Lead (Pb)-induced biochemical and ultrastructural changes in wheat (Triticum aestivum) roots

The focus of the present study was to explore lead (Pb)-induced metabolic alterations vis-à-vis ultrastructural changes in wheat roots to establish Pb toxicity syndrome at a structural level. Pb (50–500 μM) enhanced malondialdehyde (an indicator of lipid peroxidation) and hydrogen peroxide content, and electrolyte leakage, thereby suggesting reactive oxygen species-induced disruption of membrane integrity and oxidative stress in wheat roots. The activities of superoxide dismutases and catalases enhanced upon Pb exposure, whereas those of ascorbate and guaiacol peroxidases declined. Pb-induced metabolic disruption was manifested in significant alterations in wheat root ultrastructure as analyzed by transmission electron microscopy. Pb caused thinning of cell wall (at 50 μM), formation of amoeboid protrusions and folds and intercellular spaces, and appearance of lesions and nicks/breaks (at ≥250 μM Pb). Pb was deposited along the cell walls as dark precipitates. At ≤250 μM Pb, the number of mitochondria increased significantly, whereas structural damage in terms of change of shape and disintegration was observed at ≥ 250 μM Pb. Pb reduced the size of nucleoli and induced puff formation (at 250 μM), resulting in complete disintegration/disappearance of nucleolus at 500 μM. The study concludes that Pb inhibited wheat root growth involving an ROS-mediated oxidative damage vis-à-vis the ultrastructural alterations in cell membrane and disruption of mitochondrial and nuclear integrity.     

Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy

The environmental scanning electron microscope (ESEM) provides a highly relevant and controllable environment in which to study hydrated systems without the artefacts of other highly prepared specimens. The instrument facilitates control of turgor through hydration using different chamber vapour pressures. Deformation of a simple plant tissue-upper epidermal layers in Allium cepa (onion)-was observed at the scale of the two principal failure mechanisms: cell breakage; and cell separation induced by treatment with a chelating agent. Cell rupture and release of contents occurred at cellular junctions ahead of an imposed growing notch, indicating that disruption of cells occurred remotely from the creation of a new surface. Cells that separated usually maintained their turgor and the separation process took place through progressive failure of middle lamellar material seen as strands between separating cells. These mechanisms were compared with the rupture of excised Chara corallina walls that occurred by formation and breakage of strands between separating wall layers. This study provides in situ visual characterization of wall rupture and cell separation at the microscopic level in hydrated plant material.     

Morphological studies on the periostracum of the fresh-water mussel Amblema (uniondae): Light microscopy, transmission electron microscopy, and scanning electron microscopy

The structure of the periostracum in the fresh-water mussel Amblema has been described using light microscopy, transmission elec;ron microscopy, and scanning electron microscopy. The structure and evolutive course of the periostracum was studied along its entire length, from the periostracal groove until it forms the tough outer covering of the shell. At least five structurally and functionally distinct regions were identified. In addition, the periostracum itself was seen to be a multilayered structure consisting of three major layers which are themselves subdivided into minor layers. From these morphological observations, a regulatory role for the various periostracal layers in mineral trapping, nucleation, and the subsequent formation of the prismatic and nacreous layers of the shell can be postulated.     

Ultrastructure of Proechinophthirus zumpti (Anoplura,Echinophthiriidae) by scanning electron microscopy

The ultrastructure of Proechinophthirus zumpti Werneck, 1955, mainly the external chorionic features of the egg, is described through electronic microscopy techniques. This species was first cited in Argentina, infesting Arctocephalus australis (Zimmermann, 1873). The morphological adaptations of adults and nymphs are described in both species of Proechinophthirus parasitic on Otariidae: P. fluctus (Ferris, 1916) and P. zumpti.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

