GIBBERELLIC ACID-INDUCED GERMINATION OF SPORES OF ANEMIA PHYLLITIDIS: NUCLEIC ACID AND PROTEIN SYNTHESIS DURING GERMINATION

The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination. Germination is accompanied by the hydrolysis of storage protein granules and the localized appearance of cytoplasmic RNA, protein, and insoluble carbohydrates in a small area adjoining the spore wall and surrounding the nucleus. The protoplast of the spore enlarges in this region, the spore wall breaks and a protonemal cell is formed which contains many chloroplasts. A second division in the spore at right angles to the first yields a rhizoid cell. Autoradiography of ³H-thymidine incorporation has shown that DNA is synthesized both in the nucleus and in the immediately surrounding cytoplasm of the germinating spore until some time after the first division, although a strictly nuclear DNA synthesis is observed later. Synthesis of RNA and proteins is limited to the presumptive regions of the germinating spore which become the protonema and rhizoid, shifting to specific sites in these cells as germination proceeds. The nucleus of the spore continues to be biosynthetically active long after it ceases to divide.     

The Effects of Centrifugation on Asymmetric Cell Division and Differentiation of Fern Spores

We have investigated the effects of centrifugation on sporepolarity, asymmetric cell division, and rhizoid differentiationin the sensitive fern Onoclea sensibilis L. Centrifugation at10000 g for 30 min produces a random orientation of spores withstratification of the cell contents. After centrifugation atmost early stages of development, the nucleus retains its normalpattern of migration from the centre of the ellipsoidal sporeto the proximal face and then to an end of the spore, withoutregard to the orientation of stratification. This indicatesthat the polarity of the spore is stable to centrifugation.As long as the nucleus migrates to an end of the spore and asymmetriccell division occurs, the small cell differentiates into a rhizoid.The arrangement of large cytoplasmic organelles appears to haveno influence on nuclear migration, asymmetric cell division,or rhizoid differentiation. The only period during developmentwhen centrifugation blocks asymmetric cell division is immediatelypreceding and during mitosis and cytokinesis. Spores centrifugedat this stage do not complete nuclear migration, and symmetriccell division results, with neither cell differentiating intoa rhizoid. The source of the stable polarity of the spore isdiscussed. cell polarity, rhizoid differentiation, centrifugation, Onoclea sensibilis L., sensitive fern, fern spores     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

NUCLEAR MIGRATION AND ASYMMETRIC CELL DIVISION IN ONOCLEA SENSIBILIS SPORES: AN ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY

A method of preparation for electron microscopy of fern spores in early stages of germination is presented. The cytochemistry and fine structure of Onoclea spores during the early stages of germination are described. The cytoplasm of the hydrated spore is filled with lipid droplets, protein granules and chloroplasts. During the early stages of development ribosomes and mitochondria increase in the area surrounding the central nucleus, and a new peripheral wall forms around the protoplast. Microtubules and large, branching mitochondria are associated with the nucleus during migration from its original central position in the spore to the proximal face and then to one end of the spore. There is no morphological polarization of cytoplasmic organelles of the spore before migration of the nucleus.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

Cell Morphogenesis and Macromolecule Synthesis during Phytochrome-controlled Germination of Spores of the Fern, Pteris vittata

The object of this study was to characterize the pattern ofcell morphogenesis and synthesis of nucleic acids and proteinsduring phytochrome-controlled germination of spores of the fern,Pteris vittata. Phytochrome activation and germination wereinitiated in fully imbibed spores by exposure to a saturatingdose of red light. At timed intervals thereafter, spores werefixed in acrolein and embedded in glycol methacrylate for examinationin the light microscope. The first sign of germination, visiblein sections of the spore 12 h after irradiation, was the hydrolysisof storage protein granules. This was followed by a migrationof the nucleus from its central location to one side of thespore. Subsequently, the protoplast enlarged at the site ofthe nucleus and appeared outside the exine as a papillate structure.An asymmetrical division of the protoplast gave rise to a smallcolourless rhizoid cell and a large, chloroplast-containingprotonemal cell. During the early phase of germination, DNAwas synthesized both in the nucleus and cytoplasm as judgedby autoradiography of [³H]thymidine incorporation. [³H]Uridine,a precursor of RNA synthesis, was incorporated into the nucleolusand the rest of the nuclear material of germinating spores.Protein synthesis monitored by [³H]leucine incorporation occurredboth in the nucleus and cytoplasm during the early stage ofgermination, although a strictly cytoplasmic protein synthesiswas observed later. Addition of cycloheximide completely inhibitedgermination of photoinduced spores and incorporation of labelledprecursors of macromolecule synthesis into cellular components.Actinomycin D was much less effective as an inhibitor of germinationand, even in high concentrations of the drug which effectivelyinhibited DNA and RNA synthesis in spores, proteolysis and proteinsynthesis appeared normal. These findings are discussed withrespect to the regulation of nucleic acid and protein synthesisduring spore germination and the role of phytochrome in theprocess.     

