Ultrastructure of asci,ascospores, and spore release inLophodermella sulcigena (Rostr.) v. Hohn.

Summary The croziers were formed from large multinucleate cells at the base of the hysterothecium. The diploid ascus had basal and apical vacuoles and there was prominant endoplasmic reticulum near the extending tip of the ascus. The spore delimiting membranes were continuous with the plasmalemma and possibly arose from it. The spore walls were formed between the two membranes. The ascus had a simple apical ring around a thinner region of the wall which became the pore through which the spores were released. Just before spore release the outer layer of the ascospore wall became vesiculated and eventually mucilagenous. The long clavate ascospores were released one at a time, stretching the neck of the ascus as they emerged.     

SIEVE-ELEMENT ULTRASTRUCTURE IN PLATYCERIUM BIFURCATUM AND SOME OTHER POLYPODIACEOUS FERNS: THE NACREOUS WALL THICKENING AND MATURATION OF THE PROTOPLAST

Sieve elements of various ages were examined in petioles and midribs of Platycerium bifurcatum (Cav.) C. Chr. and Phlebodium aureum (L.) J. Sm., only older ones in similar parts of leaves of Polypodium schraderi Mett. and Microgramma lycopodioides (L.) Copel. Nacreous walls apparently are formed by most, if not all, protophloem and metaphloem sieve elements in all four species. In Platycerium and Phlebodium nacreous wall formation is closely correlated with the appearance of numerous membranes or vesicles in the region of the wall. These extracytoplasmic membranes apparently are derived from protrusions of the plasmalemma. After the nacreous layer is fully thickened, many endoplasmic reticulum (ER) membranes apparently end up outside the plasmalemma of Platycerium, where they degenerate and gradually intergrade in appearance with the fibrillar material comprising the nacreous thickening. In Phlebodium, Polypodium, and Microgramma the ER forms multivesicular bodies. As the cells approach maturity, the membranes delimiting the multivesicular bodies fuse with the plasmalemma and their vesicular contents, which are not discharged into the region of the wall, disappear. Gradually, the nacreous layer decreases in thickness and disappears. At maturity the enucleate sieve-element protoplasts of all four species are essentially similar. They are lined by a plasmalemma and a parietal, anastomosing network of ER and contain both plastids and mitochondria. The plastids in Polypodium and Microgramma are chloroplasts, but those in Platycerium and Phlebodium lack grana and intergrana lamellae.     

The effects of anhydrous ammonia on membrane stability ofP.hymatotrichum omnivorum

Sclerotia and mycelium ofPhymatotrichum omnivorum were exposed to anhydrous ammonia (NH₃) and then observed with an electron microscope in order to determine the effects of the NH₃ treatment on the fungal membranes. Sclerotia were exposed to four rates of NH₃: 28, 56, 84, and 112μg NH₃/ml of air for 24 hours. At 28μg/ml, the plasmalemma became wavy and the mitochondrial cristae began to swell and disperse. At 56μg NH₃/ml the plasmalemma showed breakage and formation of vesicles, and all other membrane systems within the cell were broken and distorted. All membranes were totally disrupted and no organelles were recognizable at 84μg NH₃/ml. Mycelium was exposed to 2, 4, 8, 20, and 40μg NH₃/ml for one minute. Damage to cell membranes was not observed at NH₃ conc. up to 4μg/ ml. At 8μg NH₃/ml the plasmalemma was broken and the mitochondria were disrupted. At 20μg/ ml and above, all internal organization was destroyed.     

Untersuchungen zur Anfälligkeit und Resistenz von Kopfsalat (Lactuca sativa) gegen falschen Mehltau (Bremia lactucae) III. Peroxydase-, peroxydatische Katalase-und Polyphenoloxydase-Aktivitäten

