plasmolysed in 0.4 M mannitol, but no protoplast could be induced to emerge even with the addition of up to 5% Cellulysin; probably indicating that the zygote wall composition and structure is different from that of the secondary wall of the vegetative cells, particularly in the absence of mucilage pores.     

ULTRASTRUCTURAL EVIDENCE FOR SECRETORY PHASES IN THE LIFE HISTORY OF STIGMATIC HAIRS OF COTTON: A DRY TYPE STIGMA

Stigmatic hairs of the cotton flower were studied through their developmental stages up to anthesis. Stigmatic hairs invariably develop from a densely straining band of epidermal cells opposite the transmitting tissue cells. At anthesis, these are single cell structures measuring up to 300 μm long. At the 5-mm stage of stylar length (7–10 days before anthesis), some stigmatic hair cells begin to accumulate an osmiophilic substance between the plasmalemma and the cell wall, possibly synthesized in the endoplasmic reticulum. This material is apparently never secreted outside the cell wall. Immediately following this secretory phase in some stigmatic hair cells a second secretory phase starts. A dense osmiophilic substance, different in appearance from the previous phase, accumulates in the vacuoles of each hair cell. Concomitantly, dimorphism develops in the cytoplasmic densities of stigmatic hair. Some stigmatic hair cytoplasm appears very dense and shows signs of degeneration while other cytoplasm appears normal. A third secretory phase, which begins at anthesis, occurs in the normal hair cells. This phase is characterized by enhanced activity in the cytoplasm of the endoplasmic reticulum and Golgi apparatus. Large vesicles containing granular material are seen fusing with the plasmalemma. Coincident with this activity there is dissolution of the middle layers of the cell wall and the cuticle is ruptured at various points. The dense osmiophilic substance that had accumulated in the vacuole breaks down into fine granular material. Significance of these changes is discussed in relation to the pollen germination mechanism on the dry type stigma of cotton.     

CELL DIVISION IN COSMARIUM BOTRYTIS1

Cell division in Cosmarium is described. Premitotic cells are very dense; the semicells, previously appressed to one another, separate slightly during entry into prophase. This separation coincides with deposition of a girdle of new wall material around the isthmus, where the 2 semicells are joined. Micro-tubules, abundant around the isthmus wall during interphase, all disappear during prophase; meanwhile, other microtubules proliferate outside the nuclear envelope. By metaphase, the nucleolus has dispersed, although remnants of it persist. The nuclear envelope breaks up, but some membranes coat metaphase chromosomes. The spindle, while being typical, is somewhat multipolar; microtubules, usually associated with elements of endoplasmic reticulum, are oriented toward numerous regions in the poles. During anaphase and telophase, these spindle tubules become increasingly directed toward a few discrete foci, and they persist after telophase. Meanwhile, the septum grows from the girdle of wall material to bisect the cell. After cytokinesis, some microtubules reappear near the isthmus, but only adjacent to the older, non-expanding semicell wall. Cell expansion then takes place, during which the nucleus, ensheathed in a complex microtubular system, moves into the forming semicell. Later, the chloroplast follows the nucleus and its 2 pyrenoids elongate and divide. When semicell expansion is complete, the chloroplast cleaves adjacent to the isthmus. The nucleus, now apparently not associated with microtubules, concurrently moves back into the isthmus. Continuous deposition of primary wall material accompanies cell expansion. Wall materials are apparently secreted as aggregates (perhaps derived from the contents of vesicles) adjacent to the plasmalemma, whose fibrous components become increasingly oriented in the outer layers of the wall by stretching. Late in semicell formation, this deposition ceases and during further expansion, the semi-cell develops a pattern of warts and ridges. Secondary wall deposition under the primary wall then follows, matching this pattern of ornamentation. In addition, numerous plugs of amorphous material capped by specialized regions of the plasmalemma, traverse the entire thickness of the secondary wall which becomes further thickened at these particular sites. The amorphous plugs presumably are eroded away later to form the mucilage pores of the vegetative cells. The wall microtubules gradually become more symmetrically arrayed around the isthmus as this new secondary wall thickens. These observations are discussed in comparison with other work on morphogenesis in desmids.     

