Nitellopsis Research Articles

The effect of aluminium on bioelectrical activity of the Nitellopsis obtusa cell membrane after H+-ATPase inhibition

AbstractAluminium induced membrane potential (Em) changes and potential changes during repolarization phase of the action potential (AP) in the internodal cells of Nitellopsis obtusa after blocking H+-ATPase activity by DCCD were investigated. Micromolar concentrations of DCCD are sufficient to give complete and irreversible inhibition of proton pumping. The membrane potential was measured by conventional glass-microelectrode technique. We found that the half-amplitude pulse duration differs significantly between standard conditions, after DCCD application, and after H+-ATPase blocking and subsequent Al3+ treatment: 4.9, 7.7 and 17.2 seconds, respectively. We propose that in the short term (2 hours) treatment of Al3+, the decrease in membrane potential was compensated for by H+-ATPase activity. Blocking H+-ATPase activity by DCCD can enhance the influence of Al3+ on the bioelectrical activity of cell membranes.

Biological Activity of New N-Oxides of Tertiary Amines

Potential biological properties of newly synthesized single and double alkyl chain N-oxides of tertiary amines (NTA) were studied. Individual compounds in each of the series had alkyl chains of different length. Various experiments were performed to determine a mechanism of the interaction between NTA and model and biological membranes. These were measurements of hemolytic efficiencies of NTA (pig erythrocytes), their influence on the transition temperatures (DPPC liposomes), on potassium leakage from cucumber, its growth and chlorophyll content (Cucumis sativus cv. Krak F1), and on the resting membrane potential in alga cells (Nitellopsis obtusa). Also, prevention of erythrocyte membrane lipid oxidation induced by UV irradiation was studied. Potential antioxidative properties of NTA were additionally tested in radical chromogen (ABTS) experiments in which antioxidative efficiencies of NTA were compared to that of the standard antioxidant Trolox. It was found that NTA readily interacted with erythrocyte membranes. Their hemolyzing efficiency increased with the alkyl chain length. Slightly more intensive interaction was found for double alkyl chain compounds. Similar results were obtained in DSC experiments, where incorporation of NTA into liposomal membranes shifted the main transition temperatures and caused a broadening of the main transition peaks depending on the alkyl chain length. Double alkyl chain compounds were also found more efficiently influencing the growth of cucumber. Influence of NTA on the resting membrane potential of algae cells was not quite following the alkyl chain length rule found in erythrocyte and liposome experiments. Also potassium leakage and chlorophyll content determined in physiological experiments were not following the increase of lipophilicity of compounds. Most efficiently influencing those parameters were NTA having shorter alkyl chains, and efficiencies of single alkyl chain compounds were evidently stronger. Both methods used to test the antioxidative properties of NTA showed that they depended on the alkyl chain lengths of compounds within each series, but double alkyl chain ones exhibited markedly greater efficiency.

Adaptation of the photosynthetic apparatus of Nitellopsis obtusa to changing light intensity at the molecular level: different pathways of a singlet excitation quenching

The effect of prolonged illumination (60 min) with photosynthetically active monochromatic radiation of low intensity (3 µmol m−2 s−1) and high intensity (60 µmol m−2 s−1), corresponding to the physiological conditions and light stress conditions, respectively, was studied in the algae Nitellopsis obtusa. Illumination of Nitellopsis obtusa cells with strong light was associated with activation of the xanthophyll cycle, manifested by the deepoxidation of violaxanthin and accumulation of antheraxanthin and zeaxanthin. At the same time, the efficient singlet excitation quenching in the photosynthetic apparatus was activated, as demonstrated by the decrease in the intensity of the chlorophyll a fluorescence emission by ca 50 %. The difference of the fluorescence excitation spectra recorded before and after the light treatment match the difference absorption spectrum of the xanthophyll cycle pigments. The illumination with low light intensity resulted also in the chlorophyll a fluorescence quenching but the effect was very small (less than 10 %). The fluorescence quenching is interpreted in terms of the energy transfer between the Qy energy level of chlorophyll a and the 21 Ag − energy level of zeaxanthin. The singlet energy levels of carotenoids, corresponding to the green spectral region, are also taken into consideration in the interpretation of the excitation energy exchange between the carotenoids and chlorophylls. Possible molecular mechanisms involved in the activation of the strong and the weak excitation quenching, including violaxanthin isomerization, and possible physiological functions of such pathways of energy transfer are discussed.

