Basket-shaped structures formed by F-actin in the nuclei of elongating cells of Nicotiana tabacum

1993 ◽  
Vol 71 (5) ◽  
pp. 725-731 ◽  
Author(s):  
Harry M. P. Kengen ◽  
Ton van Amstel ◽  
Bart Knuiman
Keyword(s):  
Nicotiana Tabacum ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Filamentous Actin ◽  
Suspension Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Cytoplasmic Actin ◽  
Extraction Buffer ◽  
Confocal Immunofluorescence

Phalloidin labelled with trimethyl rhodamine isothiocyanate in extraction buffer was used to stain actin in suspension cells of tobacco. Confocal immunofluorescence microscopy indicated the presence of filamentous actin containing structures in interphase nuclei of elongating cells of Nicotiana tabacum ‘Bright Yellow 2 Go’. The results were not affected by the omission of DMSO from the extraction medium. These structures, called actin baskets, were present in about 20% of the cells after induced elongation and varied in size, shape, and number per nucleus. The cytoplasmic actin array remained intact. It is proposed that the baskets have a transient character and are related to differentiation. Key words: confocal laser scanning microscopy, elongation, F-actin, nucleus, protoplasts, TRITC–phalloidin.

Imaging arbuscular mycorrhizal structures in living roots of Nicotiana tabacum by light, epifluorescence, and confocal laser scanning microscopy

2001 ◽  
Vol 79 (2) ◽  
pp. 231-237 ◽  
Author(s):  
Horst Vierheilig ◽  
Michael Knoblauch ◽  
Katja Juergensen ◽  
Aart JE van Bel ◽  
Florian MW Grundler ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Arbuscular Mycorrhizal ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Root Cells

Light and epifluorescence (blue light excitation) microscopy was used to obtain micrographs of the same sections of unstained (living roots) and stained (dead) tobacco (Nicotiana tabacum L.) roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. To visualize all mycorrhizal structures, roots were in situ stained with trypan blue. The metabolically active fungal tissue was determined by an in situ succinate dehydrogenase stain. A comparison of micrographs of unstained and stained mycorrhizal tobacco roots revealed that (i) finely branched arbuscules do not autofluoresce, but high autofluorescence was observed in clumped structures of collapsed arbuscules; and (ii) finely branched arbuscules are metabolically active, but no activity can be detected in autofluorescent collapsed arbuscules. Confocal laser scanning microscopy was used in combination with the two fluorochromes 5(6)-carboxyfluorescein diacetate or 5(6)-carboxy-seminaphthorhodafluor. Both fluorochromes administered to abraded tobacco leaves are transported via the phloem to the roots. Loading plants with 5(6)-carboxyfluorescein diacetate resulted in a fluorescence of root cells with highly branched arbuscules. After loading the phloem with 5(6)-carboxy-seminaphthorhodafluor, all fungal structures in the root (from relatively thick hyphae to finest branches of arbuscules) were clearly visible in the intact root. The transport route of compounds from the plants to arbuscular mycorrhizal fungi is discussed.Key words: Glomales, mycorrhiza, fluorescence, SDH, confocal, transport.

scholarly journals Improved Bioassay of Xylella fastidiosa Using Nicotiana tabacum Cultivar SR1

Plant Disease ◽  
2008 ◽  
Vol 92 (1) ◽  
pp. 14-20 ◽  
Author(s):  
M. Francis ◽  
E. L. Civerolo ◽  
G. Bruening
Keyword(s):  
Nicotiana Tabacum ◽  
Xanthomonas Campestris ◽  
Laser Scanning ◽  
Xylella Fastidiosa ◽  
Quantitative Polymerase Chain Reaction ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Leaf Scorch

Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fastidiosa cells by transmission and scanning electron microscopy, as well as by confocal laser scanning microscopy, in the xylem cells of inoculated plants. The pathogenicity of selected reisolated strains was confirmed by inoculation of grape plants in the greenhouse. The average levels of X. fastidiosa cells/g of tissue, estimated by QPCR, were higher for PD strains than for ALSD strains and reflected the relative titers of these strains in economic hosts. No symptoms were observed and bacteria were not detected in untreated tobacco or in tobacco inoculated with Xanthomonas campestris pv. campestris or water. Symptoms induced by Xylella fastidiosa in this bioassay were fully expressed within 2 months following inoculation. The described bioassay, under optimized environmental conditions, provides a useful system for studying X. fastidiosa strains (e.g., confirmation of pathogenicity and differentiation of PD and ALSD pathotypes) and for investigating X. fastidiosa–host interactions. N. tabacum cv. SR1 tobacco was a better bioassay host for X. fastidiosa than N. tabacum cvs. Havana, RP1, and TNN described previously.

