Subcellular localization of Cd in the root cells of<Emphasis Type="Italic">Allium sativum</Emphasis> by electron energy loss spectroscopy

The ultrastructural investigation of the root cells ofAllium sativum L. exposed to three different concentrations of Cd (100 (AM, 1 μM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes — high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.     

Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI

The ultrastructural investigation of the root cells of Allium cepa L. exposed to two different concentrations of chromium + nickel (Cr+Ni) (10 micromol/L and 100 micromol/L) revealed that toxic symptoms were induced by increasing heavy metal concentration and treatment time. Several significant ultrastructural changes were caused by 100 micromol/L Cr+Ni - deposition of electron dense material in cell walls; larger vacuolar precipitates surrounded by membranes inside vacuoles; increment of disintegrated organelles and high vacuolization in cytoplasm. The localization of the precipitates in which the metal ions were detected by electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) was investigated. Chromium and nickel were localized in the electron dense precipitates of the root cells exposed to only 100 micromol/L Cr+Ni. None were found in the root cells exposed to 10 micromol/L Cr+Ni. Higher amounts of Cr+Ni were mainly accumulated in the cell walls and vacuoles of the fourth or fifth cortical layer.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY–COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM‐coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long‐term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long‐term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.     

On the evidence of a diffusion barrier in the outer cortex apoplast of cress-roots (Lepidium sativum), demonstrated by analytical electron microscopy

To mark the apoplastic pathway of ions in the root of the dicotyledonous plant Lepidium sativum we used the heavy element lanthanum, which can be identified by analytical electron microscopy (EELS and ESI). In the front root tip, the primary walls of all meristematic cells contained lanthanum. 10-15 mm behind the root apex, lanthanum was found in the cortex cell walls up to the endodermis, but not in the stele. 20-25 mm from the tip, lanthanum was accumulated in the radial cell walls of the hypodermis, which, however, is not a complete diffusion barrier for ions, so that traces of lanthanum also were found in the cortex cell walls up to the endodermis. This study provides evidence for the presence of two apolastic diffusion barriers in the region of highest water uptake in cress roots.     

Electron energy loss spectroscopy techniques for the study of microbial chromium(VI) reduction

Electron energy loss spectroscopy (EELS) techniques were used to determine oxidation state, at high spatial resolution, of chromium associated with the metal-reducing bacteria, Shewanella oneidensis, in anaerobic cultures containing Cr(VI)O4(2-). These techniques were applied to fixed cells examined in thin section by conventional transmission electron microscopy (TEM) as well as unfixed, hydrated bacteria examined by environmental cell (EC)-TEM. Two distinct populations of bacteria were observed by TEM: bacteria exhibiting low image contrast and bacteria exhibiting high contrast in their cell membrane (or boundary) structure which was often encrusted with high-contrast precipitates. Measurements by EELS demonstrated that cell boundaries became saturated with low concentrations of Cr and the precipitates encrusting bacterial cells contained a reduced form of Cr in oxidation state + 3 or lower.     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Metal localization in water hyacinth roots from an urban wetland

PCA, principal components analysis
MDS, multidimensional scaling
STEM, scanning transmission electron microscopy

Metal localization within and around roots of water hyacinth ( Eichhornia crassipes ) growing in a wetland receiving urban run-off was studied by energy dispersive X-ray microanalysis of sections from freeze-substituted roots. Sampling randomly from an order of magnitude gradient in metal concentrations (Cu and Pb) allowed us the opportunity to identify general patterns of metal localization. Iron was present at high levels at the root surface, and this may have been a root plaque as described for wetland plants with roots anchored in flooded soils. Iron levels decreased centripetally across the root and were higher in cell walls than within cells. Trace metals (Cu, Zn and Pb) were not localized at the root surface. In contrast with iron, trace metal levels increased centripetally across the root, tended to be higher inside cells and were highest within cells in the stele. Variability of localization was high for all metals analysed. Multivariate statistical analyses (principal components analysis and multidimensional scaling) were useful for identifying overall patterns in elemental distribution.     