New occurrences of Charales in the Ukraine

The paper presents a synopsized list for eight species of Charales of the Ukraine and their new occurrences, a degree of vulnerability and category of protection indicated for rare Chara connivens Salzm. ex A. Braun, Ch. delicatula C. Agardh, Nitella capillaris (Krock.) J. Groves et Bull.-Webst., N. mucronata (A. Braun) Miq. in H.C. Hall, and Nitellopsis obtusa (Desv. in Loisel.) J. Groves.

Microbiological degradation of a spent offset-printing developer

To decontaminate spent offset-printing developer Polychrome 4003, several microorganisms were separated from the soil that has been used for developer dumping for 3 years. Two organism cultures were isolated and identified to genus Geomyces pannorum and Bacteria spp. These organisms, as well as commercial Septic Gobbler (SG) bacteria, were used to decontaminate the developer. Reduction of both the chemical oxygen demand (COD) and the amount of total identified organic compounds reached 30% after 40 day treatment of waste suspension by G. pannorum and Bacteria spp. A substantially higher degree of COD reduction by ∼80% and the total amount of identified organic compounds by ∼90% was achieved when SG bacteria have been applied for the same period. According to a rapid electrophysiological test with macrophytic algae Nitellopsis obtusa, the toxicity of spent offset-printing developer Polychrome 4003 was classified as extremely toxic (>100 toxic units, T.U.), and it remained at the same level after treatment with G. pannorum and Bacteria spp. More effective biodegradation with SG bacteria diminished toxicity substantially.

Biogeography of living and fossil Nitellopsis (Charophyta) in relationship to new finds from Morocco

AbstractAim To document the distribution of two closely related macrophyte species, the fossil Nitellopsis (Tectochara) merianii (A. Braun ex Unger) Grambast & Soulié‐Märsche and Nitellopsis obtusa (Desvaux) J. Groves, the single living species of genus Nitellopsis and to draw the relationships between first finds of N. merianii in North Africa and previously known records.Location South part of the High Atlas Range, Aït Kandoula basin, Morocco.Methods New finds of fossil charophyte gyrogonites were determined and illustrated. Using a large bibliographic database of both fossil and extant charophytes, we depict the biogeographical distribution N. merianii and N. obtusa and summarize the ecological requirements of N. obtusa as a modern analogue.Results The Aït Kandoula deposits, dated by magnetostratigraphy and vertebrate fossils to Upper Miocene, provided the first fossil populations of N. merianii in Africa. The distribution patterns of the Tertiary N. merianii and the living N. obtusa superimpose on a large area that extends from Spain to East Asia and characterize them as typically Eurasian taxa indicative for permanent, relatively cold and deep freshwater lakes. During the Quaternary, similar palaeolakes in the Sahara were colonized by N. obtusa.Main conclusions The presence of the Eurasian species N. merianii in North Africa is the result of dispersal by migratory waterbirds and attests to N–S migration occurring during the Upper Miocene (10–5 Myr ago). Gyrogonites imported from Southern Europe could germinate and fulfil their life cycle so as to form a new seed bank of gyrogonites because they had been deposited in a suitable environment consisting of a freshwater mountain lake.

