Lipoplexes formed by DNA and ferrocenyl lipids: effect of lipid oxidation state on size, internal dynamics, and zeta-potential

The effect of lipid oxidation state on the physical properties of complexes formed by plasmid DNA and the redox-active lipid bis-(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) is reported. With increasing concentration of BFDMA, the hydrodynamic sizes of complexes formed by BFDMA and DNA (in the presence of 1 mM Li₂SO₄) pass through a maximum and the ζ-potential changes monotonically from −40 mV to +40 mV. In contrast, complexes formed by oxidized BFDMA and DNA exhibit a minimum in size and maintain a negative ζ-potential with increasing concentration of BFDMA. Angle-dependent dynamic light scattering measurements also reveal the presence of relaxation processes within complexes formed by DNA and oxidized BFDMA that are absent for complexes formed by DNA and reduced BFDMA. These results, when combined, reveal that the amphiphilic nature of reduced BFDMA leads to lipoplexes with physical properties resembling those formed by classical cationic lipids, whereas the interaction of oxidized BFDMA with DNA is similar to that of nonamphiphilic cationic molecules bearing multiple charges (e.g., spermidine). In particular, the negative ζ-potential and measurable presence of DNA chain dynamics within complexes formed by oxidized BFDMA and DNA indicate that these complexes are loosely packed with excess charge due to DNA in their outer regions. These results, when combined with additional measurements performed in OptiMEM reduced-serum cell culture medium, lead to the proposition that the strong dependence of transfection efficiency on the oxidation state of BFDMA, as reported previously, is largely a reflection of the substantial change in the ζ-potentials of these complexes with changes in the oxidation state of BFDMA.     

Iron/dextran sulfate multilayered microcapsules for controlled release of 10-hydroxycamptothecin

Stable 10-hydroxycamptothecin (HCPT) microcrystals with a length of about 5-10 μm and a ζ-potential of −38.5 mV were produced by pH-induced reprecipitation in presence of a stabilizer hydroxypropylmethylcellulose. Sequential layer growth was achieved by the layer-by-layer (LbL) assembly of Fe³⁺ and dextran sulfate (DS) on the surface of HCPT microcrystals via both electrostatic interaction and chemical complexation process. The satisfactory drug loading content (67.2 ± 0.82%) as well as high encapsulation efficiency (60.56 ± 0.82%) for four bilayers of Fe³⁺/DS coating was achieved. Both in vitro and in vivo release study revealed that the release time increased as the number of deposited Fe³⁺/DS bilayers increased. These results indicated that such iron-polysaccharide multilayered microcapsules can be a promising approach for the construction of an effective controlled release delivery system of HCPT as well as other drugs with potential cytotoxicity or short half-life time.     

Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3

Silver nanoparticles (AgNPs) were obtained by solar irradiation of cell-free extracts of Bacillus amyloliquefaciens and AgNO₃. Light intensity, extract concentration, and NaCl addition influenced the synthesis of AgNPs. Under optimized conditions (solar intensity 70,000 lx, extract concentration 3 mg/mL, and NaCl content 2 mM), 98.23 ± 0.06% of the Ag⁺ (1 mM) was reduced to AgNPs within 80 min, and the ζ-potential of AgNPs reached −70.84 ± 0.66 mV. TEM (Transmission electron microscopy) and XRD (X-ray diffraction) analysis confirmed that circular and triangular crystalline AgNPs with mean diameter of 14.6 nm were synthesized. Since heat-inactivated extracts also mediated the formation of AgNPs, enzymatic reactions are likely not involved in AgNPs formation. A high absolute ζ-potential value of the AgNPs, possibly caused by interaction with proteins likely explains the high stability of AgNPs suspensions. AgNPs showed antimicrobial activity against Bacillus subtilis and Escherichia coli in liquid and solid medium.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

Redox properties of the oxygen-detoxifying flavodiiron protein from the human parasite Giardia intestinalis