A COMPARATIVE STUDY OF CELL DIVISION PATTERNS DURING GERMINATION OF SPORES OF ANEMIA,LYGODIUM AND MOHRIA (SCHIZAEACEAE)

Cell division patterns during germination of spores of Anemia (A. hirsuta, A. munchii, A. phyllitidis), Lygodium (L. circinatum, L. flexuosum, L. japonicum, L. salicifolium) and Mohria caffrorum have been examined by light microscopy of glycol methacrylate embedded materials. Spores of all species in a genus exhibited a constant pattern of division under different conditions of germination. In spores of species of Anemia, following an asymmetrical division, the proximal cell differentiated into the protonemal cell while the distal cell divided to produce the rhizoid. A similar division sequence was found in spores of Mohria caffrorum, but the fate of cells formed was reversed. In Lygodium spores, a proximal cell formed by an initial division of the spore cut off a protonemal cell, a rhizoid and a wedge-shaped cell by walls parallel to the polar axis. Our results contradict earlier observations on cell division sequence during germination of spores of these genera based on whole mount preparations.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

THE SPORE-GERMINATION PATTERN OF THELYPTEROID FERNS

Cell division patterns in germinating spores of several Thelypteris species were studied using light microscopy of sectioned material and scanning electron microscopy. All species exhibited the same basic germination pattern, characterized by an asymmetric cell division of the spore parallel to the equatorial plane to delimit a proximal rhizoid, followed by a perpendicular division of the basal cell to form the protonemal cell. While spore-germination patterns appear to be a potentially useful taxonomic character in some groups of ferns, the homogeneity in this character exhibited by the thelypteroid group impairs its usefulness in the taxonomy of Thelypteris.     

Effects of caffeine on germination and differentiation in spores of the fern, Onoclea sensibilis

During spore germination in the fern, Onoclea sensibilis L., the nucleus moves from a central position to one end, and an asymmetrical cell division partitions the spore into two cells of greatly unequal size. The smaller cell differentiates directly into a rhizoid, whereas the larger cell and its derivatives give rise to the prothallus. In the presence of 5 mM caffeine, the nuclei of most of the spores undergo mitotic replication, whereas cell wall formation is blocked. Multinucleate single cells are produced, which are capable of growth, but no rhizoid differentiation occurs. In some cases a partial cell wall is produced, but the nucleus moves through the discontinuity back to the center of the spore, and the enucleate, incompletely partitioned small “cell” fails to differentiate into a rhizoid. In less than 1% of the spores a broad protuberance, whose wall is yellow-brown, is formed in a multinucleate single cell. The color, staining reaction to ruthenium red, and ultrastructural appearance of the protuberance resemble that of the rhizoid wall. It appears that infrequently in the caffeine-treated spores, a feature which is characteristic of rhizoids is expressed, in the absence of asymmetric cell division, in a cell which otherwise is unable to produce a rhizoid. The results are interpreted to mean that the spore has a highly localized, persistent differentiated region. For rhizoid differentiation to occur, a nucleus must be confined in that region – a confinement which normally is accomplished by the geometrically asymmetric first cell division of germination.     

