Increase of Copper Toxicity to Growth of Chlorella vulgaris with Increase of Light Intensity

Abstract In the absence of inhibitory concentrations of copper, the photoautotrophic growth of Chlorella vulgaris INETI58C at 27°C exhibited a higher specific growth rate and reached a higher maximal concentration of biomass, under irradiance of 150 W m⁻², compared with 100 W m⁻². However, when the mineral growth medium was supplemented with CuSO₄ (range 40–80 μM), algal growth was significantly affected at the higher light intensity. In the presence of Cu²⁺, the increase in dry biomass was uncoupled from the increase in cell concentration since more than 16 autospores gathered together, inside the enlarged mother cell, suggesting that copper arrested the normal bursting of the mother cell wall. At the higher irradiance, growth medium supplementation with 80 μM of CuSO₄ led to bleaching of photosynthetic pigments. No growth was observed, while, under the lower irradiance, growth was only slightly inhibited. Results clearly showed that copper toxicity to growth of C. vulgaris was strongly influenced by light intensity. Higher light intensity elicits lethal or sublethal Cu²⁺ damage at concentrations lower than the threshold level for damage at lower light intensities. Cu²⁺ may elicit lethal or sublethal light damage at irradiances lower than the threshold level for unpolluted aquatic systems. Received: 17 January 1997; Accepted 15 April 1997     

COPPER SORPTION AND RELEASE BY CYCLOTELLA MENEGHINIANA (BACILLARIOPHYCEAE) AND CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Initial Cu⁺⁺ sorption by Cyclotella meneghiniana Kütz. (Cu⁺⁺-sensitive) and Chlamydomonas reinhardtii Dangeard (Cu⁺⁺-resistant) was rapid in the first 5 min of Cu⁺⁺ incubation with little sorption after 2 h. On a cell to cell basis, Cyclotella sorbed ca. five times more Cu⁺⁺ from the medium than Chlamydomonas. In MBL medium with EDTA Cyclotella and Chlamydomonas cells sorbed 21.0 and 4.41 nM Cu⁺⁺/10⁶ cells respectively in 6 h with 0.3 mg Cu⁺⁺/l in the medium. Proportionally similar quantities of Cu⁺⁺ were sorbed when the cells were Cu⁺⁺ incubated in MBL + citrate or filtered lake water. Cleaned cell walls of Cyclotella sorbed little Cu⁺⁺ (1.7 nM/10⁶ cells) as compared to living cells (17.5 nM Cu⁺⁺/10⁶ cells) in 3 h. Therefore, in living Cyclotella most of the Cu⁺⁺ taken up must be absorbed by the protoplasm or perhaps by the organic layer surrounding the silica wall. Cleaned cell walls of Chlamydomonas sorbed 3.5 nM Cu⁺⁺/10⁶ cells and living Chlamydomonas cells sorbed 2.6 nM Cu⁺⁺/10⁶ cells. This indicates that most of the Cu⁺⁺ sorbed by Chlamydomonas cells remained bound to the cell wall and probably did not readily enter into the protoplasm: When placed in Cu⁺⁺ free medium after Cu⁺⁺ incubation, Cyclotella and Chlamydomonas cells released 46 and 59% respectively of the Cu⁺⁺ sorbed.     

ENHANCEMENT OF INTRACELLULAR PROLINE LEVEL IN CELLS OF ANACYSTIS NIDULANS (CYANOBACTERIA) EXPOSED TO DELETERIOUS CONCENTRATIONS OF COPPER1

The intracellular proline level in Anacystis nidulans cells was enhanced when the cells were exposed to sublethal concentrations of Cu²⁺; the degree of enhancement was positively related to the concentration of Cu²⁺. Analysis by high-performance liquid chromatography confirmed that the enhancement of proline levels was the most pronounced change in the composition of the free amino acid pool during copper treatment. A direct supply of exogenous proline to the cultures lowered the inhibitory influence of Cu²⁺on the growth of cells. Further experiments showed that the supply of exogenous proline lowered the leakage of potassium ions from cells exposed to deleterious concentrations of Cu²⁺. The inhibition of potassium leakage was particularly pronounced when proline was supplied prior to Cu²⁺treatment. The present study suggests that enhanced proline protects cell membranes from being affected by deleterious concentrations of Cu²⁺.     

