Immunolocalization of myosin in rhizoids ofChara globularis Thuill

Summary Myosin-related proteins have been localized immunocytochemically in gravity-sensing rhizoids of the green algaChara globularis using a monoclonal antibody against the heavy chain of myosin from mouse 3T3 cells and a polyclonal antibody to bovine skeletal and smooth muscle myosin. In the basal zone of the rhizoids which contain a large vacuole, streaming endoplasm and stationary cortical cytoplasm, the monoclonal antibody stained myosin-related proteins as diffusely fluorescing endoplasmic strands. This pattern is similar to the arrangement of subcortical actin filament bundles. In the apical zone which contains an aggregation of ER membranes and secretory vesicles for tip growth, diffuse immunofluorescence was detected; the intensity of the signal increasing towards the apical cell wall. The most prominent myosin-staining was associated with the surface of statoliths in the apical zone. The polyclonal antibody produced a punctate staining pattern in the basal zone, caused by myosin-related proteins associated with the surface of drganelles in the streaming endoplasm and the periphery of the nucleus. In the apical zone, this antibody revealed myosin-immunofluorescence on the surface of statoliths in methacrylate-embedded rhizoids. Neither antibody revealed myosin-immunofluorescence on the surface of organelles and vesicles in the relatively stationary cytoplasm of the subapical zone. These results indicate (i) that different classes of myosin are involved in the various transport processes inChara rhizoids; (ii) that cytoplasmic streaming in rhizoids is driven by actomyosin, corresponding to the findings onChara internodal cells; (iii) that actindependent control of statolith position and active movement is mediated by myosin-related proteins associated with the statolith surfaces; and (iv) that myosin-related proteins are involved in the process of tip growth.     

The dynamic behavior of cytoplasmic F-actin in growing hyphae

Summary A dynamic population of cytoplasmic F-actin was observed with electroporated rhodamine phalloidin (RP) staining in growing hyphae ofSaprolegnia ferax. This central actin population was distinct from the fibrillar peripheral network previously described in chemically fixed hyphae in that it was diffuse, pervaded the entire cytoplasm and was most concentrated in the central cytoplasm 8.4 m from the tip. The peripheral network did not stain with electroporated RP. The apical concentration of central cytoplasmic actin was only present in growing hyphae and developed prior to tip extension. It co-localized with the polarized distribution of mitochondria and endoplasmic reticulum in the tip, suggesting that it functions in positioning these organelles during tip growth. Within the central actin there was a consistent apical cleft which only occurred in growing hyphae and whose position predicted the direction of tip growth. This cleft was coincident with the known accumulation of apical wall vesicles, suggesting that it is either established by vesicle exclusion of the central actin network or is permeated by a portion of the in vivo unstained peripheral network. Photobleaching studies showed that in both growing and non-growing hyphae, cytoplasmic actin continually and rapidly moved from subapical regions to the tip where it accumulated. It mostly moved forward at the rate of tip growth, while some also left the tip, presumably to populate subapical regions.Abbreviations RP rhodamine phalloidin - F-actin filamentous actin - DIC Nomarski differential interference contrast - FITC fluorescein isothiocyanate     

CYTOLOGY AND MORPHOGENESIS IN THE RHIZOMORPH OF ARMILLARIA MELLEA

Aided by the techniques of thin sectioning and electron microscopy, the apical region of the rhizomorph of Armillaria mellea has been examined. This region is composed of concentric zones of morphologically distinct tissues derived from a subapical meristematic zone designated the apical center. Meristematic activity is of two types: (1) primary, localized in the apical center, in which new hyphal elements are formed from apical initials, and (2) secondary, localized in the lateral regions of the apex, in which elaboration of the hyphal elements by means of elongation and secondary crosswall formation takes place. From these meristematic zones the tissues of the mature rhizomorph are derived and include: (a) peripheral hyphae, (b) cortex, (c) subcortex, and (d) primary and secondary medulla. The manner of differentiation of an apical initial appears unique and involves synchronous nuclear divisions accompanied by segmentation in many planes. The result of this activity is the formation of multinucleate hyphae. Apical initials are usually highly cytoplasmic and possess peculiar non-membrane-bound fibrous bundles, but in all other respects they resemble the hyphae of most Basidiomycetes thus far examined with the electron microscope.     