Ecotoxicological study of Lithuanian and Estonian wastewaters: selection of the biotests, and correspondence between toxicity and chemical-based indices

The toxicity of industrial and urban wastewater (WW) samples collected in Lithuania and Estonia was evaluated by using a suite of biological tests comprising the Algaltoxkit F™ with Selenastrum capricornutum, the Charatox with Nitellopsis obtusa, Daphtoxkit F™ with Daphnia magna, Thamnotoxkit F™ with Thamnocephalus platyurus, Protoxkit F™ with Tetrahymena thermophila and the Microtox® with Vibrio fischeri. The Charatox and Thamnotoxkit F™ tests showed highest relative sensitivity, responding to 80–90% of samples, respectively, and both expressed good discrimination capacity between samples. Principal Component and pairwise correlation analysis allowed to select test-battery consisting of Charatox, Thamnotoxkit™ and Microtox®. The WW toxicity was evaluated by means of cumulative indices such as average toxicity (AvTx) and two indices derived from the PEEP-index (Environ. Toxicol. Water Qual. 8 (1993) 115). In addition to these integrated evaluations of test-battery response, WW toxicity was evaluated according to the most sensitive test (MST) in the battery. The linear regression analysis between cumulative toxicity indices and chemical-based indices (derived from comparison of WW chemical concentrations and their respective maximum allowable concentration) revealed positive linear relationships ( r 2=0.7–0.8), while toxicity evaluation based on the MST was less positively related with chemical analysis data ( r 2=0.5–0.6). Although better coincidence between the toxicity and chemical-based assessments was achieved when information from all tests in the battery was assembled, the prediction of toxicity from chemical data was still limited. In search of suitable test-battery for the screening of certain type of WWs, a preliminary study comprising excessive suite of tests might be useful.

Linear Relation between Cell Growth and Water Channels Conducted Lp in Nitellopsis

Abstract: The effect of Lp on growth of internodes of Nitellopsis obtusa (Desv. In Lois) J. Gr. was studied, with treatments of HgCl2 and antibodies against aquaporins (anti‐RD28) on the plasmalemma, using laser scanning confocal microscopy, and simultaneous monitoring of cell growth, turgor, Em and Lp using a turgor/membrane potential probe (T‐EP probe). Laser scanning confocal immunofluorescent microscopy indicated the immunoreaction of anti‐RD28 on the plasma membrane. Experiments showed that, following treatment with 25 μM HgCl2, cell growth was entirely and Lp largely inhibited, and no growth recovery was seen even when Lp was completely reversed by replacement of HgCl2 with β‐mercaptoethanol (ME). Anti‐RD28 inhibited both Lp and growth of internodes of N. obtusa by about 80 %, and the possible influences on cell growth of changes in turgor, respiration and pH in cell walls, being excluded during treatment. It was confirmed experimentally that enlargement growth of internodes of N. obtusa was linearly related to cell Lp.

Response of the charophyte Nitellopsis obtusa to heavy metals at the cellular, cell membrane, and enzyme levels.

The responses of the freshwater macroalga Nitellopsis obtusa to heavy metal (HM) salts of Hg, Cd, Co, Cu, Cr, and Ni were assessed at different levels: whole-cell mortality (96-h LC(50)), in vivo cell membrane (45-min depolarization of resting potential, EC(50)), and enzyme in plasma membrane preparations (K+, Mg2+-specific H+-ATPase inhibition, IC(50)). To measure ATPase activity, a novel procedure for isolation of plasma membrane-enriched vesicles from charophyte cells was developed. The short-term ATPase inhibition assay (IC(50) from 6.0 x 10(-7) to 4.6 x 10(-4) M) was slightly more sensitive than the cell mortality test (LC(50) from 1.1 x 10(-6) to 2.6 x 10(-3) M), and the electrophysiological test with the end point of 45-min depolarization of resting potential was characterized by less sensitivity for HMs (EC(50) from 1.1 x 10(-4) to 2.2 x 10(-2) M). The variability of IC(50) values assessed for HMs in the ATPase assays was close to that of LC(50) values in the mortality tests (CVs from 33.5 to 83.5 and from 12.4% to 57.7%, respectively), whereas the EC(50) values in the electrophysiological tests were characterized by CVs generally below 30%. All three end points identified two separate HM groups according to their toxicity to N. obtusa: Co, Ni, and Cr comprised a group of less toxic metals, whereas Hg, Cu, and Cd comprised a group of more toxic metals. However, the adverse effects within each group were discriminated differently. For example, the maximum difference between the highest and lowest LC(50) for the group of less toxic metals in the long-term mortality test was approximately 60% of the response range, whereas the corresponding difference in IC(50) values in the ATPase assay was 30%. In contrast, the LC(50) values of the more toxic metals occupied only 10% of the response range, whereas the IC(50) values were spread over 70%. Further investigation should be done of the underlying mechanism or mechanisms responsible for the observed differences in the dynamic range of a particular end point of the groups of toxicants of varying strength.

