Showing papers in "Protoplasma in 1992"

Substructure of freeze-substituted plasmodesmata

Abstract: The substructure of plasmodesmata in freeze-substituted tissues of developing leaves of the tobacco plant (Nicotiana tabacum L. var. Maryland Mammoth) was studied by high resolution electron microscopy and computer image enhancement techniques. Both the desmotubule wall and the inner leaflet of the plasmodesmatal plasma membrane are composed of regularly spaced electron-dense particles approximately 3 nm in diameter, presumably proteinaceous and embedded in lipid. The central rod of the desmotubule is also particulate. In plasmodesmata with central cavities, spoke-like extensions are present between the desmotubule and the plasma membrane in the central cavity region. The space between the desmotubule and the plasma membrane appears to be the major pathway for intercellular transport through plasmodesmata. This pathway may be tortuous and its dimensions could be regulated by interactions between desmotubule and plasma membrane particles.

Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum

Abstract: In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial “slow” and “fast” lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.

Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells : an immunocytochemical study

Abstract: A monoclonal antibody (2 F 4) recognizing a conformational epitope of polygalacturonic acid was used for immunogold localization of pectins in walls of suspension-cultured carrot (D. carota L.) cells at the electron microscopic level. In microcolonies of “young” or “mature” cells, polygalacturonic acid was essentially located on the middle lamella material expanded at three-way junctions between cells or lining intercellular spaces but was not found in primary walls. Middle lamellae far from junction zones and intercellular spaces were not recognized. Largely esterified pectic polymers, only detected by the 2 F 4 antibodies after on-grid de-esterification treatment by pectin methyl esterases, were present within all primary cell walls. Golgi bodies and associated vesicles were also labeled by the 2 F 4 antibodies only after de-esterification treatment, which indicates that pectic polymers are synthesized and secreted in a highly esterified form. A decrease of pectin esterification, which results probably from an in situ enzymatic de-esterification of the pectic polymers of the primary walls, was observed in “senescent” cells. These results are discussed in relation to biochemical analyses showing changes of the methyl ester content of pectins during the cell-wall growth.

Inhibition of fungal growth by plant chitinases andβ-1,3-glucanases

Abstract: Plant chitinases and β-1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and β-1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.

Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophyll a/c light-harvesting complex in the diatom Phaeodactylum tricornutum.

Abstract: iserum against two polypeptides of the major fucoxanthin-chlorophylla/c light-harvesting complex of the diatomPhaeodactylum tricornutum and heterologous antiserum against purified photosystem I particles of maize were used to localize these two complexes on the thylakoid membranes ofP. tricornutum. As in many chromophyte algae, the thylakoids are loosely appressed and organized into extended bands of three, giving a ratio of 2∶1 for appressed versus non-appressed membranes. Immunoelectron microscopy demonstrated that the fucoxanthin-chlorophylla/c light-harvesting complex, which is believed to be associated with photosystem II, was equally distributed on the appressed and non-appressed thylakoid membranes. Photosystem I was also found on both types of membranes, but was slightly more concentrated on the two outer non-appressed membranes of each band. Similarly, photosystem I activity, as measured by the photooxidation of 3,3′-diaminobenzidine, was higher in the outer thylakoids than in the central thylakoid of each band. We conclude that the thylakoids of diatoms differ from those of green algae and higher plants in their macromolecular organization as well as in their morphological arrangement.

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Abstract: Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.

Purification and immunocytochemical localization of kafirins in Sorghum bicolor (L. Moench) endosperm

Abstract: Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as α-, β-, and γ-kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. β-Kafirins and γ-kafirins were relatively high in cysteine, and β-kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of α-kafirins with minor portions of β- and γ-kafirins. The dark staining regions, however, are composed primarily of β- and γ-kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of β- and γ-kafirin. Central endosperm protein bodies are also predominantly α-kafirin, but have a higher proportion of β-kafirin and γ-kafirin than the peripheral endosperm protein bodies.

Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

Abstract: A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Abstract: Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl α-D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.

The modified stomata of the floral nectary ofVicia faba L. 1. Development, anatomy and ultrastructure

Abstract: This study of the floral nectary ofVicia faba L. focusses on the modified stomata through which nectar flows, and on the question of whether they can regulate their stomatal pore aperture. The floral nectary ofV. faba consists of a disk which surrounds the gynoecium. As the flower bud matures, a tapered projection arises by cell divisions and expansion at a position opposite the free stamen. Modified stomata develop from pentagonal guard mother cells on this projection, and are often contiguous, but because development of modified stomata is asynchronous, many stages can occur in a local area. Plasmodesmata, which connect guard mother cells and guard cells of developing modified stomata with adjacent cell types, become covered by cell wall material on the guard-cell side in mature modified stomata. The numerous small vacuoles of guard mother cells and young guard cells are replaced by a single large one as the guard cells expand. Pore development occurs concurrent with this expansion as the ventral walls separate at a designated location leading to rupture of the inner, and then outer, cuticles, to reveal the pores of mature modified stomata.