土壤和叶面Pb污染对小麦生长及体内Pb分布和积累的影响

通过土壤添加Pb和叶面喷施Pb溶液的方式研究了Pb污染对小麦(Triticum aestivum L. )地上部分和籽粒干质量的影响,并对Pb污染条件下小麦体内Pb的分布和积累规律以及Pb污染浓度与籽粒Pb含量的相关性、叶片Pb含量与籽粒Pb含量的相关性进行了分析.结果表明:土壤中Pb添加量为2 000 mg·kg-1时,小麦地上部分和籽粒的干质量分别较对照下降了15.5%和13.3%,差异显著(P<0.05);叶面喷施100 mg·L-1Pb溶液,小麦地上部分和籽粒干质量分别较对照下降了10.3%和15.5%,差异显著(P<0.05).在土壤和叶面Pb污染条件下,小麦各器官的Pb含量均随Pb污染浓度的提高而增大;在土壤Pb污染条件下,小麦根中的Pb含量远高于其他器官,籽粒中的Pb含量最低;在叶面Pb污染条件下,小麦叶片中的Pb含量远高于其他器官,籽粒和根中的Pb含量较低.回归分析结果表明,小麦籽粒中的Pb含量与Pb污染浓度呈极显著正相关(P<0.001),籽粒中的Pb含量与土壤Pb总含量和叶面Pb污染浓度的曲线方程分别为: y＝0.269+0.001 05x+2.736×10-7x2-1.707×10-10x3和y＝0.465+0.013x-1.1×10-5x2+3.96×10-9x3;土壤中总的Pb毒性临界值为209.3 mg·kg-1,叶面Pb的毒性临界值为2.6 mg·L-1;在土壤和叶面Pb污染条件下,小麦籽粒中的Pb含量与叶片Pb含量间呈极显著的正相关(P<0.01),回归方程分别为:y=0.120 1x+0.076和y=0.001 6x+ 0.601 1,据此,在土壤和大气Pb污染条件下,可通过测定小麦叶片的Pb含量预测小麦籽粒中的Pb含量.     

Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.     

Plant morphophysiological and anatomical factors associated with nitrous oxide flux from wheat (Triticum aestivum)

Experiments were conducted to study the dynamics of nitrous oxide (N?O) emission from wheat varieties viz., Sonalika, HUW 468, HUW 234 and DBW 14 grown in alluvial soils of North Bank Plain Agroclimatic Zone of Assam, India. Attempts were made to find out the relationship of N?O emission with plant morphophysiological, anatomical and soil properties. N?O fluxes from wheat varieties ranged from 40 μg N?O-N m?2 h?1 to 295 μg N?O-N m?2 h?1. Soil organic carbon and soil temperature have shown significant relationship with N?O flux. The rate of leaf transpiration recorded from the wheat varieties at different growth stages exhibited a positive correlation with N?O emission suggesting that movement of N?O along with the transpirational water flow may be an important mechanism of N?O transport and emission through wheat plants. Anatomical investigation by scanning electron microscope revealed that N?O emission has relationship with stomatal frequency of leaf and leaf sheaths. Variety HUW 234 with the highest stomatal frequency of leaf and leaf sheath also recorded higher seasonal N?O emission compared to other varieties. Seasonal N?O emission (E(sif)) of the varieties ranged from 3.25 to 3.81 kg N?O-N ha?1. Significant variations in E(sif) values were recorded within the varieties.     

Protoperidinium (Dinophyceae) from Greece as seen by scanning electron microscopy

The marine dinoflagellates Protoperidinium ventricum (Abé) Balech, P. diaholus (Cleve) Balech, P. tristylum (Stein) Balech and P. divergens (Ehrenberg) Balech, collected from neritic plankton of the gulfs of Saronikos and Korinthiakos, Greece, have been examined by SEM. Morphological characteristics of their thecae are described and evaluated. Results are compared with patterns established by earlier SEM studies on related taxa.     

Morphological diversity of setae on the grooming legs in Anomala (Decapoda: Reptantia) revealed by scanning electron microscopy

The fifth pereiopods (P5) in Anomala are specialized appendages used mainly for grooming. We studied the articulated cuticular outgrowths, setae, on the distal segments of the P5 in 40 species from 18 Anomala families using light and scanning electron microscopy. Five general classes of setae can be found on the P5: serrate, serrulate and simple setae which all appear bristle-like, and tooth-like and scale-like cuspidate setae. We classified the bristle-like setae according to criteria of shape and the arrangement of distinct outgrowths – denticles and setules – on the shaft of the seta. In this way we were able to distinguish eleven mainly serrate and serrulate types of seta. Some setal types imply homology due to their distinctness and could thus help to solve problematic phylogenetic questions. One setal type, for example, is only present in pagurid hermit crabs and king crabs, which corroborates the theory that these two morphologically very dissimilar groups are, in fact, closely related.     