179 Conjugation Process of a Freshwater Green Alga, Spirogyra Varians (Zygnematales, Charophyceae Examined by Fitc-Lectins)

The conjugation processes of filamentous freshwater green alga Spirogyra varians were examined by the use of FITC-lectins. Conjugation comprised of five steps: 1) array with adjacent filaments, 2) formation of conjugation protrusion (papilla), 3) fusion of the protrusions, 4) formation of conjugation tube, and 5) formation of zygotes. Three lectins, ConA, RCA and UEA, showed considerable labeling during the progression of conjugation. FITC-ConA labeled the surfaces of filaments throughout the whole conjugation processes. No labeling of FITC-RCA was detected on the surface of vegetative filaments. FITC-RCA labeling was observed at the conjugation protrusions only after the papilla formation. Strong labeling continued until formation of zygotes even in hollow area between the conjugation tube. The labeling decreased gradually over time and disappeared when zygotes were formed. FITC-UEA showed similar labeling pattern with FITC-RCA except that the labeling did not disappear even after zygote formation. Inhibition experiments using D-galactose, L-fucose and D-mannose, which are complementary carbohydrates for the above lectins, showed considerable decrease of conjugation (<50% vs. 83% in control). Hamagglutination experiment using crude extract of Spyrogyra varians revealed existence of lectins specific for the above carbohydrates. These results suggested that the lectin-carbohydrate recognition system might be involved in the conjugation of Spirogyra varians .     

ULTRASTRUCTURAL CHANGES ASSOCIATED WITH UTILIZATION OF METABOLITE RESERVES AND TRICHOME DIFFERENTIATION IN THE PROTOCORM OF VANDA

Certain aspects of protocorm development in Vanda were examined ultrastructurally. The parenchymal cells of the protocorm accumulate substantial quantities of lipid, protein, and carbohydrate reserves which disappear gradually with the senescence of the parenchymatous region. The proteinaceous reserves appear initially as discrete bodies which become intimately associated with clusters of small tubules. The tubules eventually disperse throughout the cytoplasm and disappear following depletion of the protein bodies. The lipid reserves also appear as discrete bodies and are associated with an electron dense, laminated inclusion which appears to increase in size with the disappearance of the lipid bodies. While plastids in the meristematic cells differentiate a well-developed thylakoid system and contain little starch, those of the parenchymal cells contain large starch grains and numerous osmiophilic droplets and develop meager thylakoid systems. Membrane-bound crystalline structures of hexagonal and rhomboid cross section occur frequently in the cytoplasm of senescent parenchyma cells. Trichome initials, which differentiate from the epidermis, contain few conventional organelles and exhibit numerous membrane-bound structures containing many small crystalline inclusions. Numerous vesicles accumulate at the tips of the trichomes in spaces between the cell wall and the plasmalemma.     

CONJUGATION IN SPIROGYRA1

Conjugating filaments of Spirogyra were examined with both light and electron microscopes. Initially 2 or more filaments of Spirogyra were attached by mucilagenous material. Papillae appeared first in one filament and then in adjacent positions on the other filament. Subsequent growth of papillae separated the conjugating filaments; wall microtubules disappeared in papillae as they elongated. Golgi activity then increased markedly only in the male filament; mucilage production by these Golgi coincided with contraction of the male gamete from its cell wall and may be responsible for its subsequent migration. The end walls separating papillae dissolved to form the conjugation tube, allowing gamete union. The male protoplast then migrated through the tube and further cytoplasmic condensation formed an elliptical-shaped zygote. During the migration phase, zygote wall formation was initiated and numerous active Golgi apparently contributed material to it. Early zygote maturation was characterized by rapid wall formation and an increase in lipid droplets.     

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.     

ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE CORALLINE ALGA HALIPTILON CUVIERI(RHODOPHYTA)1

Tetrasporogenesis begins with the formation of the tetra-sporocyte, an elongate, apparently wall-less, cell containing few organelles. The tetrasporocyte rapidly elongates and a distinctive cell wall forms before the onset of meiosis. During this elongation phase there is also an increase in the number of plastids and mitochondria. The meiotic tetrasporocyte is characterized by extensive development of perinuclear endoplasmic reticulum (PNER) and peripheral endoplasmic reticulum (PER) and during the latter stages of sporogenesis by internuclear endoplasmic reticulum. Immediately next to the nuclear envelope the inter-cisternal spaces of the PNER are filled with very electron dense material and the PNER cisternae are quite narrow, while further away from the nucleus the PNER cisternae dilate. Throughout meiosis there is continued replication of plastids and mitochondria as well as synthesis of starch and the formation of Golgi-derived vesicles with very osmiophilic contents. Cytokinesis begins with the formation of striated thickenings on the inside of the tetrasporocyte wall, at the sites where the cleavage furrow, produced by infurrowing of the plasmalemma, will be formed. Early in cytokinesis the PER disappears and is replaced by osmiophilic vesicles and mitochondria. Tubular plasmalemma invaginations of 27–30 nm width also appear during the early stages of tetraspore wall formation. The ultra-structure of the early stages of tetraspore germination is also described.     