Physiological and hemolytic toxicity of some aminophosphonates.

The effect of novel synthesized aminophosphonates on membrane potential and electrical conductance of internodal cells of Nitellopsis obtusa and hemolysis of erythrocytes (RBC) was studied. It was found that those the organophosphorous compounds, when present at 10-100 microM concentrations, caused depolarization and increased electrical conductance of alga membranes. They also influenced fluidity of erythrocyte membranes. When used at higher concentrations aminophosphonates caused hemolysis of RBC. The changes observed depended on structural features of the aminophosphonates, i.e., substituents at the carbon, phosphorus and nitrogen atoms, and, most probably, may be the result of direct interaction of the aminophosphonates with the lipid phase of the plasma membrane and the induced structural changes. Two modes of interaction are proposed.

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.

Kinetics studies of ultraweak luminescence induced by ascorbic acid in Characeae cells and their structures.

Native non-stressed Nitellopsis obtusa cells emit low-intensity (10-10(4) photons s(-1) cm(-2)) electromagnetic radiation within the visible range. The intensity of emission by these cells is several times higher than the intensity of the environmental medium. This luminescence appears as a long-lived (observable not for hours but for a few days) spontaneous light emission, sensitized by chlorophyll. Cells exposed to ascorbic acid action emit ultraweak radiation, dependent on reagent concentrations. Ultraweak luminescence (UL) intensity increases with rise in concentration, reaching maximum for higher concentrations faster and also changing the kinetics of UL decay. Studies of UL kinetics for different substructures and fractions, as well as for intact cells, allow us to consider single- and double-exponential functions as decay curves, which could suggest two ways of ascorbic acid action in the cells. In the spectral distribution, the bands of chlorophyll, dimoles of singlet oxygen and NADH, FMN and Q(10), which are electron carriers of the respiration chain, can be taken into consideration. Ascorbic acid action shows an increase in the red part of spectrum, except 627 nm, and in these bands of emission, which can be associated with the electron carriers of the respiration chain and lipid peroxidation.

Phytotoxicities of Selected Chemicals and Industrial Effluents to Nitellopsis obtusa Cells, Assessed by Using a Rapid Electrophysiological Charophyte Test

The acute phytotoxicities of seven heavy metals (Cd2+, Cu2+, Hg2+, Ni2+, Zn2+, Cr6+ and Co2+), three phenolic compounds (phenol, 3,5-dichlorophenol and pentachlorophenol) and nine industrial effluents were appraised by using a rapid electrophysiological test with cells of the charophyte, Nitellopsis obtusa. The EC50 values (concentrations causing a 50% decrease in resting potential) obtained for reference chemicals were compared with those of five microbiotests (Polytox®, Microtox®, Selenastrum capricornutum growth inhibition, Daphnia magna immobilisation and Rotoxkit F™) taken from the scientific literature. The 45-minute charophyte test, the freshwater Algaltoxkit F™, Daphtoxkit F™ and Rotoxkit F™ were conducted simultaneously to assess the toxicities of effluents. The Toxkit microbiotests were typically two orders of magnitude more sensitive than the electrophysiological charophyte test to pure chemicals. The electrophysiological charophyte test was generally more sensitive than the Toxkit microbiotests to complex effluents. The rapid electrophysiological test, employing the 45-minute membrane depolarisation of N. obtusa cells as an endpoint, demonstrated similar sensitivity to heavy metals and phenolic compounds as the 20-minute bacterial Polytox® test, but less sensitivity than the 15-minute Microtox® test. Therefore, this rapid macroalgal test appears to be valuable as a sublethal toxicity screening tool for effluents.