The potentiation of industrial biocide activity with Cu2+. I. Synergistic effect of Cu2+ with formaldehyde

Earlier studies suggested that copper enhances the antimicrobial activity of some formaldehyde (FA)-condensate biocides in metalworking fluids as well as FA in laboratory media. The possible synergistic interaction between FA and Cu²⁺ in combination were tested against Pseudomonas aeruginosa in trypic soy broth, mineral salt base-glucose medium, and 0·9% NaCl solution. In all cases, Cu²⁺ enhanced the FA bactericidal activity. A sequential treatment of bacterial cultures was employed to study the increased effectiveness of the Cu²⁺ and FA combination. The cells were exposed to FA or Cu²⁺ and subsequently exposed to the alternate compound with centrifugation and washing between exposures. Results varied depending on the medium. Synergistic activity of FA and Cu²⁺ was established based on the interpretation of the results.     

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer’s disease. However, its physiological function remains elusive. Cu²⁺ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu²⁺ reduction and ⁶⁴Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu²⁺ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu²⁺ ions. Moreover, wild-type cells exposed to both Cu²⁺ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu²⁺ reductase activity and increased ⁶⁴Cu uptake. We conclude that Cu²⁺ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

Cell Wall Accumulation of Cu Ions and Modulation of Lignifying Enzymes in Primary Leaves of Bean Seedlings Exposed to Excess Copper

Copper is both a nutrient and an environmental toxin that is taken up by plants. In order to determine the subcellular localization of copper and to assess the resulting metabolic changes, we exposed 14-day-old bean seedlings to nutrient solutions containing varying concentrations of Cu²⁺ ions for 3 days. Biochemical analyses revealed that the cell wall was the major site of Cu²⁺ accumulation in the leaves of treated plants. Excess copper modified the activity of lignifying peroxidases in both soluble and ionic cell wall-bound fraction. The activity of ionic GPX (guaiacol peroxidase, EC 1.11.1.7) was increased by 50 and 75 μM CuSO_4. The activities of both ionic CAPX (coniferyl alcohol peroxidase, EC 1.11.1.4) and NADH oxidase were increased by both copper concentrations tested. While soluble CAPX activity decreased in leaves treated by all copper concentrations tested, the activity of soluble NADH oxidase remained unchanged at 50 μM and was enhanced at 75 μM. Treatment with CuSO₄ also increased the abundance of total phenol compounds and induced stimulation in the activity of PAL (phenylalanine ammonia lyase, EC. 4.3.1.5). Using histochemistry in combination with fluorescence microscopy we show that bean leaves from copper-exposed plants displayed biochemical and structural modifications reinforcing the cell walls of their xylem tissues. On the other hand, the perivascular fiber sclerenchyma appeared to be less developed in treated leaves.     

Inhibitory effects of copper on bacteria related to the free ion concentration

Cu²⁺ ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO₄.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10^–8–10^–6 M Cu²⁺ ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu²⁺ ion concentrations exist at low copper loads, there was competition of Cu²⁺ for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO₄, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu²⁺, up to 10^–3 M.     

The Effect of Copper on the mRNA Expression Profile of Xenobiotic-Metabolizing Enzymes in Cultured Rat H4-II-E Cells

Copper (Cu²⁺) is an essential element that plays important roles in physiological functions of the body. However, high Cu²⁺ levels can have toxic implications. This study aims to investigate the constitutive response to Cu²⁺ exposure of xenobiotic-metabolizing enzymes in cultured rat liver (H4-II-E) cell lines. Rat cells were exposed to copper sulfate (0–500 μM) for 24 h. The effects of Cu²⁺ on the messenger RNA (mRNA) expressions of phase I and II enzymes and regulatory elements were examined using real-time PCR. Metallothionein mRNA expression was induced in a dose-dependent manner after treatment with Cu²⁺. mRNA expressions of phase I enzymes such as cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2) were slightly induced after exposure to low concentrations of Cu²⁺; however, CYP1A1 and CYP1A2 mRNA expressions were significantly downregulated at higher Cu²⁺ concentrations. These effects corresponded with expression of aryl hydrocarbon receptor mRNA. The mRNA expressions of phase II enzymes were reduced upon exposure to Cu²⁺. In conclusion, phase I and II enzyme expressions were significantly modulated upon Cu²⁺ exposure. These results indicated that Cu²⁺ exposure had toxicological implications for cultured H4-II-E cells.     