The roles of calmodulin polar distribution during pollen hydration and germination

2004 ◽  
Vol 82 (6) ◽  
pp. 774-780 ◽  
Author(s):  
Wen-Jing Tao ◽  
Shu-Ping Liang ◽  
Ying-Tang Lu
Keyword(s):  
Nicotiana Tabacum ◽  
Laser Scanning ◽  
Fusion Gene ◽  
Pollen Grains ◽  
Pollen Tubes ◽  
Pollen Grain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Directional Migration ◽  
Pollen Hydration

Polarity patterning of pollen germination is a vital process for angiosperm fertilization. In our study a new method was employed to investigate the real-time distribution of calmodulin (CaM) in living pollen grains and pollen tubes. The CaM–GFP fusion gene was constructed under the control of the pollen-specific promoter LAT52-7 and transformed into Nicotiana tabacum L. Through confocal laser scanning microscopy, high levels of CaM were observed to accumulate in the three germinal apertures, and a tip–base gradient of CaM was detected in elongating pollen tubes. During pollen-grain hydration and germination, one of the three germinal apertures aggregated a much higher level of CaM than the other two. In addition, CaM showed a directional migration from the cytoplasm to this germinal aperture, where the pollen tube would emerge. Interestingly, CaM was not detected in the reproductive nucleus of either pollen grains or pollen tubes. Our findings indicated that the directional migration of CaM existed during pollen hydration and germination, and this movement may play a crucial role in the normal polarity establishment of pollen germination.Key words: calmodulin, polarity, pollen grain, Nicotiana tabacum.

scholarly journals Plastid tubules of higher plants are tissue-specific and developmentally regulated

2000 ◽  
Vol 113 (1) ◽  
pp. 81-89 ◽  
Author(s):  
R.H. Kohler ◽  
M.R. Hanson
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Suspension Cells ◽  
Developmentally Regulated ◽  
Mesophyll Cells ◽  
Green Fluorescent

Green fluorescent stroma filled tubules (stromules) emanating from the plastid surface were observed in transgenic plants containing plastid-localized green fluorescent protein (GFP). These transgenic tobacco plants were further investigated by epifluorescence and confocal laser scanning microscopy (CSLM) to identify developmental and/or cell type specific differences in the abundance and appearance of stromules and of plastids. Stromules are rarely seen on chlorophyll-containing plastids in cell types such as trichomes, guard cells or mesophyll cells of leaves. In contrast, they are abundant in tissues that contain chlorophyll-free plastids, such as petal and root. The morphology of plastids in roots and petals is highly dynamic, and plastids are often elongated and irregular. The shapes, size, and position of plastids vary in particular developmental zones of the root. Furthermore, suspension cells of tobacco exhibit stromules on virtually every plastid with two major forms of appearance. The majority of cells show a novel striking ‘octopus- or millipede-like’ structure with plastid bodies clustered around the nucleus and with long thin stromules of up to at least 40 (micro)m length stretching into distant areas of the cell. The remaining cells have plastid bodies distributed throughout the cell with short stromules. Photobleaching experiments indicated that GFP can flow through stromules and that the technique can be used to distinguish interconnected plastids from independent plastids.

scholarly journals Diversity in the Development of the Neuromuscular System of Nemertean Larvae (Nemertea, Spiralia)

2021 ◽  
Vol 9 ◽  
Author(s):  
Jörn von Döhren
Keyword(s):  
Nervous System ◽  
Laser Scanning ◽  
Muscle Development ◽  
Fluorescent Labeling ◽  
Comparative Data ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Filamentous Actin ◽  
Data Set ◽  
True Diversity

In studies on the development of nervous systems and musculature, fluorescent labeling of neuroactive substances and filamentous actin (f-actin) of muscle cells and the subsequent analysis with confocal laser scanning microscopy (CLSM), has led to a broad comparative data set for the majority of the clades of the superphylum Spiralia. However, a number of clades remain understudied, which results in gaps in our knowledge that drastically hamper the formulation of broad-scale hypotheses on the evolutionary developmental biology (EvoDevo) of the structures in question. Regarding comparative data on the development of the peptidergic nervous system and the musculature of species belonging to the spiralian clade Nemertea (ribbon worms), such considerable knowledge gaps are manifest. This paper presents first findings on fluorescent labeling of the FMRFamide-like component of the nervous system and contributes additional data on the muscle development in the presently still underrepresented larvae of palaeo- and hoplonemertean species. Whereas the architecture of the FMRFamide-like nervous system is comparably uniform between the studied representatives, the formation of the musculature differs considerably, exhibiting developmental modes yet undescribed for any spiralian species. The presented results fill a significant gap in the spiralian EvoDevo data set and thus allow for further elaboration of hypotheses on the ancestral pattern of the musculature and a prominent component of the nervous system in Nemertea. However, with respect to the variety observed, it is expected that the true diversity of the developmental pathways is still to be discovered when more detailed data on other nemertean species will be available.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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