Effects of heavy metals on seed germination and early seedling growth of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Seed is a developmental stage that is highly protective against external stresses in the plant life cycle. In this study, we analyzed toxicity of essential (Cu²⁺ and Zn²⁺) and non-essential heavy metals (Hg²⁺, Pb²⁺ and Cd²⁺) on seed germination and seedling growth in the model species Arabidopsis. Our results show that seedling growth is more sensitive to heavy metals (Hg²⁺, Pb²⁺, Cu²⁺ and Zn²⁺) in comparison to seed germination, while Cd²⁺ is the exception that inhibited both of these processes at similar concentrations. To examine if toxicity of heavy metals is altered developmentally during germination, we incubated seeds with Hg²⁺ or Cd²⁺ only for a restricted period during germination. Hg²⁺ displayed relatively strong toxicity at period II (12–24 h after imbibition), while Cd²⁺ was more effective to inhibit germination at period I (0–12 h after imbibition) rather than at period II. The observed differences are likely to be due in part to selective uptake of different ions by the intact seed, because isolated embryos (without seed coat and endosperm) are more sensitive to both Hg²⁺ and Cd²⁺ at period I. We assessed interactive toxicity between heavy metals and non-toxic cations, and found that Ca²⁺ was able to partially restore the inhibition of seedling growth by Pb²⁺ and Zn²⁺.     

Effect of HgCl2 on acetylcholine,carbachol, and glutamate currents ofAplysia neurons

Summary 1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl₂) on acetylcholine-, carbachol-, and glutamate-activated currents onAplysia neurons. Hg²⁺ was applied with microperfusion.2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl^– dependent and reversed at about –60 mV, while the slower component was K⁺ dependent and reversed at greater than –80 mV.3. Hg²⁺ (0.1–10 µM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl^– current in medial neurons. With only a 1-min preapplication of Hg²⁺, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 µM Hg²⁺.4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1–10 µM Hg²⁺ caused a complete and irreversible blockade of K⁺-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl₂ that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 µM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl₂ as well.5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg²⁺ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg²⁺ toxicity.     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Selective recovery of Zn2+ from waste slag from a metal-processing plant by the microscopic fungus Verticillium marquandii

The successful use of hay extract medium for growth of Verticillium marquandii in the presence of toxic black slag, resulted in selective and efficient Zn²⁺ recovery. An alkaline black waste slag originating from short rotary furnace, contained Zn²⁺ and Pb²⁺ (20% and 3.5%, respectively) in a form of compounds only slightly soluble in water. After 90 h of the culturing in bioreactor on hay extract medium with starch (50 g l^–1) and black waste (5 g l^–1) supplements, 80% of zinc and 0.5% of lead were bound by the mycelium. The pH value of the culture medium played a crucial role for limitation of the waste inhibitory effect on mycelium growth and for heavy metal recovery by the fungus.     

Effect of heavy metals on the glutathione status in different ectomycorrhizal <Emphasis Type="Italic">Paxillus involutus</Emphasis> strains

The glutathione (GSH) status and heavy metal tolerance were investigated in four Paxillus involutus strains isolated from different heavy-metal-polluted and non-polluted regions of Europe. The heavy metal burden in the habitats did not affect significantly either the heavy metal (Cr₂O₇²⁻, Cd²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Cu²⁺) tolerance and accumulation or the GSH production of the strains tested. Exposures to heavy metals increased the intracellular GSH concentrations in 12 from 24 experimental arrangements (four strains exposed to six heavy metals) independently of the habitats of the strains. The importance of GSH in heavy metal tolerance (high MIC values, ability to accumulate heavy metals and to grow in the presence of heavy metals) was thus demonstrated in this ectomycorrhizal fungus.     

Effects of Heavy Metals on Stomatal Movements in Broad Bean Leaves

How do heavy metals affect stomatal movements and whether water channels are involved in stomatal movements was investigated in broad bean (Vicia faba L.) leaves. Three-week old fully expanded leaves were harvested. Leaf epidermis was peeled off and soaked in the Mes–KOH buffer containing the salts of heavy metals. Stomatal aperture was measured under the microscope. The tested heavy metal ions, such as Hg²⁺, Pb²⁺, Zn²⁺, and La³⁺, partly inhibited stomatal opening in light or closing in darkness at submillimolar concentrations, while K⁺, Na⁺ and Mg²⁺ had no visible effects on stomatal movements. As compared to La³⁺, Hg²⁺ affected stomatal movements more significantly. Stomatal movements were almost completely inhibited under a combined Hg²⁺ and La³⁺ treatment. Apparently, La³⁺, a Ca²⁺ channel blocker, inhibits the changes in the cytosolic Ca²⁺ concentration in guard cells, thus affecting stomatal movements. The inhibitory effect of Hg²⁺ on stomatal movements may be explained by the inhibition of water channels. Like Hg²⁺, Zn²⁺ and Pb²⁺ interfered with stomatal movements. It is concluded that heavy metals at submillimolar concentrations inhibit stomatal movements. They may affect water fluxes through guard cell membranes in different ways, i.e., Hg²⁺, Pb²⁺, and Zn²⁺ inhibit water channels, whereas La³⁺ block ion channels. Water channels may be involved in stomatal movements by regulating water fluxes and play a dominant and primary role in stomatal movements.     