Flavodiiron proteins (FDPs) are enzymes identified in prokaryotes and a few pathogenic protozoa, which protect microorganisms by reducing O₂ to H₂O and/or NO to N₂O. Unlike most prokaryotic FDPs, the protozoan enzymes from the human pathogens Giardia intestinalis and Trichomonas vaginalis are selective towards O₂. UV/vis and EPR spectroscopy showed that, differently from the NO-consuming bacterial FDPs, the Giardia FDP contains an FMN with reduction potentials for the formation of the single and the two-electron reduced forms very close to each other (E₁ = −66 ± 15 mV and E₂ = −83 ± 15 mV), a condition favoring destabilization of the semiquinone radical. Giardia FDP contains also a non-heme diiron site with significantly up-shifted reduction potentials (E₁ = +163 ± 20 mV and E₂ = +2 ± 20 mV). These properties are common to the Trichomonas hydrogenosomal FDP, and likely reflect yet undetermined subtle structural differences in the protozoan FDPs, accounting for their marked O₂ specificity.     

The "electrostatic-switch" mechanism: Monte Carlo study of MARCKS-membrane interaction

The binding of the myristoylated alanine-rich C kinase substrate (MARCKS) to mixed, fluid, phospholipid membranes is modeled with a recently developed Monte Carlo simulation scheme. The central domain of MARCKS is both basic (ζ = +13) and hydrophobic (five Phe residues), and is flanked with two long chains, one ending with the myristoylated N-terminus. This natively unfolded protein is modeled as a flexible chain of “beads” representing the amino acid residues. The membranes contain neutral (ζ = 0), monovalent (ζ = −1), and tetravalent (ζ = −4) lipids, all of which are laterally mobile. MARCKS-membrane interaction is modeled by Debye-Hückel electrostatic potentials and semiempirical hydrophobic energies. In agreement with experiment, we find that membrane binding is mediated by electrostatic attraction of the basic domain to acidic lipids and membrane penetration of its hydrophobic moieties. The binding is opposed by configurational entropy losses and electrostatic membrane repulsion of the two long chains, and by lipid demixing upon adsorption. The simulations provide a physical model for how membrane-adsorbed MARCKS attracts several PIP₂ lipids (ζ = −4) to its vicinity, and how phosphorylation of the central domain (ζ = +13 to ζ = +7) triggers an “electrostatic switch”, which weakens both the membrane interaction and PIP₂ sequestration. This scheme captures the essence of “discreteness of charge” at membrane surfaces and can examine the formation of membrane-mediated multicomponent macromolecular complexes that function in many cellular processes.     

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V_1/2) values ranging from ∼−40 mV to ∼+30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes (“fast” and “slow”). “Slow” gating (τ_act = 90 ± 6 ms at +35 mV) was associated with a V_1/2 of activation of +16.6 ± 1.1 mV, whereas “fast” gating (τ_act = 4.5 ± 1.7 ms at +35 mV) was associated with a V_1/2 of activation of −18.8 ± 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate “fast” and “slow” activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished “slow” activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from “slow” to “fast”, suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A₁ (CA1) and novobiocin (NOVO). The calculated K_d values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex: 22.2 ± 8.8 μM⁻¹ s⁻¹ and 2.7 ± 0.6 s⁻¹, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The K_d values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC₅₀ values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC₅₀ for GM was 2.80 ± 0.18 μM, and the relative IC₅₀ values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.     

