Plasma-membrane rosettes involved in localized wall thickening during xylem vessel formation of Lepidium sativum L.

Developing xylem vessel elements in roots of cress, Lepidium sativum L., were freeze-fractured after rapid freezing in nitrogen slush (without cryoprotection). With the double-replica technique, both the plasmatic fracture face (PF) and the extraplasmatic fracture face (EF) of the plasma membrane were exposed. The EF revealed abundant, but rather indistinct terminal globules; whereas the PF showed numerous rosettes. Terminal globules and rosettes were localized, restricted to regions of secondary wall thickening only, and showed comparale frequencies per m², supporting the assumption that they are part of the same synthase complex. The abundance of rosettes in regions of high cellulose production supports their postulated involvement in cellulose microfibril formation. With up to 191 rosettes per m², the rosettes appear to be too densely arranged to be directly aligned on individual microtubules. This favors the channelling hypothesis of synthase movement in the plasma membrane.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Structure,function, and development of the peristome of the moss,Rhacopilum tomentosum,with special reference to the problem of microfibril orientation by microtubules

Summary The movement of the outer peristome teeth of the sporangium of the moss,Rhacopilum tomentosum, is driven by different swelling velocities of the outer (plates) and inner (ridges) wall thickenings due to suberin-like substances and wax-lamellae which enclose the ridges. The plates do not contain suberin-like material. The hydrophobic materials are secreted with the participation of smooth tubular ER.—When the local wall thickenings of the peristome teeth are formed, microtubules are concentrated along the plasmalemma in the thickening regions. They run along the crest of the developing plates (i.e., normal to the long axis of the tooth) and parallel to the long axis in the ridge cells. The wall thickenings are composed of layers of parallel microfibrils and of matrix substances. With a few exceptions microtubules and microfibrils have different directions. Golgi vesicles, subsurface ER and coated regions in the plasmalemma also are involved in cell wall formation. The function of the microtubules is discussed.     

Tip cell growth and the frequency and distribution of particle rosettes in the plasmalemma: Experimental studies inFunaria protonema cells

Summary ComparingFunaria protonema tip cells of different age and of experimentally modified growth rate (by changing the light-dark-regime, by application of colchicine and of D₂O and by plasmolysis) we found that the site and intensity of growth are related closely to the distribution and frequency of particle rosettes in the PF of the plasma membrane. The results confirm previous suggestions that the rosettes are involved in cellulose fibril formation and that they have a rather short life time (about 10–15 minutes,Reiss et al. 1984). The appearance of rosettes seems to depend on the exocytosis of Golgi vesicle containing wall matrix material. Morphometric calculations suggest that each Golgi vesicle may incorporate one rosette into the plasmalemma in caulonema tip cells.     

The supramolecular organization of red algal cell membranes and their participation in the biosynthesis and secretion of extracellular polysaccharides: a review

Summary The relationship between the supramolecular organization of red algal cell membranes and the biosynthesis and secretion of the cell wall skeletal and matrix polysaccharides is reviewed. Freeze-fracture studies have revealed that organized macromolecular structures — linear terminal complexes and tetrads — are present on the plasma membrane and on membranes of the endomembrane system. The linear terminal complexes seem to be involved in the biosynthesis, assembly, and orientation of the cellulose microfibrils and the tetrads in the synthesis of the matrix polysaccharides. It is shown how the research on the supramolecular organization of cell membranes has increased the knowledge on the biosynthesis and secretion of the extracellular crystalline and non-crystalline polysaccharides in red algae. In this review, the progress to date is discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Investigations of the turnover of the putative Cellulose-synthesizing particle “rosettes” within the plasma membrane ofFunaria hygrometrica protonema cells

Summary YoungFunaria protonemata were treated with Monensin (10^–6 M) and Cytochalasin (CB) (2×10^–5 M). The influence of the inhibitors on a) elongation growth, b) cell fine structure and c) particle rosettes within the plasma membrane after freeze fracture was observed. Monensin stopped cell growth, caused swelling of the mitochondria and plastids and inhibited the secretory activity of the Golgi apparatus within about 15 minutes. The number of rosettes in the PF of the plasma membrane was distinctly reduced after 4–5 minutes and decreased further to only very few after 30 minutes. The tip to base gradient in distribution was maintained for a long time. The effects were reversible, regeneration occurred within 3 hours. CB treatment showed no effect on elongation growth and cell fine structure. The number of rosettes, however, was strongly reduced within 3 minutes exposure time and their distribution was nearly uniform then. Number and tip to base gradient increased again after 6 minutes intoxication. The results are discussed in regard to the turn over of the rosettes.Abbreviations CB Cytochalasin B - PF protoplasmic fracture face - F-vesicle flat vesicle - F-Actin filamentous actin - G-Ac-tin globular actin     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

