Accumulation,localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata

Summary. Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.Correspondence and reprints: Department of Plant Biology, University Federico 11, via Foria 223, 80132 Naples, Italy.     

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.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

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.     

The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouse

The distribution of metals within the hepatopancreas of Oniscus asellus (Crustacea, Isopoda) from two uncontaminated sites, and two sites contaminated with zinc, cadmium and lead, has been studied by atomic absorption spectrophotometry, light microscopy, transmission and scanning electron microscopy and X-ray microanalysis. The hepatopancreas contains two types of intracellular granule. The first type, in the S cells, are spherical granules which contain copper, sulphur and calcium. In woodlice from contaminated sites, these ‘copper’ granules, also contain zinc, cadmium and lead. The second type, in the B cells, are flocculent deposits which contain iron. In woodlice from contaminated sites, these ‘iron’ granules also contain zinc and lead. Moribund woodlice from contaminated sites have large numbers of ‘copper’ and ‘iron’ granules in the hepatopancreas and a fine deposit of zinc and lead on the membranes of the cells. There are numerous microorganisms in close association with the microvillous border of the hepatopancreas of woodlice from all four sites. Within the microorganisms of Oniscus asellus from contaminated sites, there are deposits of material which contain zinc, lead, calcium and phosphorus ‘Copper’ and ‘iron’ granules could have evolved as storage sites for essential metals to be utilized when demand from the body exceeds uptake from the food. Woodlice in contaminated sites may be able to ‘detoxify’ potentially harmful amounts of essential and non-essential metals by storing them in a relatively insoluble form within these granules.     

Cadmium‐induced cell death in BY‐2 cell culture starts with vacuolization of cytoplasm and terminates with necrosis

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY‐2 cells treated with 50 µM CdSO₄. The cadmium‐induced alterations in cell morphology occurred gradually over a period of 3–4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium‐induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.     

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO₂) fixation by toxic metals such as cadmium – classified as a priority pollutant – could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO₂ and labelled ¹³C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO₂ fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (μXRF)/micro X-ray absorption near-edge structure (μXANES) at Cd L_III-edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the ¹³C/¹²C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO₂ decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.     

Localized intracellular polyphosphate formation by Desulfovibrio gigas.

The dissimilatory sulphate-reducing bacterium Desulfovibrio gigas, frequently sub-cultured, often contained spherical granules which stained metachromatically with some basic dyes. The granules were examined in situ by transmission electron microscopy of whole organisms and thin sections. The granules were isolated from broken bacteria as a water-insoluble, non-crystalline, white material containing magnesium, phosphorus and organic carbon, but devoid of sulphur and nitrogen. The molar ratio of phosphorus to magnesium (1 to 17) was close to the proportions in magnesium tripolyphosphate. Infrared absorption spectra for the white material and magnesium tripolyphosphate were similar.     

Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungusPisolithus tinctorius

Summary Polyphosphate granules are precipitated in the vacuoles of the ectomycorrhizal fungusPisolithus tinctorius (Pers.) Coker & Couch by various treatments, including conventional specimen preparation. Granules are not produced by glutaraldehyde fixation but appear at early stages of ethanol dehydration and are visible with Nomarski DIC microscopy. They show -metachromasy with toluidine blue O at low pH, are extracted by cold trichloroacetic acid and contain phosphorus and calcium as demonstrated by X-ray microanalysis. The granules are surrounded by electron-lucent areas that do not contain these elements at detectable levels. In contrast, vacuoles of freeze-substituted hyphae contain evenly dispersed flocculent material. Phosphorus and potassium are distributed more or less uniformly throughout, but calcium is not detected. This indicates that polyphosphate is present in the vacuole of living hyphae in soluble form and is precipitated to form granules by various treatments. It is thought that granules form when membranes, including the tonoplast, become leaky and there is an influx of precipitating ions such as calcium.Abbreviations DIC differential interference contrast - GMA glycol methacrylate - MMN modified Melin Norkrans - NMR nuclear magnetic resonance - P_i inorganic phosphate - STEM scanning transmission electron microscope     