Salinity Tolerance in Nitellopsis obtusa

Nitellopsis obtusa (Desv.) J. Groves collected from an oligohaline lake was subjected to long-term salinity treatments in the range of 1.1–17.6 psu (26–520 mosmol kg–1) by adding artificial sea salt to the lake water. The extent of turgor regulation and the solutes involved were estimated by examination of the vacuolar sap. Under salinity stress, N. obtusa did not show the capacity to accumulate K+ which enables euryhaline characeans to restore turgor pressure perfectly and brackish water species at least in part. The K+ concentration of the vacuolar sap remained constant at lower salinities but decreased with increasing salinity and time of exposure. An increase in πi by Na+ and Cl– could be considered an inefficient turgor response, but it is better explained as a failure to regulate osmotic potential and to inhibit influx of Na+ . Sucrose concentrations did not show clear relations to external salinity, but contributed 24% of the vacuolar πi in whorl cells and 16% in internodes. Provided that ionic ratios of Na+, K+ and Ca2+ in the water approximately correspond to seawater, N. obtusa can survive salinity fluctuations up to 17 psu for at least a week. For permanent growth, however, the distribution range of the species is restricted to oligohaline waters with salinities not exceeding 5 psu.

Temporal Relationship between Action Potential and Ca2+ Transient in Characean Cells

Temporal relationship between the action potential and the change in cytosolic Ca2+ concentration was investigated in cells of four species of Characeae, Chara corallina, Nitellopsis obtusa, Nitella flexilis and Nitella axilliformis. The Ca2+ transient was detected by light emission from Ca2+-sensitive photoprotein aequorin injected into the cytoplasm. Action potential was triggered by an outward or sometimes inward electric current pulse of 20–50 ms in most cases. In all species the action potential started at almost the same time as the time at which the light emission from aequorin began to increase. Also the peak of action potential almost coincided with that of light emission, which is in contrast with the slower Ca2+ transient in Chara reported by Thiel et al. [(1997) J. Exp. Bot. 48: 609]. A discussion was made on the origin of Ca2+ transient and the ionic processes during membrane excitation.

The Hydration-Induced Ca2+ Release Is a General Phenomenon in Characeae

When an internodal cell of Nitella axilliformis that had been treated with 10 mM KCl and 1 mM EGTA was examined for transcellular osmosis, an increase in the cytosolic concentration of Ca2+ on the endoosmotic side was observed, as was the case in Nitella flexilis even though the increase was much smaller in Nitella axilliformis. The hydration-induced calcium release (HICR) was also observed in other species of Characeae, namely, Chara corallina and Nitellopsis obtusa. HICR was also demonstrated in cytoplasm isolated from Nitella axilliformis and Chara corallina as from Nitella flexilis. Thus, it appears that HICR is not specific to Nitella flexilis but may be a general phenomenon in Characeae.

Short‐term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa

ABSTRACTIsolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m−3 NaCl solution unless more than several mol m−3 Ca2+ is added. Short‐term effects of NaCl stress on the cytosolic concentration of Ca2+ ([Ca2+]c), cytosolic pH (pHc) and vacuolar pH (pHv) were studied in relation to the external concentration of Ca2+ ([Ca2+]e). Changes in [Ca2+]c were measured with light emission from a Ca2+‐sensitive photoprotein, semisynthetic fch‐aequorin which had been injected into the cytosol. Both pHc and pHv were measured with double‐barrelled pH‐sensitive microelectrodes. When internodal cells were treated with 100 mol m−3 NaCl (0–1 mol m−3 NaCl (0.1 mol m−3 [Ca2+]e), [Ca2+]c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca2+]c was not influenced by the level of [Ca2+]c (0.1 and 10 mol m−3). In some cases, the transient increase in [Ca2+]c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m−3 NaCl (0.1 mol m−3 [Ca2+]c), pHc decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pHv increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pHc and pHv were suppressed when [Ca2+]e was raised from 0.1 to 10mol m−3. These results show that the charophyte alga Nitellopsis can regulate [Ca2+]c, pHc and pHv under NaCl stress in the short term and that the protective effect of Ca2+ on salinity stress is apparently unrelated to perturbation of Ca2+ and pH homeostasis.