Eyespot membranes in newly released zoospores of the green algaChlorosarcinopsis gelatinosa (Chlorosarcinales) and their fate during zoospore settlement

Summary Eyespot membranes in zoospores of the green algaChlorosarcinopsis gelatinosa were studied with the freeze-fracture technique. The PF of the plasmalemma overlying the eyespot lipid globules contains significantly greater numbers of intramembraneous particles (IMP; 8,200 IMP/m²) compared to other areas of the plasmalemma (2,100 IMP/m²). In the eyespot area the EF of the plasmalemma reveals no IMP, but regularly arranged depressions corresponding to the PF particles. Sizes of PF particles are not significantly different between the eyespot area and other areas of the plasmalemma. Zoospore settlement starts approximately two hours after release and involves in sequence, rounding up of the cells, retraction of the flagella and secretion of a cell wall. Eyespot membrane specializations on the PF of the plasmalemma disappear during flagellar retraction and before cell wall secretion.The functional significance of eyespot membrane specializations is discussed in accordance with the view that these membranes are engaged in photoreception and primary sensory transduction relating to green algal phototaxis.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery

Summary The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80—300 nm diameter) in their cytoplasm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre-and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 m long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.     

Paleolimnological studies using sequential lipid extraction from recent lacustrine sediment: recognition of source organisms from biomarkers

Free lipids were isolated from recent sediment of Loch Affric by solvent extraction; hydrolysis of residual sediment, initially with dilute alkali and then with mineral acid, gives two additional bound lipid components.The distribution patterns of fatty acids, hydroxyacids and total neutral constituents in these chemically-distinct lipids show that the mode of occurrence contains much information. The molecular compositions of the neutral and acidic fractions obtained from the three lipid extracts were determined by gas chromatography — mass spectrometry. Acidic and neutral free lipids show a dominance of long-chain (>C₂₀) compounds characteristic of the wax constituents of higher plants; n-alkan-2-ones and -hydroxyacids may be microbial metabolites of wax constituents. Base hydrolysis liberates C₁₆ and C₁₈ -hydroxyacids occurring widely in the cutins and suberins of higher plants together with higher homologues occurring in plant suberins. -Hydroxyacids liberated by acidic hydrolysis show a molecular size range (C₁₀-C₁₈) and abundance of branched chain compounds typically occurring in lipopolysaccharides of gram-negative bacteria. The biological sources of c. 50% of the sediment lipids were identified using this organic geochemical approach.     

Calcium Ameliorates Effects of Lead in Protonema of Funaria hygrometrica Hedw.

Two-days-old in vitro grown protonemata of Funaria hygrometrica Hedw. were treated with a mixture PbCl₂ (4 M Pb²⁺) and CaCl₂ (16 M Ca²⁺) (Ca+Pb) for 48 h. The results were compared with the control: distilled water (H₂O) and the solution of PbCl₂ (4 M Pb²⁺) (Pb). Protonemata treated with Ca+Pb were longer and contained more cells than those treated with Pb. Moreover, a lower number of cells showed apical cell deformations typical for lead toxicity: swollen tips and wall thickenings at the apex. If deformations were present they were not as extended as in Pb. In comparison with the control, however, protonemata treated with Ca+Pb were shorter, contained a lower number of cells and some apical cells in this material were altered. It can be concluded that the presence of calcium partially neutralised toxic effects of lead in Funaria hygrometrica protonemata cells.     

ζ-Potential of n-Alkane Emulsion Droplets and Its Role in Substrate Transport into Yeast Cells

The -potentials of n-alkane droplets, formed by fatty acids, were studied in model systems of the culture liquid of yeasts (Candida maltosa) capable of utilizing n-alkanes. The value of the -potential was found to depend on the droplet size. The negative -potential of submicron droplets was so high that it prevented the droplets from coagulating with cells also possessing a high negative -potential. The dominant role of submicron n-alkane droplets in the kinetics of yeast growth could be accounted for by the existence of a mechanism regulating the contact interactions of individual cells with the droplets followed by the uptake of the substrate.     