MORPHOLOGY AND PAEDOGAMOUS SEXUAL REPRODUCTION IN CHLOROGONIUM CAPILLATUM SP. NOV. (VOLVOCALES, CHLOROPHYTA)1

Morphology and sexual reproduction in Chlorogonium capillatum Nozaki, Watanabe & Aizawa sp. nov. (Volvocales, Chlorophyta) originating from Miyatoko Mire, Japan, were studied under controlled laboratory conditions. Vegetative cells of this new species were fusiform with blunt anterior and posterior ends, and they had a massive parietal chloroplast and numerous contractile vacuoles distributed throughout the protoplast. Several to many pyrenoids were randomly distributed in the chloroplast, but they disappeared under the light microscope when grown photoheterotrophically. During asexual reproduction, the first division took place transversely without a preceding rotation of the parental protoplast. In sexual reproduction, the parental protoplast divided successively to form 32 or 64 small, biflagellate isogametes. After gametogenesis, the gametes did not escape from the parental cell (gametangial) wall, within which pairs of the adjoining gametes fused to form quadriflagellate zygotes. Such zygotes were then released from the parental cell wall and developed into hypnozygotes, which at maturity developed numerous thin spines or hairs on the zygote wall. On zygote germination, four biflagellate germ cells were released from the zygote wall separately. This type of gametic union, "paedogamy," has not previously been described in the green algae except for Chlorococcum echinozygotum Starr . Chlorogonium capillatum can be clearly distinguished from other described species of Chlorogonium by its numerous contractile vacuoles and blunt anterior and posterior ends in vegetative cells as well as by its unique sexual reproduction, in which paedogamous conjugation occurs, and numerous thin spines or hairs that develop on the hypnozygote walls .     

An Ultrastructural Study of The Embryo/Endosperm Interface in the Developing Seeds of Solanum nigrum L. Zygote to Mid Torpedo Stage

The early developmental sequences in the formation of the Zoneof Separation and Secretion in a hexaploid species of Solanumnigrum L. are described. Ultrastructural changes which occurredduring the development of the embryo/endosperm interface couldbe related to the different stages in the embryo's development.The first step was the completion of the cell wall around thechalazal end of the zygote; a thin wall was formed along theendosperm cell(s) abutting the zygote. From the mature zygotestage to the quadrant stage, minute plasmalemma invaginationsoccurred along the endosperm wall facing the zygote. These invaginationsenlarged, and from the mid-globular stage onwards became filledwith a fine fibrillar material; this material accumulated betweenthe endosperm cell wall and the plasmalemma before being releasedinto the developing periembryonic and intercellular spaces tobecome the extracellular matrix. Cell wall development in theendosperm cells abutting the embryo followed an unusual path.During the quadrant stage, whilst the outer embryo wall increasedin thickness due to vesicle fusion, the endosperm cell wallfacing the embryo showed a loosening of the wall fibrils aswell as partial separation of these same endosperm cells fromeach other. From the early-globular stage, the endosperm cellwalls opposite the embryo became electron-translucent, disappearinginto the extracellular matrix. Enzymic secretions by the embryomay account for the alteration in the abutting endosperm cellwalls. Enzymic activity may also explain the development ofa homogenous electron-opaque layer over the outer embryo wallas well as the differences in the width of the fibrillar layerwhich accumulated around the cotyledons as the embryo grew throughthe Zone of Separation and Secretion. The potential roles ofthe extracellular matrix are briefly discussed. Solanum nigrum L.; embryo/endosperm interface; Zone of Separation and Secretion; embryo development; cellular endosperm     

FINE STRUCTURE OF THE ZYGOTE AND EARLY EMBRYO IN QUERCUS GAMBELII

This investigation begins with the late zygote and traces ultrastructural development to the late globular stage of the embryo. Two nucleoli and satellite nucleoli sometimes occur in the zygote nucleus. Mitochondria, dictyosomes, cytoplasmic ribosomes, rough ER, and lipid bodies are numerous in the zygote. Microbodies are occasionally seen. The cell wall becomes well developed before the first division. No plasmodesmata occur in the zygote wall. The basal cell of the proembryo and the suspensor cells of the later embryo have very dense cytoplasm with a high concentration of cytoplasmic ribosomes. The nuclei are very electron opaque. The terminal cell and the cells of the embryo proper have a fine structure similar to that of the zygote. Plastids increase in number, size, starch content, and amount of thylakoid lamellae as the embryo develops. Mitochondria are numerous and appear active at all stages. Dictyosome activity, ribosomal aggregation, and the amount of ER are highest during the late globular stage. Lipid bodies are present up to the early globular stage, then disappear. The inner cell walls of the embryo are thin and have many plasmodesmata. These walls begin to thicken at the late globular stage, and at this time the size of the embryo begins to show an increase over that of the zygote. The results show a corresponding increase in the amount and activity of the metabolic machinery as the development of the embryo progresses. Lipids are probably more important as a nutrient source in the zygote and early embryo; starch becomes more important in the late stages. Absorption of nutrient material into the embryo sac and developing embryo appears to be from the chalazal end.     