The fine structure ofPhycomyces

Summary The cytological organization of the apices of sporangiophores and hyphae ofPhycomyces Blakesleeanus was studied by means of light- and electron microscopy. The sporangiophore apex in growth stage I contains a mass of cytoplasm in which is embedded a cluster of lipid globules. Within the plug several zones are differentiated by the grouping of organelles. These zones are not separated by membranes. The most apical zone is low in nuclei and vesicles but rich in mitochondria and dense bodies. Below this zone lies a compact group containing up to several hundred nuclei. Along the midline of the cell, below these nuclei, lies an ovoid region from which vesicles, nuclei and mitochondria are excluded. In this ovoid exclusion zone lies the cluster of lipid globules mentioned above. Lateral to the exclusion zone (i.e. in the peripheral region of the cell) the cytoplasm is rich in nuclei, mitochondria, dense bodies, and especially in developing autophagic vesicles. Of these vesicles, the most mature are found farthest from the cell apex. The region between the exclusion zone and the upper end of the cell's large central vacuole is occupied largely by mature, swollen autophagic vesicles. In addition to the zonal organization described above, microtubules are found to run along the cylindrical cell's axis at a distance from the cell wall, and extend to the extreme apex of the cell. Similar tubules occur in growing hyphae, together with dense bodies, and the hyphal apex contains non-autophagic vesicles that increase in size with distance from the hyphal tip. The hyphae lack the zonation shown by sporangiophore apices. Perinuclear masses of cisternae are described and related to the dictyosomes of higher plants. The findings are discussed in relation to the function of the apices in tip growth and sporulation.This work was supported in part by a National Science Foundation Graduate Fellowship to the author, and in part by grant No. GB 3241 from the National Science Foundation to ProfessorKenneth V.Thimann.     

Localized membrane-wall adhesions inPelvetia zygotes

Summary Membrane-wall adhesions in zygotes of the brown algaPelvetia were visualized following plasmolysis. Strands of cytoplasm remained firmly attached to the cell wall at discrete adhesion sites during plasmolysis. Adhesion sites were uniformly distributed in ungerminated zygotes, but were concentrated in the apical 5 m of the elongating rhizoid in germinated zygotes. Few adhesions were detected along the flanks of the rhizoid or in the thallus region of germinated zygotes. The structure, physiology and function of apical adhesions in the rhizoid were characterized. F-actin was found at adhesion sites in plasmolyzed zygotes labeled with rhodamine phalloidin, and disruption of cortical F-actin reduced the number of adhesions. Manipulation of cytosolic H⁺ and Ca²⁺ activities also disrupted adhesions. On the extracellular surface, the number of adhesions was reduced by inhibition of cellulose synthesis, protease cleavage of wall proteins, and changes in extracellular H⁺ and Ca²⁺ activities. Chronic treatment with the synthetic peptide RGDS, which prevents cell adhesion in fibroblasts, also reduced adhesion number. The number of adhesions per cell did not correlate with growth rate, but was inversely correlated with the ability to establish new rhizoid growth sites. The results indicate that membrane wall adhesions containing F-actin on the cytoplasmic face are localized in the growing rhizoid apex. The adhesions may be structurally related to focal adhesions in animal cells.Abbreviations ASB actin-stabilizing buffer - ASW artificial seawater - DCB 2,6-dichlorobenzonitrile - EGTA ethyleneglycol bis-(amino-ethyl ether) N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - Pipes piperazone-N,N-bis-(2-ethanesulfonic acid) - Tris tris(hydroxymethyl)amino-methane     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

Microtubule organization in the differentiating transfer cells of the placenta inLilium spp.

Summary Placental cells in the ovarian transmitting tissue ofLilium spp. are organized as transfer cells with inbuddings facing the ovarian locule. A detailed analysis of microtubule (MT) organization during development of these polarized cells is reported here. Formation of wall projections occurs at the apical part of the cell starting on the day of anthesis, and a fully mature secretion zone is found four days after anthesis. MTs are organized into distinct cortical and central arrays. The cortical array undergoes a unique transition at anthesis. MTs in the basal half of the cell remain in longitudinal bundles while in the apical half of the cell their longitudinal orientation is replaced by a transverse alignment. One day after anthesis, these transverse bundles become a meshwork of short, randomly organized MTs, while MTs in the basal half of the cell retain their longitudinal alignment. The realignment of MTs in the apical half of the cell coincides with the deposition of the secondary cell wall. The central array is composed of short, randomly arranged strands of MTs in the cytoplasm between the nucleus and the apical and basal periclinal walls of the cell. This array first appears as solitary strands in the apical part of the cell one day before anthesis. The central array extends during development and is eventually seen in the basal half of the cell. We propose that MTs in the cortical region near the apical wall act as templates for the deposition of cellulose microfibrils in the secondary cell wall. MTs in the central array in these transfer cells may be involved in the trafficking of vesicles and/or positioning of organelles near the secretion zone.Abbreviations MT microtubule - daa day after anthesis - dba day before anthesis     