Chronic exposure to uranium leads to iron accumulation in rat kidney cells

After it is incorporated into the body, uranium accumulates in bone and kidney and is a nephrotoxin. Although acute or short-term uranium exposures are well documented, there is a lack of information about the effects of chronic exposure to low levels of uranium on both occupationally exposed people and the general public. The objective of this study was to identify the distribution and chemical form of uranium in kidneys of rats chronically exposed to uranium in drinking water (40 mg uranium liter(-1)). Rats were killed humanely 6, 9, 12 and 18 months after the beginning of exposure. Kidneys were dissected out and prepared for optical and electron microscope analysis and energy dispersive X-ray (XEDS) or electron energy loss spectrometry (EELS). Microscopic analysis showed that proximal tubule cells from contaminated rats had increased numbers of vesicles containing dense granular inclusions. These inclusions were composed of clusters of small granules and increased in number with the exposure duration. Using XEDS and EELS, these characteristic granules were identified as iron oxides. Uranium was found to be present as a trace element but was never associated with the iron granules. These results suggested that the mechanisms of iron homeostasis in kidney could be affected by chronic uranium exposure.     

Element Distribution in Mycelium of Pisolithus arrhizus Treated with Cadmium Dust

Mycelium of Pisolithus arrhizus cultured on agar media containingcadmium dust (collected from industrial electrofilters) andon media without the dust was analysed by electron spectroscopicimaging (ESI) and electron energy loss spectroscopy (EELS) connectedwith the transmission electron microscope. Complementary investigationsof polysaccharide (PATAg) and cysteine rich protein localizationwere carried out. The analyses revealed abundant depositionof glycogen in the form of net-like material in the cytoplasmof the dust-supplemented mycelium, accompanied by increasedlevels of Ca. Heavy metals like Cd, Cu and Fe accumulated mainlyin electron opaque granules and in the outer pigmented layerof the cell wall, both characterized by the presence of polysaccharidesand cysteine-rich proteins.Copyright 1994, 1999 Academic Press Pisolithus arrhizus (Pers.) Rausch., mycorrhizal fungi, heavy metals, cytochemistry, element localization     

Cadmium,nickel, copper,and zinc influence on microfilament organization in Arabidopsis root cells

The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd²⁺, Ni²⁺, Zn²⁺, and Cu²⁺ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO₄, NiSO₄, CuSO₄, and ZnSO₄ were used in concentrations of 5–20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals.     

Elemental imaging by EELS and EDXS in the analytical electron microscope

Electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) can be used to obtain elemental maps from thin biological samples in the analytical electron microscope. The EELS is particularly sensitive for the low-atomic-number elements, including C, N, and O, as well as other elements with favorable ionization cross-sections, such as Fe. The EDXS is useful for a complementary range of atoms, such as P, S, K, and Ca. A system is described for obtaining elemental distributions in an analytical electron microscope operated in the scanning transmission mode at 100–200 keV beam energy. The spatial resolution is typically limited to 10–20 nm when a conventional source is used. A satellite microcomputer controls acquisition of EELS and EDXS data from successive pixels in an image. These data are processed “on-the-fly” by a host computer to remove the noncharacteristic background intensity. Resulting images are stored on disk and can be analyzed by means of an image display system controlled by interactive software. The technique is demonstrated with elemental maps from two samples: alveolar macrophages containing respirable particles; and pancreatic beta cells that secrete insulin.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

Cu2+ Reduction by Tomato Root Plasma Membrane Vesicles

Reduction of Cu2+ by plasma membrane vesicles isolated from tomato (Lycopersicon esculentum Mill.) roots was investigated. Plants were grown in hydroponic culture with complete nutrition for 4 weeks or were deprived of Fe for the last 7 d. Plasma membrane vesicles were prepared by aqueous two-phase partitioning. Reduction of Cu, Fe, and ferricyanide by plasma membrane vesicles was measured. An increase in the activity of all three pyridine-nucleotide-dependent activities was noted in plasma membrane preparations from Fe-deficient, compared to Fe-sufficient, plants. Solubilization and chromatographic separation of two plasma membrane electron transport systems indicated that the Fe-chelate reductase was probably responsible for reduction of Cu. Assays used a variety of Cu chelates, and for each the Cu activity in the assay was determined by the program Geochem PC. The rate of reduction of Cu correlated with the level of Cu activity, and results support the idea that free Cu2+ and not Cu chelates may serve as the true substrate for reduction. Reduction was observed only in assays in which Cu activity was equivalent to Cu-enriched or Cu-toxic soils. These results suggest that reduction of Cu by tomato root may have little or no physiological relevance under conditions experienced by the root in the soil.     