Stem Elongation and Cell Wall Proteins in Flowering Plants

Abstract: The growth of stems (hypocotyls, epicotyls) and stem-like organs (coleoptiles) in developing seedlings is largely due to the elongation of cells in the sub-apical region of the corresponding organ. According to the organismal concept of plant development, the thick outer epidermal wall, which can be traced back to the peripheral cell wall of the zygote, creates a sturdy organ sheath that determines the rate of stem elongation. The cells of the inner tissues are the products of secondary partitioning of one large protoplast; these turgid, thin-walled cells provide the driving force for organ growth. The structural differences between these types of cell walls are described (outer walls: thick, sturdy, helicoidal cellulose architecture; inner walls: thin, extensible, transversely-oriented cellulose microfibrils). On the basis of these facts, current models of cell wall loosening (and wall stiffening) are discussed with special reference to the expansin, enzymatic polymer remodelling and osmiophilic particle hypothesis. It is concluded that the exact biochemical mechanism(s) responsible for the coordinated yielding of the growth-controlling peripheral organ wall(s) have not yet been identified.     

分株紫萁卵发生的超微结构

用透射电镜对蕨类植物分枝紫萁(Osmunda cinnamamae L. var. asiatica Fernald)卵发牛进行了超微结构的研究。卵发生过程中,许多泡囊不仅移向细胞周围,而且在细胞质膜内排为一列,并通过胞吐作用聚集在细胞质膜外,它们释放或分泌嗜锇物质。观察到少数泡囊内含片层状结构的嗜饿物质紧贴于细胞质膜,似乎将其冲破。与此同时,在卵细胞和颈卵器壁之问形成分离腔,其宽度大于以往报道的真蕨类,在卵细胞质膜外出现额外的卵膜,其宽度大于蕨属和鳞毛蕨属。造粉体被大型常呈三角状半圆形或近椭圆形的淀粉粒所充满,当卵成熟时逐渐减少。核大型平扁状,核内出现2-3对平行的双层膜,紧贴核膜。未发现核外突。线粒体一度似不发育,最后恢复正常。     

Studies on conjugation of <Emphasis Type="Italic">Spirogyra</Emphasis> using monoclonal culture

We succeeded in inducing conjugation of Spirogyra castanacea by incubating algal filaments on agar plate. Conjugation could be induced using clone culture. The scalariform conjugation was generally observed, while lateral conjugation was rarely. When two filaments formed scalariform conjugation, all cells of one filament behaved as male and those of other filament did as female. Very rarely, however, zygospores were formed in both of pair filaments. The surface of conjugation tube was stained with fluorescently labeled-lectins, such as Bandeiraea (Griffonia) simplicifolia lectin (BSL-I) and jacalin. BSL-I strongly stained the conjugation tubes, while weakly did the cell surface of female gamete first and then that of male gamete. Jacalin stained mainly the conjugation tubes. Addition of jacalin inhibited the formation of papilla, suggesting some important role of jacalin-binding material at the initial step of formation of the conjugation tubes.     

分株紫萁卵发生的超微结构

用透射电镜对蕨类植物分枝紫其(Osmunda cinnamamae L. var.asiatica Fernald)卵发生进行了超微结构的研究.卵发生过程中,许多泡囊不仅移向细胞周围,而且在细胞质膜内排为一列,并通过胞吐作用聚集在细胞质膜外,它们释放或分泌嗜锇物质.观察到少数泡囊内含片层状结构的嗜饿物质紧贴于细胞质膜,似乎将其冲破.与此同时,在卵细胞和颈卵器壁之间形成分离腔,其宽度大于以往报道的真蕨类,在卵细胞质膜外出现额外的卵膜,其宽度大于蕨属和鳞毛蕨属.造粉体被大型常呈三角状半圆形或近椭圆形的淀粉粒所充满,当卵成熟时逐渐减少.核大型平扁状,核内出现2~3对平行的双层膜,紧贴核膜.未发现核外突.线粒体一度似不发育,最后恢复正常.     