Investigations on the susceptibility of head lettuce (Lactuca sativa) to downy mildew (Bremia lactucae) III. Activities of peroxidase, catalase and polyphenoloxidase Host cell walls in contact with intercellular hyphae of Bremia lactucae stain electron positively in susceptible and incompletely resistant varieties of lettuce after appropriate electron microscopy preparation for peroxidase activity. The outer membranes of the mitochondria of the parasite also stained darkly in susceptible varieties whereas in incompletely resistant plants Bremia innermost mitochondrial membranes and host cell mitochondria were darkly stained. This latter observation suggests increased respiration and could be explained as a resistance reaction. Catalase activity was observed in the microbodies of susceptible, in incompletely resistant and healthy varieties. There were no differences in stain intensity in the three kinds of varieties suggesting that catalase activity is not involved in resistance reactions. Polyphenoloxidase activity was infrequently observed on the host cell wall in susceptible and healthy plants, whereas strong activity in incompletely resistant varieties was observed in vesicles in the haustorial sheath. These vesicles were not surrounded by unit membranes and therefore could not have originated from the unit membranes of the extrahaustorial matrix or from the host plasmalemma. They may have been derived from the host protoplast and involved in inactivation of parasite produced toxins thereby contributing to resistance.     

A study of the fine structure of ascosporogenesis in Saccobolus kerverni

Summary Observations of ascospore fromation in KMnO₄-fixed Saccobolus kerverni apothecia with the electron microscope reveal the following sequence. Ascus formation is preceded by the development of croziers whose fine structure differs little from that of vegetative hyphae. Following fusion of the two nuclei in the ascus mother cell, the resultant ascus elongates, and two large vacuoles appear, first below and later above the fusion nucleus. These vacuoles soon occupy dominant positions at the tip and bottom of the ascus and assume a flocculent appearance. Nuclear blebbing occurs during meiosis, mitosis, and the subsequent spore delimitation process in the central cytoplasmic portion of the ascus. Each spore initial is surrounded by two membranes, the plasma and investing membranes, between which the spore wall is deposited in two layers, an inner primary wall and an outer secondary wall. Following primary wall deposition the spores clump; secondary wall deposition begins outside the primary wall at the places where the spores are contiguous. Interdigitation of these walls and disappearance of the investing membranes in the sutures lead to the envelopment of all eight ascospores in a common secondary wall. A flocculent material in the epiplasmic vacuoles aggregates around the mature spore balls.Based on a portion of a dissertation presented to the Faculty of the Graduate School of the University of Texas in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Ultrastructural Studies of the Zygote and Oocyst Wall Formation of Eimeria truncata of the Lesser Snow Goose1

ABSTRACT. Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer, of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Comparison of the biological activity of fusicoccin in higher plants with its binding to plasma membranes

The concentration dependences of the binding of fusicoccins (FCs) A, B, C, D, J and H to plasma membranes isolated from maize (Zea mays L.) roots have been studied in parallel with the effects of these compounds on elongation and ⁸⁶Rb transport in detached maize roots. The dissociation constants obtained showed a good correlation between the affinity of the FCs for the plasmalemma and their biological activity. However, the range of physiologically active FC concentrations proved to be about two orders of magnitude higher than that calculated from the dissociation constants. It was also shown that Vicia faba L. mesophyll protoplasts, unlike isolated plasma membranes, have two FC-binding sites, one with a K _D similar to that of the isolated plasmalemma while the other has a substantially higher K _D , apparently corresponding to the physiologically active state of the FC-binding proteins.Abbreviation FC fusicoccin     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

Role of the Plasmalemma in Controlling the Gravitropic Response of Wheat Coleoptiles

The changes in the specific binding of ³H-IAA to the plasmalemma from segments of wheat (Triticum aestivumL.) coleoptiles and the physiological activity of the IAA–protein complexes thus formed in dependence on the duration of gravitational stimulation (GS) (1 g) were studied. The overall inhibition of the formation of IAA–protein complexes was accompanied by a transverse polarization of their functional activity occurring as early as within two minutes after the onset of GS. The pretreatment of plasmalemmal vesicles with 0.1 M CaCl₂prevented the in vitroIAA–protein complex formation in the plasmalemma. It is suggested that the GS results in an increase in the plasmalemma permeability for Ca²⁺, which reduces the capacity of the plasmalemma to bind IAA at the early stages of the gravitropic response.     