A HISTOCHEMICAL STUDY OF THE SEEDLING SHOOT APICAL MERISTEM OF PINUS LAMBERTIANA

Vernalized seeds of Pinus lambertiana were scarified and planted in perlite. At 5, 8, 10, 13 and 16 days after planting, seedlings were selected for morphological examination and histochemical study. The shoot apical meristem consisted of a relatively homogeneous population of cells at 5 days. Cytohistological zonation was observed in the meristem by the eighth day and needle primordia initiation began at this time. Acid phosphatase (AP) activity was high in the extreme tip of the apex at 5 days. At 8 days AP activity was intense in the peripheral zone but weak in the apical initial and central mother cell zones. The apical meristem of the 10–16-day-old seedlings exhibited high AP activity in the peripheral zone only during the early stages of needle primordia initiation. The distribution of cytoplasmic and nuclear protein-bound SH was correlated with cytohistological zonation. Protein-bound SH was distributed relatively uniformly at 5 days, but by the eighth day the 4 cytohistological zones contained differential quantities. Succinic dehydrogenase (SD) activity was observed throughout the apex at 5 days, but by the eighth day the apical initial and central mother cell zones exhibited differentially greater levels of SD activity. Irradiation with 500 R of X-rays at 7 days after planting completely inhibited needle primordia initiation and disrupted the cytohistological zonation of the apex. Correlated with the inhibition of needle primordia initiation was the loss of SD activity in the apical initial and central mother cell zones. Irradiation also resulted in the gradual loss of protein-bound SH from the cytoplasm of the apical initial, central mother cell and peripheral zone.     

A MORPHOMETRY STUDY OF THE ULTRASTRUCTURE OF ECHINOCEREUS ENGELMANNII (CACTACEAE). II. THE MATURE,ZONATE SHOOT APICAL MERISTEM

The shoot apical meristems of adult Echinocereus engelmannii plants are zonate and have a tunica, central mother cells, a peripheral zone, and a pith-rib meristem. An ultrastructural, stereological study showed that each zone has its own distinct ultrastructure, but that the differences between the zones are quite small, both on a protoplasmic basis and on a cytoplasmic basis. Furthermore, the ultrastructure present in the adult apices differed only slightly from that which had been found in seedling apices, demonstrating a long-term stability of structure. The standard deviations found in the sample were small, indicating little variability from one plant to the next and suggesting that there are little or no cyclic changes during the plastochron or a 24-hr photoperiod. The ultrastructures found in the shoot apical meristems differed significantly and markedly from mature tissues of the same plants.     

ZONATION IN THE APICAL CELL OF ZONARIA

Light and electron microscopic techniques were used to study the row of apical cells that form the meristem of the dictyotalean brown alga, Zonaria farlowii. A distinctly polar pattern occurs in the cells. Four ultrastructurally different cytoplasmic zones have been seen: an apical zone, a nuclear zone, a compound vacuolar zone, and a vegetative zone. The apical cell of Zonaria is a differentiating cytoplasmic unit where striking intracellular gradients occur.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Cytoplasmic vesicles in germinating spores ofGilbertella persicaria

Summary Germ tube apices ofGilbertella persicaria contain cytoplasmic vesicles, similar to the secretory vesicles found at the tips of vegetative hyphae. The vesicles are present at all stages of development, from the time of germ tube initiation to the establishment of branched hyphae. In contrast to the abundant vesicles at tips of established hyphae, the germ tubes have only a few apical vesicles in a layer next to the plasma membrane. When germinated spores are treated by washing and centrifuging prior to fixation, the cytoplasm is often disrupted near the apex, and the clusters of apical vesicles disappear. The findings indicate the delicate nature of hyphal tips and the necessity of avoiding prefixation stresses in order to preserve the apical apparatus of growing hyphae.     

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.     

PLASMOLYSIS,HECHTIAN STRAND FORMATION,AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID,CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.     