Patch clamp study of the voltage-dependent anion channel in the thylakoid membrane.

Measurements of single channel currents were performed on isolated membrane patches from osmotically swollen thylakoids of the Charophyte alga Nitellopsis obtusa. A channel with a high selectivity for anions over cations and a conductance of 100 to 110 pS (114 mm Cl-) was revealed. The channel has a bell-shaped voltage-dependence of the open probability, with a maximum at about 0 mV. This dependence was explained by two gating processes, one causing channel closure at positive and one at negative potentials. The steepness of the voltage-dependence corresponded to approximately 2 elementary charges to be transferred across the entire membrane in each of the two gating processes. The analysis of the anion channel kinetics in the millisecond time domain revealed an e-fold increase of mean open and decrease of mean closed times when the membrane voltage was made more positive by 20 and 36 mV, respectively. Concert transitions of two identical anion channels between open and long inactivated states were observed, while the millisecond closed-open transitions of the two channels within a burst of activity were kinetically independent.

Relationship between biomass and surface area of six submerged aquatic plant species

Mathematical relationships relating the biomass to the surface area of Elodea canadensis Michx., Myriophyllum spicatum L., Nitellopsis obtusa (Desv.) J.Gr., Potamogeton lucens L., Potamogeton pectinatus L. and Potamogeton perfoliatus L. were developed. The methods leading to these relations are presented for plants from Lake Geneva (Switzerland). Results include three sets of data for each species: leaves, stems and whole plant. Differences were evidenced according to plant species; for a same biomass E. canadensis and M. spicatum offered the highest surface area, P. pectinatus and N. obtusa the lowest. For 1.0 g dry weight of whole plant, the surface area was 1255 cm 2 for E. canadensis, 1205 cm 2 for M. spicatum, 560 cm 2 for N. obtusa, 653 cm 2 for P. lucens, 500 cm 2 for P. pectinatus, and 762 cm 2 for P. perfoliatus. Plants with dissected morphology did not necessarily offer the largest surface area per unit biomass.

Wastewater toxicity assessment using the electrophysiological response of a charophyteNitellopsis obtusa

A biotesting method for the assessment of acute sublethal toxicity to charophyte cells of Nitellopsis obtusa was developed and applied for the study of Vilnius and Visaginas wastewaters (WW). This computer-assisted testing procedure was based on the recording of the electrophysiological response of up to 16 single algal cells simultaneously. Membrane parameters of living cells were measured according to the K+-anaesthesia method modified for multichannel recording with extracellular electrodes. Concentration-dependent monotonical decrease of cell resting potential, induced by toxic WW action, was used to determine an effective concentration (EC) values. A multistep time-saving procedure, when the cell group was exposed to subsequent treatment with increasing WW concentrations (30 min per treatment), proved to be adequate for biotesting. The ECs obtained by this procedure were in good agreement with those obtained alternatively by undertaking one-step testing (i.e., 60 min exposure per concentration per cell group). The relative sensitivity of the cellular electrophysiological response, as a toxicity end point, was confirmed by comparing it with lethal concentration (LC) responses in 96- and 192-h exposure tests conducted on the same N. obtusa cells. For Vilnius WW samples taken on different dates, the EC ranged from 27 to 46% WW and corresponded to 96-h LC of 28–50% WW. In contrast, Visaginas WWs sampled before and after chlorination showed no toxic effect either at the membrane or whole cell level. © by John Wiley & Sons, Inc.