Effects of ozone on the plasma membrane proteins in Arabidopsis thaliana (L.) leaves

The protein pattern of leaf plasma membranes from Arabidopsis thaliana (L.) Landsberg erecta was analysed in order to detect changes induced by acute short-term ozone treatment. Plasma membranes were isolated 0, 3 and 8 h after the end of a 2 h fumigation of the plants with 500 nmol mol^?1 of O₃. Proteins extracted from plasma membranes were separated by high-performance two-dimensional polyacrylamide gel electrophoresis. Eight hours after the end of fumigation, one new protein appeared and the amounts of two other proteins increased significantly. The reported study is a first step towards the identification of plasmalemma proteins altered by ozone and to a more detailed characterization of structural changes occurring in the plasma membrane after ozone exposure.     

Osmotic Water Permeability of Vacuolar and Plasma Membranes Isolated from Maize Roots

The method of stopped flow was used to follow the changes in light scattering by the vesicles of plasmalemma and tonoplast isolated from maize (Zea maysL.) roots and treated by osmotic pressure. In both membrane preparations, the rate of the process depended on the osmotic gradient and was described with the simple exponential function. The rate constants derived from these functions were the following: the coefficient of water permeability in the tonoplast (P= 165 ± 7 m/s) exceeded by an order of magnitude the corresponding index for plasmalemma (11 ± 2 m/s). The presence of HgCl₂(1.6 nmol/g membrane protein) decreased the tonoplast water permeability by 80%. Microviscosity studies of the hydrocarbon zone in the isolated membranes by using a fluorescent diphenylhexatriene probe demonstrated that the two membranes do not differ in the phase state of their lipid bilayer. The authors conclude that the observed difference in water permeability does not depend on the state of the lipid phase and probably reflects the dissimilar functional activity of plasmalemma and tonoplast aquaporins.     

Integration of sperm and egg plasma membrane components at fertilization

Studies examining the integration of the sperm and egg plasma membranes, subsequent to gamete fusion in the surf clam, Spisula solidissima, were carried out employing the concanavalin A-horseradish peroxidase-diaminobenzidine procedure (Con A-HRP-DAB). When unfertilized Spisula eggs were incubated in Con A, either prior to or after aldehyde fixation and reacted with HRP-DAB, enzymatic precipitate was found associated with the vitelline layer and plasmalemma. The plasma membranes of sperm treated in a similar manner failed to stain. The plasma membranes of fertilized eggs reacted with Con A-HRP-DAB and examined by 1 min postinsemination were associated uniformly with enzymatic precipitate except at sites of sperm incorporation. These portions of unstained plasma membrane were derived from the spermatozoon and delimited the contents of the fertilization cone. From 2 to 4 min postinsemination, HRP-DAB reaction product became associated with the plasma membrane delimiting the fertilization cone. By 4 min postinsemination no difference in staining of the plasma membranes derived from the egg or the sperm (plasmalemma delimiting the fertilization cone) was detected. Evidence is presented suggesting that the acquisition of HRP-DAB reaction product by the former sperm plasmalemma is due to the movement of Con A binding sites from the egg plasma membrane.     

In vitro modification of spinach plasmalemma thickness

Floral induction in the long day plant spinach (Spinacia oleracea) has been shown to be accompanied by a thickening of plasmalemma. This change was observed at early evocation, in both shoot apices and leaves, as well as after inducing GA₃ treatment. To get further information on this thickening, plasma membranes from spinach leaves were isolated, in the present study, using aqueous two phase partitioning and the effect of variousin vitro treatments on their thickness was investigated. The average plasmalemma thickness was unaffected by Na⁺ and K⁺ ions. It was increased upon the effect of either Ca²⁺ or gibberellic acid. A thickening of plasmalemma was also observed when plasma membranes from vegetative plants were incubated with a cytosolic preparation from photoinduced plants. The results were discussed in relation with the plasmalemma modifications previously reported in spinach.     

Phytochrome and phosphotungstate-chromate-positive vesicles from Cucurbita pepo L.