Hyphal tip organization inAllomyces arbuscula

Summary Light and electron microscopic observations on vegetative hyphae ofAllomyces arbuscula revealed the specialized organization of the tip. There were some minor differences related to culture conditions, but the main ultrastructural features common to all hyphal tips disclosed a special type of organization distinct from that of other fungi. A crescent-shaped apical zone consisted of vesicles and membrane cisternae embedded in a granular matrix. Vesicles fused with the apical plasmalemma and presumably contributed to its expansion and to wall growth. The apical zone contained few ribosomes and generally no other organelles. Mitochondria were concentrated in the immediate subapical zone and scattered through the remainder of the hyphae, as were microbodies. Microtubules formed an asterlike structure with its center in the apical zone. Proximally of the apex, microtubules were axially oriented. Nuclei occurred only a certain distance from the tip. The elements of the apex may maintain the polarity of the hyphae via a gradient and hold it in a state of vegetative growth.     

UV microirradiations elicit Ca2+-dependent apex-directed cytoplasmic contractions in hyphae

Summary Polarized tip-ward cytoplasmic contractions were induced in hyphae ofSaprolegnia ferax with ultraviolet microirradiations. These unidirectional contractions were similar in appearance and ionic requirements to those previously induced in hyphae ofBasidiobolus magnus, suggesting that the observed inherent cytoplasmic polarity is a general phenomenon. During growth the cytoplasm is continually moving forward with respect to the lateral cell wall and plasma membrane in order to maintain its position in the tip. These contractions may be an exaggerated form of this cytoplasmic migration. F-actin was most concentrated in the contracted cytoplasm, implying that it may be involved in generating the contraction. Contractions were enhanced by external Ca²⁺ and by irradiating the tip region which is rich in Ca²⁺ sequestering organelles, suggesting that flooding of the cytoplasm with Ca²⁺ caused the contractions. H⁺ did not affect contraction frequency. Neither the change in cytoplasmic consistency that preceded contraction, the contraction itself, nor the F-actin damage induced were confined to the microirradiated zone. This is in keeping with irradiation-induced damage to a network under tension or a flux of diffusible ions causing the response. Thus Ca²⁺ may regulate actin-myosin interactions that generate cytoplasmic migration.Abbreviations EGTA ethyleneglycol-bis-(-amino-ethyl ether) N, N-tetra-acetic acid - F-actin filamentous actin - PIPES piperazine-N N-bis-(2-ethanesulfonic acid) - wavelength     

Stellate Chlamydospores and Thromboplerous Hyphae in the Mycelium of the Agaric <Emphasis Type="Italic">Lepista flaccida</Emphasis>

Development and morphology of mycelial chlamydospores and thromboplerous hyphae of the agaricoid Hymenomycete Lepista flaccida are described from laboratory cultures. The chlamydospore mother cell wall produces finger-like projections that become empty, stellate appendices of the mature spore. The fully developed wall is multi-layered. The cytoplasm contains two nuclei and one or two big oil drops. In thromboplerous hyphae, initial grains of the deuteroplasm condense into a solid, homogeneous mass.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

AN ULTRASTRUCTURAL BASIS FOR HYPHAL TIP GROWTH IN PYTHIUM ULTIMUM

Hyphae of the fungus Pythium ultimum extend by tip growth. The use of surface markers demonstrates that cell expansion is limited to the curved portion of the hyphal apex. Growing and non-growing regions are reflected in internal organization as detected by light and electron microscopy. The young hypha consists of three regions: an apical zone, a subapical zone and a zone of vacuolation. The apical zone is characterized by an accumulation of cytoplasmic vesicles, often to the exclusion of other organelles and ribosomes. Vesicle membranes are occasionally continuous with plasma membrane. The subapical zone is non-vacuolate and rich in a variety of protoplasmic components. Dictyosomes are positioned adjacent to endoplasmic reticulum or nuclear envelope, and vesicles occur at the peripheries of dictyosomes. A pattern of secretory vesicle formation by dictyosomes is described which accounts for the formation of hyphal tip vesicles. Farther from the hyphal apex the subapical zone merges into the zone of vacuolation. As hyphae age vacuolation increases, lipid accumulations appear, and the proportional volume of cytoplasm is reduced accordingly. The findings are integrated into a general hypothesis to explain the genesis and participation of cell components involved directly in hyphal tip growth: Membrane material from the endoplasmic reticulum is transferred to dictyosome cisternae by blebbing; cisternal membranes are transformed from ER-like to plasma membrane-like during cisternal maturation; secretory vesicles released from dictyosomes migrate to the hyphal apex, fuse with the plasma membrane, and liberate their contents into the wall region. This allows a plasma membrane increase at the hyphal apex equal to the membrane surface of the incorporated vesicles as well as a contribution of the vesicle contents to surface expansion.     

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.