星星草营养器官适应盐胁迫的结构特征

采用0．6％Na2CO3胁迫处理星星草[Puccinellia tenuiflora（Turcz．）Scribn．et Merr．]幼苗,光镜和电镜观察其根和叶的显微和超微结构。结果表明,星星草根的表皮向外突出形成密集的根毛;外皮层由1～2层细胞组成,排列较紧密;中皮层薄壁细胞排列疏松,形成发达的通气组织;内皮层呈典型的五面加厚;中柱鞘排列紧密,其壁加厚;初生木质部与初生韧皮部相间排列,初生木质部为5～7原型,中央为后生木质部导管,无髓存在。叶的表皮有表皮毛和丰富的蜡质层;叶上表皮泡状细胞数目较少,且深陷;气孔下陷,其下有较大的气室;叶脉有大、中、小3种维管束,大、中型维管束为C3型,小型维管束为C4型。星星草可能是介于C3和C4植物之间的类型,具有耐盐碱及耐干旱特征。     

Toxicities of soluble Al, Cu, and La include ruptures to rhizodermal and root cortical cells of cowpea

Elevated levels of many metals are toxic to plant roots, but their modes of action are not well understood. We investigated the toxicities of aluminium (Al), copper (Cu), and lanthanum (La) in solution on the growth and external morphology of 3-d-old cowpea (Vigna unguiculata L.) roots for periods of up to 48 h. Root elongation rate decreased by 50% at ca. 30 μM Al, 0.3 μM Cu, or 2.0 μM La, accompanied by a decrease in the distance from the root tip to the proximal lateral root. Kinks developed in some roots 2.0 ± 0.4 mm from the root apex on exposure to Al or La (but not Cu). Light and scanning electron microscopy showed that soluble Al, Cu, or La caused similar transverse ruptures to develop > 1 mm from the root apex through the breaking and separation of the rhizodermis and outer cortex from inner-layers. The metals differed, however, in the range in concentration at which they had this effect; developing in solutions containing 54 to‑600 μM Al, but only from 0.85 to 1.8 μM Cu or 2.0 to 5.5 μM La. These findings suggest that Al, Cu, and La bind to the walls of cells, causing increased cell wall rigidity and eventual cell rupturing of the rhizodermis and outer cortex in the elongating zone. We propose that this is a major toxic effect of Al, and that Cu and La also have additional toxic effects.     

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.     

Production of concentrated polyphenols by the root cap cells ofCorylus associated withTuber: ultrastructural study and element localization using electron energy loss spectroscopy and imaging

In mycorrhizae, root cap cell differentiation is a rapid event. Dark deposits abruptly invade the cytoplasm and the vacuoles. Finally the vacuoles fill with phenolic deposits and the cytoplasm is moribund. After degradation of the cell wall and cytoplasm, hyphae make contact with polyphenols and penetrate the polyphenols. The polyphenols are incorporated into the fungal mantle. Microanalysis by means of an electron-energy-loss spectrometer (EELS) and an electron imaging system (ESI) reveals the presence of calcium and nitrogen in the polyphenolic material just incorporated into the apical mantle. More distant polyphenols no longer contain calcium but nitrogen is still locally present.     

Ultrastructural distribution of terbium across capillary endothelium: detection by electron spectroscopic imaging and electron energy loss spectroscopy

We used terbium as an intravital tracer of permeability pathways across the walls of capillaries in the rete mirabile of the eel swimbladder and in frog mesentery. Terbium was detected in unstained ultra-thin sections by electron density using electron spectroscopic imaging (ESI) and by electron energy loss spectroscopy (EELS). Enhancement of intrinsic contrast in zero loss images (elastically scattered electrons) permitted imaging of membrane-bound compartments and terbium within them which might otherwise have been undetected in counterstained sections. Element-selective imaging with EELS indicated that terbium was associated with heavy electron-dense deposits, but the terbium mass:volume of sections in areas of lighter deposition was insufficient to obtain a terbium signal. In the rete capillaries, terbium was deposited on the luminal surface, throughout vesicular profiles, and in the interstitium, but could not be traced through interendothelial junctions. Fine terbium deposits were detectable throughout apparent vesicular connections across the endothelium. In the frog mesentery, terbium penetrated some but not all interendothelial clefts, and was detectable in small quantities within luminal and abluminal vesicular profiles and in the interstitium. The results indicate that in the rete capillaries, terbium permeates the capillary via a transcellular route. This route may be provided by transient fusions of luminal and abluminal vesicular compartments.     

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H₂O₂)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.