Enterochromaffin cells in the chicken duodenum during prenatal development

Summary The development of enterochromaffin cells (EC) was studied in the duodenum of the chicken during prenatal development.Basally granular cells, i.e. EC were first found at the 14th incubation day and thereafter their number increased greatly during further development, as did also the number of specific osmiophilic granules in the cells.First empty or pale granules were found in the cytoplasmic area showing various kinds of saccules and vacuoles, possibly representing the poorly developed Golgi area. The primitive granules seemed to turn rapidly in to granules with high affinity for osmium tetroxide.The differentiation of EC occurred in the epithelial lining of the intestinum and no morphologically similar cells were found in other intestinal layers.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki. The electron microscopic observations were made in the Electron Microscope Laboratory, University of Helsinki; the skillful assistance of Mr. M. Nyholm is acknowledged.     

Consumption of lipid granules and formation of vacuoles in the pollen tube ofTradescantia reflexa

Summary The ultrastructure in pollen tubes ofTradescantia reflexa was studied with special attention to the consumption of lipid granules and the formation of vacuoles. Electron dense, thin vesicles (EDTV) were developed from the small spherical vesicles. ER-associated lipid granules attached to both surfaces of the EDTV. Then, the EDTV became bottle-shaped, surrounding a part of cytoplasm. In the compartment surrounded by the membranes of EDTV, lipid granules were decomposed and finally disappeared. Protuberances were formed at the terminals of the expanded membranes of the EDTV and at the delimiting membranes of the compartment. These protuberances were pinched off from the membranes to form vesicles. These vesicles were transported from the site of origin to destinations in the pollen grain where they fused with each other to form vacuoles.Abbreviation EDTV electron dense, thin vesicle(s)     

Fine structure of antheridia and oogonia of Phytophthora macrospora,the downy mildew fungus of rice plants

In the present paper fine structure of antheridium and oogonium ofPhytophthora macrospora (Sacc.)S. Ito etI. Tanaka, the downy mildew fungus of rice plants was discussed.Before the fertilization some nuclei and a large number of mitochondria were scattered in the cytoplasm of the antheridium. Many lipid granules were observed in the peripheral region, but vacuoles did not appear at this stage of antheridium. Many mitochondria were associated in the neighborhood of the fertilization pore. The wall at the pore was very thin, but the wall surrounding the pore was slightly swollen towards the inside.In the oogonium, many nuclei, mitochondria and cytoplasmic matrix were observed at the peripheral part. A large number of lipid granules was found in the oogonium, but they were more numerous in the peripheral region. The vacuoles developed as the oogonium matured. They were enveloped by tonoplast and contained vacuolar matrix. Many electron dense granules were in contact with the tonoplast or free in the vacuoles, and they were larger in the central part. As stated above, wall at the fertilization pore was thin. However, the oogonial wall surrounding the pore swelled protruding into the oogonium. An electron-dense layer was recognized between the antheridial and oogonial wall, and the walls of both the organs were closely in contact with each other.Contribution No. 252.     

Biogenesis of Protein Bodies in Embryonic Axes of Soybean Seeds (Glycine max. cv. Enrei)

Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.     

Location of copper in larvae of Plectrocnemia conspersa (Curtis) (Trichoptera) exposed to elevated metal concentrations in a mine drainage stream

Using histological techniques together with transmission electron microscopy and X-ray microanalysis, copper-containing granules (which also contain significant amounts of sulphur) have been observed in the cells of the malpighian tubules and in the subcuticular region of larvae of Plectrocnemia conspersa. It is suggested that the granules in both these regions are primarily pigment granules, which provide a mechanism for taking potentially toxic concentrations of copper (and possibly other metals) out of circulation. This mechanism of metal tolerance may in part account for the reported occurrence of larvae of P. conspersa in waters with elevated concentrations of trace metals.     