Morphological changes in the isolated rat liver perfused in a non-recirculating system: scanning and transmission electron microscopy

Isolated perfused rat livers have been used for various studies, but detailed investigation into the structural integrity of hepatocytes of this system is lacking. In this study, isolated rat livers were perfused in vitro with oxygenated Krebs-Ringer bicarbonate buffer solution, for 2 minutes and 1, 2, 3, and 4 hour(s) at 37 degrees C, using a non-recirculating perfusion system. The perfused livers were processed for semithin section light microscopy, transmission electron microscopy, and scanning electron microscopy. Sectional areas of cell deaths were measured by a camera-tracing assembly from 1.5 microns thick Araldite sections stained with toluidine blue. Progressive nuclear and cytoplasmic changes, leading to cell death, occurred in the hepatocytes of the centrilobular zone, during the 2nd, 3rd, and 4th hour of the perfusion at a rate of 9.03% +/- 1.5%, 38.7% +/- 2.7%, and 55.1% +/- 5.9% (mean +/- standard deviation) of the total sectional areas respectively. Midzonal hepatocytes showed normal basophilic staining but exhibited loss of glycogen granules, loss of microvilli, development of aqueous vacuoles and formation of blebs. The fine structures of cell organelles, glycogen granules, microvilli and plasma membrane of the cells in the periportal zone were well preserved throughout the experimental period. For further quantitative, metabolic and functional studies using isolated rat liver perfused with Krebs-Ringer solution, it is evident from the present investigation that the periportal zone represents the functional region of the hepatic lobule. Whilst progressive changes, leading to cell death, occurred in the centrilobular zone.     

The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).     

External sense organs in freshwater oligochaetes (Annelida, Clitellata) revealed by scanning electron microscopy

Freshwater oligochaetes have at least two kinds of external sense organs: multiciliate organs of short cilia (also present in earthworms) and sense organs with one to three long cilia (unknown in earthworms and possibly acting as rheoreceptors). Ciliate sense organs of freshwater oligochaetes are distributed over their entire body surface, including the clitellum. They are scattered on the prostomium and pigidium and are arranged into a transversal chaetal row and dispersed or forming a few other discrete transversal rows on chaetal segments. Three species display very prominent sense organs (sensory buds in Protuberodrilus tourenqui and papillae in Ophidonais serpentina and Spirosperma velutinus). The number of cilia per organ at the prostomium of freshwater families appears to be fewer than that of terrestrial ones. It is suggested that the total number of cilia at the prostomium of the freshwater species could be related to their habitat, evolving from an epibenthic to an endobenthic way of life.     

A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM)

This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.     

Characters in the book lungs of Scorpiones (Chelicerata,Arachnida) revealed by scanning electron microscopy

The fine structure of the book lungs in 29 species representing ten monophyletic taxa of the Scorpiones (Arachnida) was investigated using scanning electron microscopy (SEM). Scorpion lungs are not homogeneous across the group. Here we describe and score three sets of phylogenetically informative characters: (1) the surface ornament of the lung lamellae, (2) the distal margins of the lamellae and (3) the fine structure of the spiracle margin. Provisional results suggest that reticulation on the surface of the lung lamellae is characteristic of the Buthidae. By contrast, non-buthid scorpions maintain the air space between adjacent lamellae using projecting trabeculae. Typically they are simple struts, but the trabeculae are distally branched in all investigated Scorpionidae, plus at least one species belonging to the Liochelidae. Simple thorns on the lamellar margins probably represent the plesiomorphic condition, while more complex, branched, arcuate morphologies appear to be homoplastic, occurring sporadically in numerous scorpion sub-groups. The tightly packed, hexagonal pillars around the posterior margin of the spiracle support a close relationship between Scorpionidae and Liochelidae, to the exclusion of the Urodacidae.     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Uptake of Lysine by Wild-Type and S-(2-Aminoethyl)-L-cysteine-Resistant Suspension-Cultured Cells of Triticum aestivum

The kinetics of uptake of L-lysine in wheat (Triticum aestivumcv. Chinese Spring) were analyzed in wild-type cells and inAEC-1 variant cells that are resistant to S-(2-aminoethyl)-L-cysteine(AEC). Uptake of lysine by AEC-1 cells was considerably slowerthan that by the wild-type cells. In the presence of carbonylcyanidem-chlorophenylhydrazone, the rates of uptake by both types ofcell were reduced to a similar linear component. Fitting theuptake data to one linear (diffusional) component and one Michaelis-Menten(active) system showed that, as compared to wild-type cells,AEC-1 cells have a reduced V_max and an increased K_m with respectto the active component, byt they have a similar diffusionalcomponent. Inhibition experiments with various amino acids indicatedthat the active component represents a carrier specific forbasic amino acids, which was competitively inhibited by AEC.The AEC-1 cells also showed reduced uptake of several neutraland acidic amino acids, but the rate of uptake of 3-O-methylglucosewas somewhat higher than that by wild-type cells. (Received May 16, 1989; Accepted September 4, 1989)