离体培养鼠疟原虫动合子形成过程的电子显微镜观察

本文报道了用透射电子显微镜观察离体培养的鼠疟原虫配子体到动合子的发育过程。 鼠疟原虫配子的发生是由嗜锇小体趋向配子体表面开始。雌配子体从红细胞中逸出后,嗜锇小体消失。雄配子体微管形成和鞭毛轴丝集合是从红细胞中逸出前出现的。合子转变为动合子由致密内膜及膜下微管形成时开始,继之形成顶端复合物,随着突起增大,表膜复合物逐渐向后延伸,最后包绕整个虫体,即完成动合子的发育。疟原虫生活史第一次核分裂可能发生在动合子形成期间。本文证实了离体培养的动合子与蚊体内发育的动合子结构相同。     

Fine Structure of Germinating Botrytis fabae Sardina Conidia

The fine structure of germinating Botrytis fabae conidia wasstudied using both chemically stained sections and freeze-etchedreplicas. Germinating conidia have fewer organelles than restingconidia, glycogen is absent, and prevacuoles have disappeared.Endoplasmic reticulum which occurs as small strands close tothe cell wall of resting conidia becomes, on germination, multiplesheets surrounding the nuclei. A cross wall is formed at thebase of the germ tube soon after germination commences. Thenew wall material which appears to be continuous with this septalwall is produced, at least partly, from a new wall layer laiddown in the centre of the old conidial wall. An apical corpuscleis present at the apex of young germ tubes. Freeze-etched preparationsshow the formation of lomasomes by the passage of vesicles throughthe plasmalemma of conidia and germ tubes. In young hyphae lomasomescontain a complex arrangement of branching tubules. Some ofthe particles on the outer plasmalemma of young hyphae are arrangedin a geometrical pattern.     

Ultrastructure of Gum-resin Ducts in Cashew (Anacardium occidentale)

In Anacardium occidentale L., the gum-resin ducts in the primaryphloem of the stem develop schizogenously. Appearance of anintercellular space amongst the densely stained cells signalsthe initiation of a duct. During this process, the middle lamella,appears in places, as an electron-opaque area. The epitheialcells bordering the duct are oval and have convex inner tangentialwalls which are without any plasmodesmata, although they areabundant on radial and outer tangential walls. The cytoplasmof the epitheial cells is rich in rough endoplasmic reticulum(ER), free ribosomes, polysomes, mitochondria with swollen cristae,plastids with occasional osmiophilic inclusions, dictyosomesand vesicles. Osmiophilic material has been observed in thedilation of the cisternae of dictyosomes and also in vesicles.Sometimes, the osmiophilic material aggregates and forms largemembrane-bound globules. The globules fuse with the plasmalemmaat the inner tangential wall, and presumably the contents aredeposited in the space between the protoplast and the wall.This material passes through the loose matrix of the wall intothe duct. Some of the epitheial cells of the mature duct show‘dark’ cytoplasm, degraded organelles and occasionallyhigh vacuolation; ultimately they are lysed and their remainscollect in the duct. Anacardium occidentale L., cashew, gum-resin ducts, epithlial cell, dictyosome, osmiophilic material, ultrastructure     

薄荷头状腺毛分泌过程的超微结构研究

电镜观察表明,刚形成的薄荷头状腺毛的头部细胞,细胞核较大细胞质浓,有一些小液泡,质体和线粒体最显著,分泌前期,内质网及高尔基体数量明显     

CELL DIVISION AND FILAMENT FORMATION IN THE DESMID BAMBUSINA BREBISSONII (CHLOROPHYTA)1

Cell division and semicell expansion in the filamentous desmid Bambusina brebissonii Kütz. were investigated using transmission and scanning electron microscopy. Interphase cells are typical of desmids, containing a full complement of organelles and a cell wall penetrated by complex pores, but the cells lack a well-defined median constriction. Cell division involves an open spindle and the centripetal growth of a primary septum formed by the fusion of small, dark-staining vesicles probably derived from dictyosomes. Telophase nuclei are separated by a system of interzonal microtubules and numerous large, lighter-staining vesicles also derived from the dictyosomes. Following cell division, an elaborate replicate cross wall is formed which consists of both primary and secondary wall layers. During semicell expansion, a portion of the primary wall splits apart as the new semicells evaginate and expand to their full size. The primary wall stops splitting at a thick ring of secondary wall material leaving the cells united by the remaining common layer of primary wall. When semicell expansion is completed, the primary wall is not shed from the lateral walls of the new semicells, and pores through both primary and secondary wall layers begin to produce sheath material. However, pores in the end walls of cells do not function unless the filament is broken. The intact primary wall between cells and the absence of sheath production between cells comprise the mechanism serving to hold the cells of Bambusina brebissonii together in long filaments.