THE ASSOCIATION OF ROSETTE AND GLOBULE TERMINAL COMPLEXES WITH CELLULOSE MICROFIBRIL ASSEMBLY IN NITELLA TRANSLUCENS VAR. AXILLARIS (CHAROPHYCEAE)1,2

A freeze-fracture investigation of the putative cellulose synthesizing complex (terminal complex) morphology in Nitella translucens var. axillaris (A. Br.) R.D.W. internodal cells revealed single solitary EF globules and PF rosettes on the plasma membrane. The average density of rosettes in elongating internodal cells was 5.6 μm^?2 with slight spatial variation observed. In only three other algal genera (all zygnematalean) have rosette / globule terminal complexes been observed, while this characteristic is common to all vascular plants and one moss thus far investigated. This evidence strongly suggests that the rosette type of terminal complex morphology is an additional characteristic of charophycean algae and lends further support to the hypothesis that this group of algae represents the evolutionary line that gave rise to vascular plants. Observations were also made from the freeze-fracture of Nitella internodal cells concerning the orientation of cell wall microfibrils and cytoskeletal elements near the plasma membrane. The pattern of microfibril orientation in growing internodal cells is initially transverse to the cell long axis, becoming progressively axial presumably due to the strain of elongation. In mature internodal cells, the pattern of microfibril orientation is helicoidal. Microtubules appressed to the inner surface of the plasma membrane are oriented parallel to the most recently formed microfibrils in elongating and mature internodal cells.     

Modulation of swimming behavior in the medicinal leech

Expression of swimming in the medicinal leech (Hirudo medicinalis) is modulated by serotonin, a naturally occurring neurohormone. Exogenous application of serotonin engenders spontaneous swimming activity in nerve-cord preparations. We examined whether this activity is due to enhanced participation of swim motor neurons (MNs) in generating the swimming rhythm. We found that depolarizing current injections into MNs during fictive swimming are more effective in shifting cycle phase in nerve cords following serotonin exposure. In such preparations, the dynamics of membrane potential excursions following current injection into neuronal somata are substantially altered. We observed: 1) a delayed outward rectification (relaxation) during depolarizing current injection, most marked in inhibitory MNs; and 2) in excitor MNs, an enhancement of postinhibitory rebound (PIR) and afterhyperpolarizing potentials (AHPs) following hyperpolarizing and depolarizing current pulses, respectively. In contrast, we found little alteration in MN properties in leech nerve cords depleted of amines. We propose that enhanced expression of swimming activity in leeches exposed to elevated serotonin is due, partly, to enhancement of relaxation, PIR and AHP in MNs. We believe that as a consequence of alterations in cellular properties and synaptic interactions (subsequent paper) by serotonin, MNs are reconfigured to more effectively participate in generating and expressing the leech swimming rhythm.Abbreviations AHP Afterhyperpolarizing potential - DCC Discontinuous current clamp - DE Dorsal excitor motor neuron - DI Dorsal inhibitor motor neuron - IPSP Inhibitory postsynaptic potential - MN Motor neuron - PIR Postinhibitory rebound - VE Ventral excitor motor neuron - VI Ventral inhibitor motor neuron     

Structural and ultrastructural characteristics of the vascular apparatus of the sensitive plant (Mimosa pudica L.)

Summary 1. In motor organs ofMimosa pudica xylem contains living fibriform elements limited by a thick lignified highly pitted wall, whereas in other parts of the plant (stem, petiole, rachis), xylem and protoxylem vessels are closely associated with parenchyma cells which possess wall ingrowths. These ingrowths, at the apex of which the plasmalemma and the tonoplast touch, are localized like those of transfer cells of C type described byGunning andPate. Nevertheless, xylem parenchyma cells differ from cells of C type in several characteristics. Moreover, in motor organs, phloem contains cells characterized by wall ingrowths, less abundant on the parts adjacent to the sieve tubes; these cells which are localized near collenchyma cells of primary phloem, look like transfer cells of A type defined byGunning andPate; they are absent from internodes, petioles and rachides. 2. In motor organs, three types of vascular cells (companion cells, living xylem fibriform elements and protoxylem parenchyma cells) are characterized by reduced vacuolar volumes and well developed membrane systems, as compared with homologuous cells belonging to other parts of the plant. 3. A symplastic continuity holds from the middle of motor organs to their cortex: it is provided by the presence, in xylem and phloem respectively, of living fibriform elements and collenchyma cells bearing numerous pit fields containing large numbers of plasmodesmata. Several ultrastructural features suggest that the vascular apparatus ofMimosa pudica would be the site of intensive lateral transfer at different levels, specially in motor organs. Possible functions of certain structures observed are discussed in relation to some hypotheses relative to excitatory conduction pathways.     