Vesicle production and fusion during lobe formation in Micrasterias visualized by high-pressure freeze fixation

Abstract: Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the “dark vesicles” (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.

Photoinduction of formation of circular structures by microfilaments on chloroplasts during intracellular orientation in protonemal cells of the fern Adiantum capillus-veneris

Abstract: Changes in the organization of cortical actin microfilaments during phytochrome-mediated and blue light-induced photoorientation of chloroplasts were investigated by rhodamine-phalloidin staining in protonemal cells of the fernAdiantum capillusveneris. Low- and high-fluence rate responses were induced by partial irradiation of individual cells with a microbeam of 20 μm in width. In the low-fluence rate responses to red and blue light, a circular structure composed of microfilaments was induced on the chloroplast concentrated in the irradiated region, on the side facing the plasma membrane, as already reported in the case of the low-fluence rate response induced by polarized red or blue light. Such a structure was not observed on the chloroplasts located far from the microbeam. Time-course studies revealed that the structure was induced after the chloroplasts gathered in the illuminated region and that the structure disappeared before chloroplasts moved out of this region when the microbeam was turned off. In the high-fluence rate response to blue light, chloroplasts avoided the irradiated site but accumulated in the shaded area adjacent the edges of microbeam. The circular structure made of microfilaments was also observed on the chloroplasts gathered in the area and it showed the same behavior with respect to its appearance and disappearance during a light/dark regime as in the case of the low-fluence rate response. However, no such circular structure was observed in the high-fluence rate response to red light, in which case the chloroplasts also avoided the illuminated region but no accumulation in the adjacent areas was induced. These results indicate that the circular structure composed of microfilaments may play a role in the anchorage of the chloroplast during intracellular photo-orientation.

Effect of the floury-2 locus on protein body formation during maize endosperm development

Abstract: The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. These proteins, collectively called zeins, are translocated into the lumen of the rough endoplasmic reticulum, where they assemble into protein bodies. Protein body formation in normal genotypes occurs via an ordered deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about 1 μm. These structures consist of a central core that contains predominantly α-zein; this central region is surrounded by a peripheral layer of β- and γ-zeins, and the entire structure is bounded by rough endoplasmic reticulum.

Actin and the elongation of plant cells

Abstract: We have investigated in parallel the effects of different types of inhibitors on elongation of oat coleoptile cells in IAA and on the integrity of the longitudinally oriented actin-containing microfilaments present in control cells as detected by rhodamine phalloidin (RP) staining. Where growth was 50% inhibited by cytochalasin D (CD), we observed extensive to complete breakdown of the microfilaments (MFs) with the appearance of new RP staining in a few nuclei and markedly along the cross walls. When the CD-treated coleoptiles were held at 4°C the nuclei were uniformly strongly stained and cross wall staining was not seen, suggesting that translocation to the nuclei may be an intermediate step in final disposition of the actin. The divalent ions calcium and magnesium both inhibited growth in a dose dependent way, with calcium giving 50% inhibition at 65 mM and magnesium at 25 mM. KCl was not inhibitory and did not reverse the inhibition by divalent ions even at 250 mM. At 50% inhibition by either ion, the long MFs in many cells were replaced either by short fragmented MFs and small brightly staining granules (calcium) or by short usually twisted MFs and large, less intensely staining masses (magnesium). Iodoacetate at 2mM inhibited growth almost completely and resulted in short, fragmented, twisted or curled MFs in most of the cells. Abscisic acid also caused replacement of some MFs with faintly fluorescent bodies somewhat larger than those in CaCl2; occasionally granules similar to those in CaCl2 were also seen. Only mannitol and galactose, which inhibit growth by their osmotic effect, did not cause breakup of the MFs; indeed the MFs in mannitol appeared if anything wider and thicker. The results show that under the influence of three types of growth inhibitors the actin-containing MFs in the cells are broken down to different extents resulting in new structures. The results support the idea that the integrity of the MF bundles is linked, perhaps causally, to the elongation of theAvena cells.

Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea

Abstract: The penetration peg is the structure used byMagnaporthe grisea to pierce the surface of rice leaves or very hard nonbiodegradable substrates. Penetration pegs produced by appressoria in vitro were examined by electron microscopy and immunofluorescence microscopy using various fluorophore labeled anti-actins. Freeze-substitution preparation of appressoria at early stages of substrate penetration showed that peg cytoplasm consisted primarily of a zone of exclusion, excluding even ribosomes, and was continuous with a similar region in the appressorium. Apical vesicles were, however, observed in short, presumably elongating pegs. Immunofluorescence microscopy was used to demonstrate binding of a monoclonal anti-actin to penetration peg cytoplasm, following “permeabilization” of appressoria by means of a brief sonication. Occasional filaments and ca. 300 nm diameter plaques were labeled in appressorial cytoplasm. Western blot analysis of germ tube extracts showed that the monoclonal probe bound predominantly to a single band with a molecular weight similar to that of rabbit muscle actin. Preincubation of the antibody with actin virtually eliminated peg labeling. We conclude that the penetration peg contains actin which may play a role in the formation of the zone of exclusion.

Molecular probes for the plant cell surface

Abstract: The precise deposition of the plant cell wall, in terms of orientation and timing, dictates the anatomy of a plant and the form of all plant structures. The varied cell fates within a developing structure, such as the root apex, can be seen at the earliest developmental stages by distinctive cell shapes, i.e., distinctive patterns of cell surface positions. In later developmental stages the importance of the cell surface is again emphasized by walls of distinctive form and chemistry associated with cell types. Understanding the form and functions of the plant celt surface will aid greatly our understanding of the developmental mechanisms underlying multicellular plant structures. Cell number and cell size increase within the closely adhered collective of a meristem, without slipping or sliding of neighbouring cells. Very little is known of the molecular mechanisms underlying the ceil cell in-

Ultrastructural immuno-localization of pectins and glycoproteins in Arabidopsis thaliana pollen grains

Abstract: With the monoclonal antibodies MAC 207 and JIM 8 we could demonstrate the presence and localization of arabino-galactan glycoproteins in pollen grains ofArabidopsis thaliana, with JIM 5 the presence and localization of unesterified pectins. MAC 207 preferentially labels the peripheral plasma membrane of the vegetative cell, whereas JIM 8 specifically labels the area of the sperm cell “wall”. Unesterified pectins are present in the inline layer near the plasma membrane, and in the outer intine region which contains remnants of cytoplasmic channels.

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Abstract: Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves of Solanum tuberosum L

Abstract: Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.

Spatio-temporal reorganization of intracytoplasmic microtubules is associated with nuclear selection and differentiation during the developmental process in the ciliateTetrahymena thermophila

Abstract: Indirect immunofluorescence has revealed various intracytoplasmic microtubular structures, which are transiently polymerized in specific subcellular locations during the developmental process of conjugation in the ciliateTetrahymena thermophila. These structures include: (1) micronuclear spindles, (2) perimicronuclear microtubules, (3) microtubular baskets surrounding migrating pronuclei, and (4) microtubules interconnecting the pronuclei with the conjugants' junctional zone. Furthermore, a peripheral network of intracytoplasmic microtubules related to the cell cortex is present in both vegetative cells and in conjugants. Comparative observations made on cells undergoing normal conjugation and defective conjugation (occurring either spontaneously or induced by taxol) has revealed some rules governing the pattern of deployment of conjugation-specific microtubules. The presence of perinuclear microtubular arrays during early postmeiotic stages of development is strictly limited to more anteriorly located nuclei which includes the selected haploid nucleus that further divides to form the stationary and migratory pronuclei. These perinuclear microtubules may be involved in the positional control of nuclear fates leading to effective nuclear selection. Microtubular bundles associated with pronuclei and connecting the junctional zone are only formed in the presence of functional pronuclei, and may be involved in the guidance of pronuclei leading to their fusion. The mechanism of cytoplasmic control of nuclear differentiation of derivatives of the zygotic nucleus appear to be associated with a coordinate action of two microtubular arrays: spindle microtubules of the second postzygotic division and the peripheral intracytoplasmic network of microtubules, leading to a proper subcortical positioning of the postzygotic nuclei at opposite poles of the cell.

Immunolocalization of H + -ATPases in the plasma membrane of pollen grains and pollen tubes of Lilium longiflorum

Abstract: A heterogeneous distribution of H+-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H+-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H+-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H+-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.

Hydrostatic pressure mimics gravitational pressure in characean cells.

