Thomas E. Jensen

Uptake of heavy metals by Plectonema boryanum (cyanophyceae) into cellular components, especially polyphosphate bodies: An X-ray energy dispersive study

Abstract In situ X-ray energty dispersive analysis has been used to study heavy metal uptake in Plectonema boryanum . Air-dried cells observed in the STEM mode of the SEM show numerous polyphosphate bodies in the cells. By using this system as a probe to analyse cell sectors it was determined that the heavy metals Cd, Co, Cu, Hg, Ni, Pb and Zn are all taken up and sequestered in the cell sectors with polyphosphate bodies. Smaller amounts of the metals Cd, Cu, Hg, Pb and Zn are also concentrated in the cell sectors without polyphosphate bodies. Control polyphosphate bodies cell sectors generate high P and K signals and a low Mg signal. Sometimes a Ca peak is also generated. In Cd-exposed cells the K and Mg signal is lost from the polyphosphate body cell sectors. In Cu-treated cells the Mg and K peaks are lost from the polyphosphate body cell sectors. In Pb-treated cells the K signal is reduced from both the bodies and the cell sectors without bodies. In Hg-treated cells the K signal is lost and the Ca signal is enhanced. The other metals did not significantly affect the elemental peaks generated in the polyphosphate body cell sectors or the cell sectors without polyphosphate bodies. The sequestering of heavy metals in the polyphosphate bodies is suggested as serving a dual purpose in cells by providing a storage site for essential metals and acting as a detoxification mechanism.

Fine structure of abscission zones : I. Abscission zones of the pedicels of tobacco and tomato flowers at anthesis.

SummaryElectron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 mμ in width while the main groove averages approximately 200 mμ in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 mμ in thickness and are spaced at 4 mμ intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 mμ and 1,500 mμ in profile. In tomato they average 300 mμ and 500 mμ. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 μ in diameter in chloroplasts ranging from 1.6 μ to 2.2 μ in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 mμ are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.

Utilization of morphometric analysis in evaluating response ofPlectonema boryanum (Cyanophyceae) to exposure to eight heavy metals

The techniques of morphometric analysis were used to study the ultrastructural changes induced inPlectonema boryanum (Cyanophyceae) by heavy metal exposure. Four of the test metals (Mn, Zn, Hg, and Cd) produced no changes in overall cell dimensions. Two metals (Pb and Cu) produced significant increases in cell size and two other metals (Co and Ni) caused significant decrease in cell size. Mn, Pb, Co, Ni, and Cd caused significant increases in the surface area of the cells thylakoids and Ni, Co, Zn, and Hg caused a significant reduction in the volume of the intrathylakoidal spaces. All tested metals except Pb and Mn caused the production of extra intracellular membrane whorls. Pb, Mn, Co, Zn, Hg, Cd, and Ni caused coalescence of cellular lipid while Cu caused a reduction in cellular lipid content. Co caused a reduction in the number of polyhedral bodies and Cd caused an increase in the volume of the cells polyphosphate bodies. The physiological significance of these findings are discussed in terms of potential toxic action of cations and the cellular mechanisms for the detoxification of cations once they enter the cell. Common mechanisms of both procaryotic and eucaryotic algae are discussed and the utilization of stereological techniques for electron microscopic morphometric analysis is established in studies of metal toxicity at the cellular level.

The toxicological response of the algaAnabaena flos- aquae (cyanophyceae) to cadmium

The toxicological response of the cyanophycean algaAnabaena flos- aquae to cadmium was investigated by three integrated approaches: 1) the determination of the incipient lethal concentration of the metal, 2) study of metal incorporation and cellular compartmentalization using X-ray energy dispersive analysis, and 3) the quantification of intracellular structural changes, after metal exposure, using morphometric analysis. After 96 hr of exposure, the incipient lethal concentration was calculated to be 0.118 ± 0.04 μM cadmium. At concentrations three orders of magnitude higher than the incipient lethal concentration, cadmium was incorporated into both the cellular cytoplasm and the cells polyphosphate bodies. Cadmium also caused the polyphosphate bodies to lose Mg and Ca, resulting in ionic changes in the elemental composition of these cellular inclusions. The utilization of stereological techniques for electron microscopic morphometric analysis established that all concentrations of cadmium tested caused significant reductions in the surface area of the cells thylakoids. Cadmium induced changes in the numbers and relative volume of the cell occupied by polyhedral bodies, polyphosphate bodies, lipid inclusions, cyanophycin granules, membrane limited crystalline, inclusions, and changes in the volume of the cell wall layers were also documented. The physiological significance of these findings are discussed in terms of the toxic action of cadmium and the cellular mechanisms for detoxification of cations once they enter the cell.

An x-ray energy dispersive study of cellular compartmentalization of lead and zinc in Chlorella Saccharophila (Chlorophyta), Navicula incerta and Nitzschia closterium (bacillariophyta)

Abstract The compartmentalization of the heavy metals Pb and Zn was studied in Chlorella saccharophila, Navicula incerta and Nitzschia closterium using the STEM with dispersive X-ray spectrometer. Cells were exposed to the metals, at various concentrations, for 96 hr and then air dried on formvar coated grids. Lead was found to be concentrated in cell sectors with polyphosphate bodies in C. saccharophila at 13.3 ppm and in N. incerta and N. closterium at 10 ppm. Lead was also detected when the probe was placed on the cell margin (cell wall sector) of C. saccharophila at 25 ppm and 5.6 ppm in N. closterium . No Pb was detected in the cell margin sector (cell wall) of N. incerta at metal concentrations up to 10 ppm. In cells exposed to Zn, the metal was detected in polyphosphate body cell sectors in C. saccharophila starting at 1.0 ppm and in N. incerta starting at 2.3 ppm. Small amounts of Zn were detected in cell sectors away from polyphosphate bodies in C. saccharophila starting at 2.3 ppm. No Zn was detected in N. closterium at metal concentrations up to 2.3 ppm. It is suggested that the compartmentalization of the heavy metals Pb and Zn into polyphosphate bodies and the cell wall may be a means by which some algae reduce the toxicity of these cations. These means of sequestering heavy metals may also be a significant way in which heavy metals move, in large amounts, in the food chain.