The phosphotungstic acid-chromic acid (PTA-CrO₃) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from Cucurbita hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTA-CrO₃-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.Abbreviations PTA-CrO₃ phosphotungstate-chromate - RNP ribonucleoprotein     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Aquaporin Content in Cell Membranes of Mesembryanthemum crystallinum as Affected by Plant Transition from C3 to CAM Type of Photosynthesis

Western-blot analysis was used to determine the contents of aquaporin isoforms MIP A, MIP B, and MIP C in cell membranes isolated from roots and leaves of Mesembryanthemum crystallinum plants with C₃ and Crassulacean acid metabolism (CAM) types of photosynthesis. These membrane preparations were also used to assess osmotic water permeability; to this end, the rate of osmotic vesicle shrinking was registered as the light scattering intensity by the method of stopped flow. The cell membranes represented by the plasmalemma and the tonoplast-enriched fraction were obtained by separating the microsomes in a two-phase polymer system. Plant transition from C₃ to CAM-photosynthesis occurred in the course of plant development or was induced by salinization. All three isoforms under study were found in the plasma membranes of roots and leaves of the C₃ plants, whereas in the CAM plants, independent of the transition-inducing factor, the aquaporin contents notably decreased in the leaf membranes and remained unchanged in the roots. In the membranes isolated from roots and leaves of the C₃ plants, the values of osmotic water permeability exceeded two–threefold the corresponding indices characteristic of the CAM plants. The authors believe that aquaporin isoforms in M. crystallinum are under the organ- and tissue-specific control.     

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.     

The ultrastructure of the ascus apical apparatus of some Dermateaceae (Helotiales)

The ascus apical apparatus ultrastructure of 12 species of the Dermateaceae (Helotiales, Ascomycota) was studied. Its development and details are described on the basis of transmission electron microscopy micrographs for all studied species. A majority of them, 8 species belonging to the genera Belonopsis, Mollisia, and Pyrenopeziza, show considerable similarity in the structure of their ascus apical apparatus. Two species, Mollisia melaleuca and Mollisia ramealis, show some deviations from this type, suggesting that the genus Mollisia is not fully homogeneous. Two other species, Podophacidium xanthomelum and Pezicula cinnamomea represent two ascus apical apparatus structure types of their own, which are remarkably different from the Mollisia type. The possible taxonomic implications arising from ascus apical apparatus diversity in the Dermateaceae are discussed.     

Transport properties of mobile charges in algal membranes: Influence of pH and turgor pressure

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. If the tonoplast and plasmalemma in series are charged to voltages of the order of 10mV, the decay of the initial voltage with time can be described by the sum of two or three exponential relaxations. It is not possible to explain the exponential decay of the voltage by twoRC-circuits in series (e.g. tonoplast and plasmalemma), because this would lead to unreasonable values for the specific capacities of the two membranes. The exponential relaxations might be attributable to the transport of mobile negative charges present in both membranes, possibly as a part of a transport system. From an analysis of the experimental results in terms of the proposed model, the translocation rate constantk and the total surface densityN _t of the mobile charges in one membrane could be evaluated. On averagek is of the order of 600 sec^–1 andNt is about 5×10^–12 mol cm^–2 (average turgor pressure 1.6 bar). The transport properties of the mobile charges within the tonoplast and plasmalemma were studied as a function of different parameters such as external pH, glutardialdehyde, electrical breakdown and turgor pressure. When the pH is lowered from 8.2 to 4 or 5 the mobile charges disappear completely, presumably as the result of protonation of the anionic groups. This pH effect was found to be completely reversible. Electrical breakdown causes a reversible disappearance of the relaxation with the longer half-time due to the decrease in membrane resistance. The value of the electrical breakdown voltage determined by injection of charge pulses of 300-sec duration into the cell is pH-independent and therefore is consistent with the mobile charge model and with results previously reported (U. Zimmermann & R. Benz.J. Membrane Biol 53:33–43, 1980). Addition of glutardialdehyde leads also to a disappearance of the mobile charges probably due to cross-linkage. Increase of the turgor pressure from 0.05 bar to 2 bar results in an increase ink by a factor of 2 and inNt by about 30%. The increase ink is in reasonable agreement with that expected on the basis of the assumed compressibility of the membranes. The elastic compressive modulus perpendicular to the membrane plane calculated from the pressure dependence of the translocation rate constantk is in very good agreement with that derived from electrical breakdown experiments (14 and 13 bar, respectively). The presence of charges within the membranes as well as the compressibility of the membranes are discussed in terms of a possible turgor-pressure-sensing mechanism.