Abstract: Hydrostatic pressure applied to one end of a horizontal Chara cell induces a polarity of cytoplasmic streaming, thus mimicking the effect of gravity. A positive hydrostatic pressure induces a more rapid streaming away from the applied pressure and a slower streaming toward the applied pressure. In contrast, a negative pressure induces a more rapid streaming toward and a slower streaming away from the applied pressure. Both the hydrostatic pressure-induced and gravity-induced polarity of cytoplasmic streaming respond identically to cell ligation, UV microbeam irradiation, external Ca2+ concentrations, osmotic pressure, neutral red, TEA Cl-, and the Ca2+ channel blockers nifedipine and LaCl3. In addition, hydrostatic pressure applied to the bottom of a vertically-oriented cell can abolish and even reverse the gravity-induced polarity of cytoplasmic streaming. These data indicate that both gravity and hydrostatic pressure act at the same point of the signal transduction chain leading to the induction of a polarity of cytoplasmic streaming and support the hypothesis that characean cells respond to gravity by sensing a gravity-induced pressure differential between the cell ends.

Senescence of hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) in primary culture

Abstract: In order to evaluate the suitability of isolated fish hepatocytes for toxicological studies, hepatocytes were isolated from rainbow trout (Oncorhynchus mykiss) by liver collagenase perfusion. Isolated hepatocytes were investigated for seven days in primary culture on uncoated petri dishes using light and electron microscopy. Viability of isolated hepatocytes, as estimated from trypan blue exclusion, declined from >90% at the beginning of the incubation to ⩽80% after eight days of primary culture. Survival of hepatocytes was best at an incubation temperature of 14°C, and addition of fetal calf serum failed to improve cell performance. Freshly isolated hepatocytes appeared as solitary spherical cells with numerous microvilli at the outer surface. Except for a 30% reduction in cell size due to stress-induced glycogen reduction, the ultrastructure of freshly isolated hepatocytes closely resembled that of rainbow trout hepatocytes in vivo, which is characterized by distinct cytoplasmic segregation into a perinuclear portion containing rough endoplasmic reticulum (RER) arranged in extensive stacks, mitochondria, peroxisomes and the peribiliary complex (dictyosomes, lysosomes), and large peripheral glycogen fields occasionally interspersed with small lipid inclusions. Within one day of culture, about 60–80% of the isolated hepatocytes sedimented to form a monolayer attached to the culture dishes, whereas up to 20% remained in suspension forming hepatocyte aggregates. Cell adhesion was weak, and during prolonged culture increasing amounts of cell detached, whereas the floating cell accumulations grew to aggregates of more than 100 cells. Cell viability and ultrastructure was similar in monolayers and spheroids, and only from the fifth day in culture, did hepatocytes in the centre of floating aggregates become necrotic. From day 1 to day 5, hepatocytes in primary culture displayed only minor cytological alterations. Excellent cytoplasmic compartmentation, restoration of hepatocytic glycogen stores, high secretion rates of very low density lipoproteins by dictyosomes, establishment of cell-to-cell contacts, restitution of cellular polarity and the epithelial character of the cells, as well as formation of bile canaliculi documented recovery of the hepatocytes in primary culture. From day 5 in culture, an increasing number of cells detached from the substratum, and cell senescence was indicated by a marked increase in ultrastructural heterogeneity, with progressive vesiculation and fractionation of the RER, transformation of RER stacks into huge membrane whorls, aggregation and proliferation of peroxisomes and SER, lack of dictyosomal VLDL production, drastic accumulation of lysosomes, myelinated bodies and autophagic vacuoles, as well as successive exhaustion of cellular glycogen deposits. Whereas with conventional methods for assessing cell viability, most hepatocytes appeared intact for up to ten days in culture, cytological investigations revealed severe deterioration of cellular integrity from day 7. However, for incubation periods of up to five days, isolated rainbow trout hepatocytes can be recommended as an excellent model for physiological and toxicological studies.

Somatic embryogenesis, formation of morphogenetic callus and normal development in zygotic embryos of Arabidopsis thaliana in vitro

Abstract: Zygotic embryos ofArabidopsis thaliana showed three different types of developmental response, when cultured in vitro: (1) normal development, (2) formation of morphogenetic callus, and (3) somatic embryogenesis. Early zygotic embryos were mechanically isolated and inoculated into different volumes of various culture media. It was possible to isolate embryos to the octant stage. Survival and further development in culture were observed in embryos to the early globular stage. Culture success increased with the initial size of the cultivated embryos. Neither the volume of the culture medium nor its composition were found to significantly influence the proportion of embryos developing in vitro. Whereas normal development from stages beyond 35 μm diameter was possible without phytohormones, callus formation was frequently observed in the presence of phytohormones, even if used at very low concentrations. Embryos smaller than 35 μm formed callus even without added phytohormones, and the proportion of embryos undergoing callus formation decreased with increasing embryo size at the time of culture initiation. Shoot morphogenesis was easily induced in embryo derived callus. Somatic embryogenesis was reliably observed during the culture of embryos from later stages (post heart-shaped) in liquid medium on a shaker.