A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO4 resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.

Deposition of condensed phosphate as an effect of varying sulfur deficiency in the cyanobacterium Synechococcus sp. (Anacystis nidulans)

Uptake of orthophosphate and deposition of condensed phosphate were investigated in cells of Synechococcus sp. (Anacystis nidulans) deficient in phosphorus or sulfur. When phosphorus was restored to phosphorus-starved cells, uptake was rapid and immediate, with the greatest accumulation occurring within the first hour. Uptake was optimum in the pH 7.5–8.5 range. Long-term (6-day) studies of uptake and deposition with cells exposed to a wide range of sulfur deficiency showed that both processes were greatest when the level of exogenous sulfur was reduced to zero. The increase in cellular phosphorus as determined chemically was in agreement with the increased number and size of polyphosphate bodies at the ultrastructural level. Possible mechanisms for the control of phosphorus uptake and condensed phosphate formation by exogenous sulfur are discussed.

A morphometric and x-ray energy dispersive approach to monitoring pH-altered cadmium toxicity inAnabaena flos-aquae

Cadmium toxicity and uptake as influenced by different pH values have been studied in the freshwater cyanobacteriumAnabaena flos-aquae, using the techniques of morphometric analysis, x-ray energy dispersive analysis and atomic absorption spectrophotometry. A general reduction in cell dimension, thylakoid surface area, number and volume of polyhedral bodies, polyphosphate bodies, cyanophycin granules, lipid bodies, membrane limited crystalline inclusions, volume and number of wall layers and mesosomes was observed. These reductions were more pronounced in both acidic and alkaline medium than at pH 7.2. At 0.12 μM Cd, the uptake increased with alkaline pH values, and uptake was greater at pH 7.2 than at either acid or alkaline pHs. Lysis of cell wall at 1.18 μM Cd showed the following decreasing trend: pH 4.0 > pH 5.5 > pH 10.0 > pH 9.0 > pH 7.2. There was a total loss of lipid bodies at 1.18 μM Cd at all pH values listed.It is suggested that these techniques can be successfully employed for bioassay studies of metal toxicity to algae. In particular, cell wall lysis and loss of lipids by algae are good indicators of pH effects and metal toxicity in the aquatic ecosystem.

Morphometric analysis of the response ofAnabaena flos-aquae andAnabaena variabilis (Cyanophyceae) to selected concentrations of zinc

The toxicological responses of the freshwater cyanophycean algaeAnabaena flos-aquae andA. variabilis, to selected concentrations of the essential cation zinc, were quantified by the techniques of stereological electron microscopic morphometry. The cation concentrations used were 20.6, 52.0, and 114.7 μM, which represented the 96-hr EC50 dose forA. variabilis and an equal bisection of a log scale above and below this concentration. The results indicated that the surface area of the thylakoids ofA. flos-aquae were significantly increased after exposure to 114.7 μM Zn, and that there was a significant increase in the number of lipid bodies per cell. All test concentrations caused a significant reduction in both the numbers and relative volume of the cells cyanophycin granules, but resulted in an increase in the cells membrane limited crystalline inclusions. Both 52.0 and 114.7 μM Zn caused a significant increase in the surface area of the thylakoids ofA. variabilis, but only the highest concentration caused an increase in the volume of the interthylakoidal spaces. The relative volume and numbers of lipid bodies ofA. variabilis cells were increased by exposure to 52.0 μM Zn, and 114.7 μM Zn caused a significant increase in the relative volume and numbers of the cells polyphosphate bodies. The significance of these results are discussed in terms of potential cellular detoxification mechanisms, which are important in the cells ability to cope with the anthropogenic introduction into the aquatic environment of metal and industrial pollutants.

Fine structure of abscission zones : IV. Effect of ethylene on the ultrastructure of abscission cells of tobacco flower pedicels.

SummaryThe effect of ethylene on abscission of flower pedicels of tobacco plants has been investigated. For the first 2 h of exposure to C2H4, the pedicels bend rather than break in response to applied force, but after 2.5 h exposure they break at the abscission zone under an applied force of 40 g. The break strength of the abscission zone decreases exponentially with time to 5 g at 5 h after beginning of the C2H4 treatment. An examination of the tissue at the fine structural level 2 h after exposure to C2H4 reveals the accumulation of rough endoplasmic reticulum (RER) in the abscission cells. Rough ER becomes increasingly abundant by 3–5 h exposure of the tissue to C2H4. There is approximately a 30 fold increase in RER by 5 h of exposure, as compared to untreated tissue.Loss in the integrity of the membranes of microbodies occurs by 5 h exposure of the tissue to C2H4. As cell wall degradation proceeds, fibrous material, vesicular structures, and electron dense bodies—the latter often appearing striated—develop in the disintegrating wall. Little change is seen in the structure of nuclei, mitochondria, chloroplasts and in the crystalloid cores of microbodies during the first 5 h of exposure of the tissue to C2H4. However, disorganization of cytoplasmic components does occur in cells where cell wall breakdown is at an advanced stage.