Ecology of aquatic Ranunculus communities under the Mediterranean climate

The Iberian Peninsula encompasses more than 80% of the species richness of European aquatic ranunculi. The floristic diversity of the phytocoenosis characterised by aquatic Ranunculus and the main physical–chemical factors of the water were studied in 43 localities of the central Iberian Peninsula. Four aquatic Ranunculus communities are found in most of the aquatic environments. These are species-poor and have an uneven distribution: three species of Batrachium are heterophyllous and their communities are distributed in different aquatic ecosystems on silicated substrates; one species is homophyllous and its community occurs in various aquatic ecosystems with carbonated waters. In the Mediterranean climate, Ranunculus species are present in different habitats, as shown by the results of all the statistical analyses. Ranunculus trichophyllus communities occur in base-rich waters with a high buffering capacity (2273.44 ± 794.57 mg CaCO₃ L⁻¹) and a high concentration of cations (Ca²⁺, 121 ± 33.12 mg L⁻¹; Mg²⁺, 71.64 ± 82.77 mg L⁻¹), nitrates (2.89 ± 4.80 mg L⁻¹), ammonium (2.19 ± 1.36 mg L⁻¹) and sulphates (216.25 ± 218.54 mg L⁻¹). Ranunculus penicillatus communities are found in flowing waters with a high concentration of phosphates (0.48 ± 0.6 mg L⁻¹) and intermediate buffering capacity (683.66 ± 446.76 mg CaCO₃ L⁻¹). Both Ranunculus pseudofluitans and Ranunculus peltatus communities grow in waters with low buffering capacity (R. pseudofluitans, 385.91 ± 209.2 mg CaCO₃ L⁻¹; R. peltatus, 263.3 ± 180.36 mg CaCO₃ L⁻¹), and a low concentration of cations (R. pseudofluitans: Ca²⁺, 12.57 ± 9.42 mg L⁻¹; Mg²⁺, 3.42 ± 1.67 mg L⁻¹; R. peltatus: Ca²⁺, 15 ± 18.26 mg L⁻¹; Mg²⁺, 6.26 ± 8.89 mg L⁻¹) and nutrients (R. pseudofluitans: nitrates, 0.23 ± 0.2 mg L⁻¹; phosphates, 0.09 ± 0.1 mg L⁻¹; R. peltatus: nitrates, 0.19 ± 0.21 mg L⁻¹; phosphates, 0.09 ± 0.12 mg L⁻¹); the first in flowing waters, the latter in still waters.     

Species-dependence of the redox potential of the primary quinone electron acceptor QA in photosystem II verified by spectroelectrochemistry

The redox potentials E_m(Q_A/) of the primary quinone electron acceptor Q_A in oxygen-evolving photosystem II complexes of three species were determined by spectroelectrochemistry. The E_m(Q_A/) values were experimentally found to be −162 ± 3 mV for a higher plant spinach, −171 ± 3 mV for a green alga Chlamydomonas reinhardtii and −104 ± 4 mV vs. SHE for a red alga Cyanidioschyzonmerolae. On the basis of possible deviations for the experimental values, as estimated to differ by 9-29 mV from each true value, plausible causes for such remarkable species-dependence of E_m(Q_A/) are discussed, mainly by invoking the effects of extrinsic subunits on the delicate structural environment around Q_A.     

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E_550-535) of 19.7 ± 6.3 mM⁻¹ cm⁻¹ and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 ± 122 μmol min⁻¹ mg⁻¹ protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

The effect of flow turbulence on plant growth and several growth regulators in Egeria densa Planchon

The effects of turbulence velocity on Egeria densa Planchon was studied for 12 weeks using mechanically oscillating grid-generated turbulence without mean flow. The root-mean-square of the turbulence velocity fluctuations (u′) ranged from 1.62 ± 0.44 to 2.86 ± 0.8 cm s⁻¹ (high turbulence), 1.36 ± 0.2 to 1.86 ± 0.78 cm s⁻¹ (medium turbulence) and 0.67 ± 0.12 to 0.81 ± 0.16 cm s⁻¹ (low turbulence). The control was subjected to gentle manual mixing once a day. Shoot elongation was significantly reduced with increasing turbulence intensity, and the endogenous indole acetic acid (IAA) concentration was significantly decreased with increasing turbulence intensity and exposure time. The plants exposed to high turbulence showed a 64.6% decrease in endogenous IAA concentration compared to the control, while it was decreased only 26.9% in plants exposed to low turbulence. IAA and cytokinin catabolism was increased, and there was an increase in the hydrogen peroxide concentration of the tissues, which triggered peroxidase activity. The total chlorophyll and chlorophyll a content decreased with the time of exposure. Although the flow turbulence negatively affected plant growth and metabolism, all of the plants survived for the experimental period.     