The role of reflecting pigment cells in the turnover of crayfish photoreceptors

Summary The photoreceptors of the crayfish Procambarus clarkii undergo an extensive cycle of turnover in the late afternoon. Quantitative light and electron microscopy reveal a sharp increase in the fractional volume (i.e., density) of reflecting-pigment-cell granules and vacuoles shortly following late-afternoon photoreceptor turnover. The reflecting pigment cells (RPCs), which permanently reside within the crayfish retina, are shown to serve much the same function as the vertebrate pigment epithelium. The RPCs phagocytose partially digested photoreceptive microvilli and the ingested debris is degraded further into the granules and vacuoles which characterize these cells. Phagosome degradation appears to be mediated by Golgi complexes. Acid phosphatase appears to be involved in the initial rhabdom breakdown but not in the final reduction of RPC granules.     

Electron microscopic observations of the phagocytosis and subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils in vitro

Neutrophils isolated from the blood of Atlantic salmon (Salmo salar L.) were studied by electron microscopy at various time intervals after being incubated with opsonised and non-opsonised Aeromonas salmonicida strains, namely, the avirulent MT004 (A-layer negative) and the virulent MT423 (A-layer positive). After 15 min incubation with all four groups of bacteria (virulent, avirulent, opsonised or non-opsonised) a large number of neutrophils showed an elongated shape with the nucleus and all the organelles being located in one pole of the cell. Small vacuoles and clumping of glycogen granules were also observed. Neutrophils devoid of granules were noted after 30 min incubation, the majority containing engulfed bacteria. Degenerate neutrophils were also found in all the groups incubated with bacteria. Phagocytosis of bacteria was observed after 15 min incubation. The number of intracellular bacteria was very low, usually one or two per cell, although some neutrophils incubated with the opsonised avirulent strain MT004 contained a larger number of engulfed bacteria. Ingestion of bacteria was usually accompanied by the formation of phagocytic vacuoles containing an amorphous material of moderate electron-density as well as granule discharge into the vacuole. Both strains (MT004 and MT423), opsonised and non-opsonised, underwent morphological alterations after 3-7 h incubation suggesting that both A. salmonicida strains were killed by the neutrophils.     

Phosphorus distribution in perichromatin granules and surrounding nucleoplasm as visualized by electron spectroscopic imaging

Summary— The in situ distribution of phosphorus in perichromatin granules (PCGs), and in the surrounding nucleoplasm was investigated in rat liver cells by means of electron spectroscopic imaging of unstained preparations. A 2–3 nm fibril containing high concentration of phosphorus was found to be the main substructural feature of the PCGs revealed in the maps of phosphorus. This fibril is folded within the PCG with no apparent order. Fibrils of similar diameter and phosphorus content were also found in both the halo surrounding the PCG and dispersed in the nucleoplasm. Some of such fibrils are in continuity with those occurring within PCGs. Sometimes these fibrils are grouped forming a stalk connecting the PCG to chromatin. Some stalked PCGs are U-shaped or kidneyshaped, resembling Balbiani ring granules in the process of formation as observed in Chironomus salivary gland cell nuclei. The external fibrils are interpreted as perichromatin fibrils considered to be precursors of PCGs.     

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Blood Cell Ultrastructure of the Ascidian Botryllus schlosseri L. II. Pigment Cells

Colonies of Botryllus schlosseri L., bred in the laboratory and genetically selected as regards the blue and/or reddish pigments, were used. The following phenotypes were investigated under the electron microscope: (a) blue colonies without reddish pigment; (b) reddish colonies without blue pigment; (c) colonies with both blue and reddish pigments; (d) colonies with neither blue nor reddish pigments. In the pigmented colonies, a specialized blood pigment cell type was recognized that, in giant membrane-limited vacuoles, contained a great number of granules. In general, the granules were similar in size, not individually limited by a membrane and were made up with electrondense material often arranged in concentric rings. Although there could be some variability within the same cell, in each phenotype the granules displayed a characteristic pattern so that the differences in colour of the granules, as seen in vivo, were paralleled by differences in the ultrastructural architecture. In the unpigmented colonies also, granulated vacuolar cells, rare in number but morphologically comparable to the pigment cells, were seen. On the basis of these results, the hypothesis of the existence of a prospective pigment cell and of a common origin for all the pigment cells of B. schlosseri is discussed.