Metal-binding sites in germinating fern spores (Onoclea sensibilis)

Summary During germination of the spore of the sensitive fernOnoclea sensibilis L. the nucleus migrates from a central position to the proximal face and then to one end of the ellipsoidal spore. An asymmetric cell division follows giving rise to a small cell which differentiates immediately into a rhizoid, and a large cell which divides further to give rise to the prothallus. The proximal face of the spore coat is differentiated from the remainder of the spore by its ability to bind nickel ions under certain conditions and by its staining with a sulfide-silver procedure which localizes heavy metals. The inner portion of the exine at the proximal face is differentiated from the outer part by its ability to stain with sulfide-silver at specific periods during germination. The exine at the proximal face also contains pore-like structures 50 nm in diameter which extend from the inner layer of the exine to the outer surface. Sulfide-silver staining material appears to be extruded through the pores at specific periods during germination. The percentage of spores showing nickel-binding and sulfide-silver stainability increases sharply during the first two to four hours of imbibition, then decreases sharply during the following two hours. This is followed by a second rise in staining at 8 to 12 hours of imbibition.The role of the ion-binding sites in the exine is discussed in relation to the stable polarity of the spore.Publishing prior to 1984 asAlix R. Bassel     

NUCLEAR MIGRATION IN GERMINATING SPORES OF ONOCLEA SENSIBILIS: THE PATH AND KINETICS OF MOVEMENT

Nuclear migration was observed in individual germinating spores of Onoclea sensibilis from the onset of movement to the completion of mitosis. About 16 hr after the initiation of germination, the nucleus migrated from its initial position in the center of the spore to the proximal side. It then appeared to migrate along the raphe to one end of the spore where an asymmetric division occurred. The average velocity of migration was measured at 0.256 ± 0.065 μm/min. During migration the nucleus underwent changes in shape. No migrations or movement other than that by the nucleus were observed.     

竹叶蕨配子体发育的培养观察

首次在光学显微镜下观察竹叶蕨孢子及其萌发、丝状体发育、片状体和生长点的形成及分化、原叶体细胞形态、假根及性器的发育等方面所表现出的显微特征。初步讨论竹叶蕨科从鳞始蕨科中分立出来的合理性,以及原叶体边缘细胞的形态、叶绿体对光的敏感性、假根的形态和精子器的形成及分化的系统学意义。     

2种培养基下紫萁配子体发育及孢子体形成的研究

采用光镜技术对不同培养基下紫萁配子体发育和孢子体形成进行了研究.结果表明:(1)紫萁孢子绿色,四面体形,具三裂缝,孢子两极萌发分别产生假根和原叶体细胞,原叶体细胞形成球形体,球形体产生分生细胞发育为片状体和原叶体.(2)原叶体可为雄性、雌性或两性;雄原叶体较小,其两翼基部产生多数精子器;雌原叶体大,具有明显的中肋,在中...     

Spore Germination and Rhizoid Differentiation in Onoclea sensibilis: A Two-Dimensional Electrophoretic Analysis of the Extant Soluble Proteins

Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two-dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells.     

Spore Germination of Ceratopteris thalictroides (L.) Brongn

Germination of the spores of Ceratopteris thalictroides wasexamined by light and scanning electron microscopy. Spores germinateby scission of the spore coat at the trilete markings. The initialcell divisions produce a proximal prothallial initial with rhizoidsthat are distal and lateral. This represents a reverse orientationof the more common situation in ferns. Evidence is presentedthat rhizoid initials divide with the most lateral of the daughtercells differentiating into a rhizoid. Rhizoids are long andunbranched. The uniseriate protonema is transient with bi-dimensionalgrowth established quite early. About a week following germination,the gametophyte is broad at the basal region and narrow at theanterior region. Further growth of cells in the basal regionis by elongation and the anterior region broadens to producean elongate-ovate gametophyte.     