Migration and retention of dissolved iron in three mesocosm wetlands

Three mesocosm wetlands (250 cm × 100 cm × 100 cm) with different wetland plants (Calamgrostis angustifolia, CA, Carex lasiocarpa, CL, and C. angustifolia/C. lasiocarpa mixture, AL, respectively) and hydrologic regimes were set to test migration and retention of exogenous dissolved iron ((NH₄)₂Fe(SO₄)₂of 40 mg Fe(II) L⁻¹) in the Sanjiang Plain Wetland in northeast China. The experiment was designed as two stages: open migration period (OMP) for 1.5 d and close retention period (CRP) for 28.5 d. Based on the outflow Fe(II) concentration during the OMP, retention efficiencies (RE) and iron retention fluxes adjusted by area (RF_ad) in the three mesocosm wetlands were calculated, and the migration of iron were modeled using the first-order kinetic model. Outflow pH decreased gradually from a weak alkaline condition to a weak acid condition during the OMP, and then increased during the CRP, while outflow Eh and DO decreased during the experiment. The three mesocosm wetlands had considerable RE ranging from 75% to 98%, with the averaged RF_ad of 4.31 ± 0.17, 4.20 ± 0.16, and 4.37 ± 0.13 g m⁻² h⁻¹ for CA, CL, and AL, respectively. The reduction conditions in the mesocosm wetlands developed after 4 d or 12 d and the former retained iron during the OMP became mobile and discharged primarily in the form of Fe(III). The first-order kinetic model could simulate the outflow concentration of dissolved iron during the OMP (R² = 0.91, 0.69, and 0.68 for CA, CL, and AL, respectively), while the outflow dissolved iron during the CMP was difficult to model because the changed pH and Eh conditions in the mesocosm wetlands cause the former precipitated iron to be mobile after several days.     

Effects of inorganic nitrogen forms on growth, morphology, nitrogen uptake capacity and nutrient allocation of four tropical aquatic macrophytes (Salvinia cucullata, Ipomoea aquatica, Cyperus involucratus and Vetiveria zizanioides)

This study assesses the growth and morphological responses, nitrogen uptake and nutrient allocation in four aquatic macrophytes when supplied with different inorganic nitrogen treatments (1) NH₄⁺, (2) NO₃⁻, or (3) both NH₄⁺ and NO₃⁻. Two free-floating species (Salvinia cucullata Roxb. ex Bory and Ipomoea aquatica Forssk.) and two emergent species (Cyperus involucratus Rottb. and Vetiveria zizanioides (L.) Nash ex Small) were grown with these N treatments at equimolar concentrations (500 μM). Overall, the plants responded well to NH₄⁺. Growth as RGR was highest in S. cucullata (0.12 ± 0.003 d⁻¹) followed by I. aquatica (0.035 ± 0.002 d⁻¹), C. involucratus (0.03 ± 0.002 d⁻¹) and V. zizanioides (0.02 ± 0.003 d⁻¹). The NH₄⁺ uptake rate was significantly higher than the NO₃⁻ uptake rate. The free-floating species had higher nitrogen uptake rates than the emergent species. The N-uptake rate differed between plant species and seemed to be correlated to growth rate. All species had a high NO₃⁻ uptake rate when supplied with only NO₃⁻. It seems that the NO₃⁻ transporters in the plasma membrane of the root cells and nitrate reductase activity were induced by external NO₃⁻. Tissue mineral contents varied with species and tissue, but differences between treatments were generally small. We conclude, that the free-floating S. cucullata and I. aquatica are good candidate species for use in constructed wetland systems to remove N from polluted water. The rooted emergent plants can be used in subsurface flow constructed wetland systems as they grow well on any form of nitrogen and as they can develop a deep and dense root system.