In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes

In vitro selection of Zn²⁺-dependent RNA-cleaving DNAzymes with activity at 90°C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn²⁺ over Pb²⁺, Ca²⁺, Cd²⁺, Co²⁺, Hg²⁺, and Mg²⁺. The Zn²⁺-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn²⁺-binding dissociation constant of 234 μM and that its activity increases with increasing temperatures from 50–90°C. A fit of the Arrhenius plot data gave E_a = 15.3 kcal mol⁻¹. Surprisingly, the selected Zn²⁺-dependent DNAzymes showed only a modest (∼3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn²⁺ is present. Since this effect is highly specific for Zn²⁺, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.     

Competitive adsorption of Pb, Cd and Zn ions onto Eichhornia crassipes in binary and ternary systems

A batch sorption technique was used to study the biosorption of Pb²⁺, Cd²⁺ and Zn²⁺ ions onto the vastly abundant water hyacinth weed, Eichhornia crassipes biomass in binary and ternary systems at a temperature of 30 °C and pH 4.84. Mutual interference effects were probed using equilibrium adsorption capacity ratios, , where the prime indicates the presence of one or two other metal ions. The combined action of the metals was found to be antagonistic, and the metal sorption followed the order Pb²⁺  Cd²⁺  Zn²⁺. The behaviour of competitive biosorption for Pb–Cd and Pb–Zn combinations were successfully described by the Langmuir Competitive Model (CLM), whilst the model showed poor fitting to the Cd–Zn data. In conclusion, Pb²⁺ ions could still be effectively removed from aqueous solution in the presence of both Cd²⁺ and Zn²⁺ ions, but removal of the Cd²⁺ and Zn²⁺ ions would be suppressed in the presence of Pb²⁺.     

Responses of elongation growth rate,turgor pressure and cell wall extensibility of stem cells of Impatiens balsamina to lead,cadmium and zinc

Elongation growth rate of stem cells of Impatiens balsamina was inhibited by the heavy metals Pb²⁺, Cd²⁺ and Zn²⁺ due to their suppression on cell wall extensibility. Effective turgor was also inhibited by Pb²⁺ and Cd²⁺ but it played a secondary role in reducing the stem cell elongation growth rate. The major rate-limiting factor for cell elongation growth was the cell wall extensibility. Furthermore, Cd²⁺ was found to be more toxic than Pb²⁺, while Pb²⁺ was more toxic than Zn²⁺.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Effects of Cadmium, Lead, Mercury and Zinc on °-Aminolevulinic Acid Dehydratase Activity from Radish Leaves

The purpose of the present study was to investigate the in vitro and the in vivo effects of cadmium, zinc, mercury and lead on -aminolevulinic acid dehydratase (ALA-D) activity from radish leaves. The in vivo effect of these metals on growth, DNA and protein content was also evaluated. The results demonstrated that among the elements studied Cd²⁺ presented the highest toxicity for radish. 50% inhibition of ALA-D activity (IC₅₀) in vitro was at 0.39, 2.39, 2.29, and 1.38 mM Cd²⁺, Zn²⁺, Hg²⁺ and Pb²⁺, respectively. After in vivo exposure Cd²⁺, Zn²⁺, Hg²⁺ and Pb²⁺ inhibited ALA-D by about 40, 26, 34 and 15%, respectively. Growth was inhibited by about 40, 10, 25, and 5% by Cd²⁺, Zn²⁺, Hg²⁺, and Pb²⁺, respectively. DNA content was reduced about 35, 30, 20, and 10% for Cd²⁺, Zn²⁺, Hg²⁺, and Pb²⁺, respectively. The metal concentration in radish leaves exposed to Cd²⁺, Zn²⁺, Hg²⁺, and Pb²⁺ was 18, 13, 6, and 7 mol g^–1, respectively. The marked ability of radish to accumulate Cd²⁺ and Zn²⁺ raises the possibility of using this vegetable as a biomonitor of environmental contamination by these metals.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb²⁺) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 μM to 200 μM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 μM Pb²⁺. In tobacco plants growing for 6 weeks in soil polluted with Pb²⁺ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb²⁺-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb²⁺, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb²⁺ accumulation in the above-ground parts may explain the lower levels or the absence of Pb²⁺-induced DNA damage in leaves.     

Accumulation of Cd2+ and Pb2+ in the suspension cultures of Agave amaniensis and Costus speciosus and the determination of the culture's growth and phytosteroid content

Cell suspension cultures of Agave amaniensis and Costus speciosus were grown in media containing Cd^{2 +} up to 25 and 20 mg l^–1, respectively, and Pb²⁺ up to 40 mg l^–1. The cultures hyper-accumulated Cd²⁺ up to 900 and 530 g g^–1 and Pb²⁺ up to 1390 and 1170 g g^–1 dry wt. in their respective biomasses. Increasing Pb²⁺ up to 30 mg l^–1 increased the biomass production and total sitosterol content of Costus speciosus by up to 1.7- and 1.3-fold, respectively.     