DNA Synthesis and Cell Division During Spore Germination in Lygodium japonicum

This paper describes the ontogenetic sequence of cell divisionsand associated DNA synthetic patterns observed in sectionedspores of Lygodium japonicum (Thunb.) Sw., collected at differentstages of germination. Following exposure to a saturating doseof red light, the spore undergoes an asymmetric division toform a basal cell, which retains nearly all of the storage inclusions,and an apical cell which expands and protrudes from the rupturedsporoderm. Division of the apical cell results in formationof a protonemal cell and an intermediate cell. Subsequently,the latter cell divides to form the primary rhizoid and a wedgecell adjacent to the protonemal cell. Secondary rhizoids mayarise from later divisions of either the basal cell or the wedgecell. In addition, the wedge cell appears to have the capacityto form a secondary prothal-lial filament. Histochemical localizationof cell constituents indicates an increasing concentration ofcytoplasmic RNA and protein in the presumptive protonemal regionof the spore cell prior to division. Autoradiography of ³H–thymidineincorporation has shown that synthesis of nuclear DNA precedeseach cell division. Although strictly nuclear DNA synthesisoccurs during early stages of germination, extra-nuclear DNAsynthesis increases greatly following division of the sporecell. The results are discussed in relation to earlier studieson cell division patterns seen in whole mount preparations ofgerminating spores of different species of Lygodium. Lygodium japonicum, spore germination, cell division, DNA synthesis     

ULTRASTRUCTURAL CHARACTERISTICS OF SPORE GERMINATION IN THE MOSS DAWSONIA SUPERBA

The spore wall of Dawsonia superba has characteristics that, in many respects, are similar to those of other mosses except for the exine, which is layered in Dawsonia. Imbibed spores have a well-developed endoplasmic reticulum with dilated cisternae that are associated with vesicles at the periphery of the cell. Ribosomes on the surface of the vesicles suggest that many vesicles originate from the endoplasmic reticulum. Two types of protein storage bodies are observed: membrane bound protein bodies with a homogeneous matrix which gradually becomes vesicular, and densely stained and non-membrane bound bodies consisting of crystalline arrays of fibrils. As in spores of higher plants, the protein reserves disappear during germination and may be converted to starch and other materials needed for development of the gametophyte.     

Development of the Mycetozoan Echinosteliopsis oligospora*

SYNOPSIS. The mycetozoan genus Echinosteliopsis, resembling the myxomycete Echinostelium in some of its features, is described. The single species, E. oligospora Reinhardt & Olive, forms small sporocarps which consist of a basal disk, stalk and a sporangium with only 1–8 spores. Spores form progressively, not simultaneously, by segmentation. The spores germinate to release non-flagellate amebae which, in liquid, assume a characteristic broad, fan shape. Each ameba has one or more nuclei. The nucleus is distinctive because of refractile, globular to elongate peripheral bodies which cytochemical tests indicate to be primarily RNA. At the time of nuclear division the characteristic RNA bodies disappear and, as observed with the phase microscope and in stained preparations, optically dense material accumulates in the middle area of the nucleus. Threads, either a spindle or actual chromatin, can be seen attached to the nuclear membrane. The threads separate to opposite poles as the nucleus elongates. During this division process the nuclear membrane apparently remains intact. Synchronous binucleate divisions, as well as a tripolar nuclear division, have been observed. Uninucleate and synchronous binucleate divisions may or may not be followed by cytokinesis. The absence of cell division after nuclear division leads to the production of cells with varying numbers of nuclei. Nuclear divisions in early sporangial stages and in spores have not been observed. The spores are uni- to multinucleate. In 8-spored sporangia and in most 4-spored sporangia there is a characteristic small “stalk spore” at the apex of the stalk. The stalk spore germinates slowly, if at all, but the larger spores germinate readily. No evidence of a sexual process has been found.     

Spore Germination Patterns in the Ferns, Cyathea and Dicksonia

Cell division patterns during germination of spores of Cyatheaaustralis, C. cooperi and Dicksonia antarctica were examinedby light microscopy of sectioned materials and by the scanningelectron microscope. In C. australis and C. cooperi the rhizoidwas traced to a small cell formed by an asymmetric divisionof the spore by a wall parallel to its equatorial plane. Incontrast, the rhizoid was formed by a division of the sporeparallel to its polar axis in D. antarctica. In spores of bothgenera, a second division wall oriented in a plane perpendicularto the first gave rise to the protonemal cell. Certain aspectsof germination described here in spores of Cyathea and Dicksoniaare in conflict with the published accounts of spore germinationin these genera. Cyathea, Dicksonia, spore germination, cell division pattern