ENHANCEMENT OF SENDAI VIRUS-MEDIATED CELL FUSION BY CUPRIC IONS

The effect of divalent cations on cell fusion by concentrated Sendai virus, inactivated by beta-propiolactone, was investigated using Vero and mouse L-929 cells in monolayers. With both cell lines, which are normally resistant to exogenous viral fusion, Cu²⁺ in sublethal concentrations was found to promote polykaryon formation to a marked degree. The simultaneous presence of Cu²⁺ and virus was required for this effect, which was thought to be related to the cytotoxic action of Cu²⁺ on the cell membrane. Accordingly, under standard conditions and in the absence of virus, leakage of isotopically labeled intracellular protein was shown to bear a quantitative relationship to Cu²⁺ concentration. Concomitant changes in the membrane were seen electron microscopically to consist of loss of microvilli and the appearance of numerous vesicles on, or adjacent to, the membrane. The relationship of enhanced fusibility to these toxic changes was not further elucidated. The fusion-promoting effect of Cu²⁺ far exceeded that of Ca²⁺; and other cations tested had no effect.     

Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture,hormone distribution,lignin accumulation and mineral profile

Growth, in particular reorganization of the root system architecture, mineral homeostasis and root hormone distribution were studied in Arabidopsis thaliana upon copper excess. Five-week-old Arabidopsis plants growing in hydroponics were exposed to different Cu²⁺ concentrations (up to 5 μM). Root biomass was more severely inhibited than shoot biomass and Cu was mainly retained in roots. Cu²⁺ excess also induced important changes in the ionome. In roots, Mg, Ca, Fe and Zn concentrations increased, whereas K and S decreased. Shoot K, Ca, P, and Mn concentrations decreased upon Cu²⁺ exposure. Further, experiments with seedlings vertically grown on agar were carried out to investigate the root architecture changes. Increasing Cu²⁺ concentrations (up to 50 μM) reduced the primary root growth and increased the density of short lateral roots. Experiment of split-root system emphasized a local toxicity of Cu²⁺ on the root system. Observations of GUS reporter lines suggested changes in auxin and cytokinin accumulations and in mitotic activity within the primary and secondary root tips treated with Cu²⁺. At toxic Cu²⁺ concentrations (50 μM), these responses were accompanied by higher root apical meristem death. Contrary to previous reports, growth on high Cu²⁺ did not induce an ethylene production. Finally lignin deposition was detected in Cu²⁺-treated roots, probably impacting on the translocation of nutrients. The effects on mineral profile, hormonal status, mitotic activity, cell viability and lignin deposition changes on the Cu²⁺-induced reorganization of the root system architecture are discussed.     

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture

Aims: To determine the effects of supplemented copper (Cu²⁺) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. Methods and Results: Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. Conclusions: Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. Significance and Impact of the Study: This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.     

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I,II, and IV Leads to Increased ROS Production

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu²⁺ overloading states. Besides, mitochondria are one of the most important targets for Cu²⁺, an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu²⁺. Our results showed that Cu²⁺ induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu²⁺-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu²⁺ treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu²⁺ could interact with respiratory complexes (I, II, and IV). This suggests that Cu²⁺-induced liver toxicity is the result of metal’s disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu²⁺-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Copper toxicity to five Parmelia lichens in vitro

Treatment of Parmelia caperata, P. perlata, P. subrudecta, P. sulcata and P. tiliacea with CuSO₄ resulted in a time- and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus, indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached intracellular sites in the thallus, and K⁺/Cu²⁺ exchange was not electroneutral. After 5 h of exposure to copper, the extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with copper absorption levels, but only at 0.05<P<0.10, suggesting that membrane damage was proportional to the amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall. Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic, and usnic acids. Titration of the extracts with copper and assay of the free Cu²⁺ concentration revealed the presence of copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu²⁺ to each of the lichen substances detected in the extracts. However, no significant correlation (P>0.10) was found between the Cu²⁺-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a simple function of the Cu²⁺-binding properties of the lichen substances present in the thallus. Several hypotheses were formulated to interpret the results.     

Physiological responses induced by copper bioaccumulation in <Emphasis Type="Italic">Eichhornia crassipes</Emphasis> (Mart.)