Uptake of heavy metals by <Emphasis Type="Italic">Stylonychia mytilus</Emphasis> and its possible use in decontamination of industrial wastewater

The heavy metal resistant ciliate, Stylonychia mytilus, isolated from industrial wastewater has been shown to be potential bioremediator of contaminated wastewater. The ciliate showed tolerance against Zn²⁺ (30 μg/mL), Hg²⁺ (16 μg/mL) and Ni²⁺ (16 μg/mL). The metal ions slowed down the growth of the ciliate as compared with the culture grown without metal stress. The reduction in cell population was 46% for Cd²⁺, 38% for Hg²⁺, 23% for Zn²⁺, 39% for Cu²⁺ and 51% for Ni²⁺ after 8 days of metal stress. S. mytilus reduced 91% of Cd²⁺, 90% of Hg²⁺ and 98% of Zn²⁺ from the medium after 96 h of incubation in a culture medium containing 10 μg/mL of the respective metal ions. Besides this, the ciliate could also remove 88% of Cu²⁺ and 73% Ni²⁺ from the medium containing 5 μg/mL of each metal after 96 h. The ability of Stylonychia to take up variety of heavy metals from the medium could be exploited for metal detoxification and environmental clean-up operations.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Modulation of Na+ transport across leech skin by pesticides and heavy metals

The aim of the present study was to investigate the effects of environmental pollutants, such as heavy metals and pesticides on ion transport across the skin of the leech (Hirudo medicinalis). We wanted to examine the suitability of this epithelium as a model system for studies concerning the mechanisms of toxic action caused by environmental pollutants. For this purpose we performed Ussing chamber experiments to test three representative heavy metals and pesticides, respectively, for their effects on current flow across leech dorsal integument. Two representatives of each substance class showed distinct effects on ion transport across this epithelium. The heavy metal ions Pb²⁺ and Hg²⁺ produced a significant inhibition of amiloride-sensitive Na⁺ transport across leech skin in concentrations below or close to their limiting values in waste water. Therefore, it seems feasible to use leech skin for future investigations of the toxic actions of these heavy metals. The fact that Pb²⁺ and Hg²⁺ exerted their effects only when applied apically points to a specific action of these divalent cations on ion channels in the apical membrane. However, this inhibition does not seem to be a general feature of divalent cations because Cd²⁺ did not influence ion transport across leech skin at all. Since current flow through amiloride-sensitive Na⁺ channels in typical vertebrate tight epithelia is stimulated by numerous divalent cations, the pronounced inhibition of amiloride-sensitive Na⁺ channels in leech skin by Pb²⁺ and Hg²⁺ might lead to a further differentiation of amiloride-sensitive Na⁺ channels. The two widespread pesticides lindane and promecarb exerted their effects only at comparativ high concentrations. This low sensitivity restricts the usefulness of leech skin as a subject for further analysis of toxicity mechanisms, at least for these two pesticides.     

A halophilic thermotolerant Bacillus isolated from a marine hot spring able to produce a new exopolysaccharide

A halophilic, thermotolerant Bacillus strain (B3-15), isolated from water of a shallow, marine hot spring at Vulcano Island (Eolian Islands, Italy), produced an exocellular polysaccharide at 165 mg l^–1. It grew on kerosene as sole carbon source and was resistant to Cd²⁺, Zn²⁺, As²⁺ and Hg²⁺. From 16S rDNA analysis, strain B3-15 was related to B. licheniformis. The exopolysaccharide was a tetrasaccharide repeating unit essentially constituted by sugars having a manno-pyranosidic configuration.     

Zinc and lead uptake by mycelium and regenerating protoplasts of Verticillium marquandii

The ability of the filamentous fungus Verticillium marquandii for Zn²⁺ and Pb²⁺ uptake from aqueous solution was studied. The 24-h-old living mycelium bound Zn²⁺ and Pb²⁺ (206.2 and 324.5 mg/g dry weight, respectively) effectively, in contrast to a very low Zn²⁺ uptake by autoclaved mycelium (20.2 mg/g). The most effective results were noted when the metals were introduced as acetates and incubated with mycelium for 24 h in case of Zn²⁺ while Pb²⁺ achieved the maximum level of metal binding after as early as 3 h. The cell wall was the main site of effective Zn²⁺ and Pb²⁺ binding by V. marquandii mycelium (91.0–93.6% of metals were located in cell wall after 24 h of exposure). The metabolic inhibitors: antimycin A and sodium azide had a strong limitation effect on Zn²⁺ uptake by a 24-h-old living mycelium, whereas Pb²⁺ binding did not decrease to a large extent. The freshly obtained protoplasts accumulated Zn²⁺ and Pb²⁺ on a low level in comparison with cells at different stages of cell wall regeneration. The use of regenerating protoplasts showed that resynthesis of cell wall was necessary for high binding of Zn²⁺, whereas Pb²⁺ uptake on the significant level took place during cell wall regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.