Immature erythroid cells with novel morphology and cytoskeletal organization in adultXenopus

Summary Nucleated erythrocytes of non-mammalian vertebrates are a useful model system for studying the correlation between changes in cell shape and cytoskeletal organization during cellular morphogenesis. They are believed to transform from spheres to flattened discs to ellipsoids. Our previous work on developing erythroblasts suggested that pointed cells containing incomplete, pointed marginal bands (MBs) of microtubules might be intermediate stages in the larval axolotl. To test whether the occurrence of such pointed cells was characteristic of amphibian erythrogenesis, we have utilized phenylhydrazine (PHZ)-induced anemia in adultXenopus. In this system, circulating erythrocytes are destroyed and replaced by erythroblasts that differentiate in the blood, making them experimentally accessible. Thus, we followed the time-course of morphological and cytoskeletal changes in the new erythroid population during recovery. During days 7–9 post-PHZ, pointed cells did indeed begin to appear, as did spherical and discoidal cells. The percentage of pointed cells peaked at days 11–13 in different animals, subsequently declining as the percentage of elliptical cells increased. Since degenerating old erythrocytes were still present when pointed cells appeared, we tested directly whether pointed ones were old or new cells. Blood was removed via the dorsal tarsus vein, and the erythrocytes washed, fluorescently tagged, and re-injected. In different animals, 2–8% of circulating erythrocytes were labeled. Subsequent to induction of anemia in these frogs, time-course sampling showed that no pointed cells were labeled, identifying them as new cells. Use of propidium iodide revealed large nuclei and cytoplasmic staining indicative of immaturity, and video-enhanced phase contrast and anti-tubulin immunofluorescence showed that the pointed cells contained pointed MBs. The results show that pointed cells, containing incomplete, pointed MBs are a consistent feature of amphibian erythrogenesis. These cells may represent intermediate stages in the formation of elliptical erythrocytes.Abbreviations MB marginal band - MS membrane skeleton - PHZ phenylhydrazine     

Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts

A systematic investigation covering a wide diversity of yeast species was made on the appearance of respiratory deficient (petite) mutants after treatment with acriflavine. Petite mutants were obtained from certain species only, but in these species all strains were found to have in common the property of giving rise to petite mutants; such species were designated as petite positive. Species failing to give rise to petite mutants were accordingly called petite negative. The primary action of acriflavine, namely the inhibition of the synthesis of the respiratory system, was shown to occur not only in petite positive yeasts, but also in petite negative ones. Some implications of the results are discussed.     

Vom Einfluß des Phytochroms auf die Xylemdifferenzierung im Hypokotyl des Senfkeimlings (Sinapis alba L.)

Zusammenfassung P₇₃₀, das aktive Phytochrom, bewirkt eine vermehrte Bildung von Gefäßen (Tracheen und Tracheiden) im Hypokotyl des Senfkeimlings. Das Differenzierungsmuster der Leitbündel und der Verlauf der Leitbahnen sind im belichteten und im etiolierten Keimling gleich. Es wird geschlossen, daß auch bezüglich der Ausbildung der Leitbahnen das P₇₃₀ lediglich im Rahmen einer sekundären Differenzierung als Auslöser wirkt. Die primäre Differenzierung (vgl. Wagner und Mohr, 1966 b) wird durch P₇₃₀ offenbar nicht beeinflußt.

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Phytochrome-mediated control of xylem differentiation in the hypocotyl of the mustard seedling (Sinapis alba L.)

Summary P₇₃₀, the active phytochrome, causes an increased formation of xylem elements (tracheids and vessel elements) in the hypocotyl of the mustard seedling (Figs. 3,4). On the other hand, the pattern of differentiation of the bundles and the course of the bundles within the hypocotyl (Figs. 1,2) are the same in etiolated as well as in illuminated seedlings.—It has been concluded that in connection with bundle differentiation P₇₃₀ acts only as a trigger at the level of secondary differentiation. The pattern of differentiation is laid down in the course of primary differentiation which apparently is not influenced by P₇₃₀. The same problem has been dealt with more in detail in a foregoing paper (Wagner and Mohr, 1966b).