Eichhornia crassipes (Mart.) has strong ability to remove Cu²⁺ from copper-contaminated water. Physiological responses in E. crassipes exposed to known concentrations of Cu²⁺ were examined in this study, and demonstrated that E. crassipes could accumulate 314 mg kg⁻¹ dry weight of Cu when exposed to 5 mg l⁻¹ of Cu²⁺ for periods up to 14 d. However, there were marked changes in physiology of the plant commencing at Cu²⁺ concentrations of 1 mg l⁻¹. Results of this study showed that E. crassipes could tolerate moderate concentrations (i.e. 0.5 mg l⁻¹) of Cu²⁺, without significant changes in photosynthetic pigment concentrations, while high concentrations (i.e. 5 and 10 mg l⁻¹) of Cu²⁺ resulted in substantial loss in pigment concentrations. Increases in malondiadehyde (MDA) content were also demonstrated in plant exposure to high Cu²⁺ concentrations. Soluble protein content increased to a level slightly higher than the control at <0.5 mg l⁻¹ of Cu²⁺, but then decreased with exposure to >1 mg l⁻¹ of Cu²⁺. Our results suggest that E. crassipes has a substantial capacity to accumulate copper when cultivated at moderate concentrations of Cu²⁺, without marked changes in its physiology. The findings indicate that E. crassipes is a promising possibility for phytoremediation of moderately Cu-contaminated water bodies. Handling editor: S. M. Thomaz     

The co-effect of copper and lipid vesicles on Aβ aggregation

Both metal ions and lipid membranes have a wide distribution in amyloid plaques and play significant roles in AD pathogenesis. Although influences of different metal ions or lipid vesicles on the aggregation of Aβ peptides have been extensively studied, their combined effects are less understood. In this study, we reported a unique effect of copper ion on Aβ aggregation in the presence of lipid vesicles, different from other divalent metal ions. Cu²⁺ in a super stoichiometric amount leads to the rapid formation of β-sheet rich structure, containing abundant low molecular weight (LMW) oligomers. We demonstrated that oligomerization of Aβ40 induced by Cu²⁺ binding was an essential prerequisite for the rapid conformation transition. Overall, the finding provided a new view on the complex triple system of Aβ, copper ion and lipid vesicles, which might help understanding of Aβ pathologies.     

Removal of copper using marine brown alga, Undaria Pinnatifida modified with oxime

Summary As oxime is selective for Cu²⁺, oxime groups were introduced to the cell wall of alga by glutaraldehyde. Such modified biomass showed high affinity for Cu²⁺, which resulted in the increase of copper sorption capacity about 4.5 times higher than that of natural alga. For pH range from 2.5 to 3.0, only Cu²⁺ were removed by alga biomass modified with oxime, while other heavy metal ions such as Ca²⁺,Cd²⁺,Pb²⁺ were not adsorbed. By changing pH, selective recovery of Cu²⁺ was achieved.     

Multifunctional peptide‐based fluorescent chemosensor for detection of Hg2+, Cu2+ and S2− ions

A novel multifunctional fluorescent peptide sensor based on pentapeptide dansyl‐Gly‐His‐Gly‐Gly‐Trp‐COOH (D‐P5) was designed and synthesized efficiently using Fmoc solid‐phase peptide synthesis (SPPS). This fluorescent peptide sensor shows selective and sensitive responses to Hg²⁺ and Cu²⁺ among 17 metal ions and six anions studied in N‐2‐hydroxyethylpiperazine‐N‐2‐ethane sulfonic acid (HEPES) buffer solution. The peptide probe differentiates Hg²⁺ and Cu²⁺ ions by a ‘turn‐on’ response to Hg²⁺ and a ‘turn‐off’ response to Cu²⁺. Upon addition of Hg²⁺ or Cu²⁺ ions, the sensor displayed an apparent color change that was visible under an ultraviolet lamp to the naked eye. The limits of detection (LOD) of DP‐5 were 25.0 nM for Hg²⁺ and 85.0 nM for Cu²⁺; the detection limits for Cu²⁺ were much lower than the drinking water maximum contaminant levels set out by the United States Environmental Protection Agency (USEPA). It is noteworthy that both D‐P5‐Hg and D‐P5‐Cu systems were also used to detect S^2? successfully based on the formation of ternary complexes. The LODs of D‐P5‐Hg and D‐P5‐Cu systems for S^2? were 217.0 nM and 380.0 nM, respectively. Furthermore, the binding stoichiometry, binding affinity and pH sensitivity of the probe for Hg²⁺ and Cu²⁺ were investigated. This study gives new possibilities for using a short fluorescent peptide sensor for multifunctional detection, especially for anions.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.