Relationship of actin organization to growth in the two forms of the dimorphic yeast Candida tropicalis

Abstract: Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.

Sperm cells in pollen tubes ofNicotians tabacum L.: three-dimensional reconstruction, cytoplasmic diminution, and quantitative cytology

Abstract: The organization of the sperm cells and vegetative nucleus (male germ unit) ofNicotiana tabacum was examined 18 h after semivivo pollination using transmission electron microscopy, computerassisted serial section reconstruction and quantitative cytology. Based on a measurement of 11 cellular parameters in nine reconstructed sperm cell pairs, there are no statistically significant differences between the two cells. The Svn is characterized by a strapshaped cytoplasmic extension that is physically associated with the surface of the vegetative nucleus. The nucleus is located adjacent to the sperm crosswall, with sperm organelles being distributed between the nucleus and the extension. The Sua is a tapered cell with cytoplasmic areas at both poles and deep axial invaginations near the crosswall. This cell has a centrally-located nucleus and a largely polar distribution of organelles. Three mechanisms for cytoplasmic diminution were observed that appear to contribute actively to the loss of cytoplasmic volume and organelles: (1) enucleated cytoplasmic body production in the Sua; (2) vesiculation at the tip of the cytoplasmic projection of the Svn; and (3) vesicle-containing body accumulation in the periplasm of both the Svn and Sua.

Pollen development in orchids 4. Cytoskeleton and ultrastructure of the unequal pollen mitosis in Phalaenopsis

Abstract: The unequal first mitosis in pollen ofPhalaenopsis results in a small generative cell cut off at the distal surface of the microspore and a large vegetative cell. No preprophase band of microtubules is present, but polarization of the microspore prior to this critical division is well marked. A generative pole microtubule system (GPMS) marks the path of nuclear migration to the distal surface, and the organelles become unequally distributed. Mitochondria, plastids and dictyosomes are concentrated around the vegetative pole in the center of the microspore and are almost totally excluded from the generative pole. The prophase spindle is multipolar with a dominant convergence center at the GPMS site. The metaphase spindle is disc-shaped with numerous “minipoles” terminating in broad polar regions. In anaphase, the spindle becomes cone-shaped as the spindle elongates and the vegetative pole narrows. These changes in spindle architecture are reflected in the initial shaping of the telophase chromosome groups. F-actin is coaligned with microtubules in the spindle and is also seen as a network in the cytoplasm. An outstanding feature of orchid pollen mitosis is the abundance of endoplasmic reticulum (ER) associated with the spindle. ER extends along the kinetochore fibers, and the numerous foci of spindle fibers at the broad poles terminate in a complex of ER.

Ultrastructure of root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Abstract: Individuals of the plant-parasitic nematodeCriconemella xenoplax, monoxenically cultured on root expiants of clover, carnation, and tomato, fed continuously for up to 8 days from single cells in the outer root cortex. Individual cortical cells parasitized by nematodes were modified into discrete “food cells” in all hosts examined. The nematode's stylet penetrated between epidermal cells and frequently through a subepidermal cortical cell. Electron-transparent callose-like material continuous with the cell wall enveloped the portion of the stylet that traversed subepidermal cortical cells. Food cells were typically located in the first or second cell layers of the cortex. The stylet penetrated 5–6 μm through the wall of the food cell without penetrating the plasma membrane. Electron-transparent callose-like deposits formed between the invaginated plasma membrane and stylet, except at its aperture. The plasma membrane of the food cell was appressed tightly to the wall of the stylet aperture creating a 130–160 nm hole in the membrane. This opening provided continuity between the lumen of the stylet and the food cell cytosol for ingestion of nutrients by the nematode. Ribosomes were dissociated from the cisternae of the endoplasmic reticulum in food cells and accumulated with other cell organelles in a zone of modified cytoplasm around the stylet. A fibrillar material appeared to form a barrier in the cytosol around the stylet aperture that limited movement of cell organelles toward the aperture. Electron-dense secretory components were secreted into the food cell by the nematode. Clusters of putative nematode secretory components consisting of 20–40 nm diameter, electron-dense particles were dispersed in the densely particulate zone of cytoplasm around the stylet tip. The cytosol immediately around the stylet aperture in the center of the modified cytoplasm was finely granular.

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Abstract: In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

Abstract: To assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.