Radovan Popovic

Resolution of the photosystem I and photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature

Green leaves illuminated with photosynthetically active light emit red fluorescence, whose time-dependent intensity variations reflect photosynthetic electron transport (the Kautsky effect). Usually, fluorescence variations are discussed by considering only the contribution of PSII-associated chlorophyll a, although it is known that the fluorescence of PSI-associated chlorophyll a also contributes to the total fluorescence [Aust. J. Plant Physiol. 22 (1995) 131]. Because the fluorescence emitted by each photosystem cannot be measured separately by selecting the emission wavelength in in vivo conditions, the contribution of PSI to total fluorescence at room temperature is still in ambiguity. By using a diode array detector, we measured fluorescence emission spectra corresponding to the minimal (F(O)) and maximal (F(M)) fluorescence states. We showed that the different shapes of these spectra were mainly due to a higher contribution of PSI chlorophylls in the F(O) spectrum. By exciting PSI preferentially, we recorded a reference PSI emission spectrum in the near far-red region. From the F(O) and F(M) spectra and from this PSI reference spectrum, we derived specific PSI and PSII emission spectra in both the F(O) and F(M) states. This enables to estimate true value of the relative variable fluorescence of PSII, which was underestimated in previous works. Accurate separation of PSI-PSII fluorescence emission spectra will also enable further investigations of the distribution of excitation energy between PSI and PSII under in vivo conditions.

Polymer coating of copper oxide nanoparticles increases nanoparticles uptake and toxicity in the green alga Chlamydomonas reinhardtii.

Copper oxide nanoparticles (CuO NPs) are frequently used in a polymer-coated form, to be included in paints or fabrics for antimicrobial properties. Their application in antifouling paints may lead to the contamination of aquatic ecosystems. However, the toxicological risk of NPs in the environment is hard to evaluate due to a lack of knowledge on the mechanisms of NP interaction with biological systems. In this study, we investigated the effect of polymer coating on CuO NP toxicity in the green alga Chlamydomonas reinhardtii by comparing bare and polymer-coated CuO NPs prepared from the same CuO nanopowder. Both CuO NP suspensions were toxic to C. reinhardtii after 6 h treatment to concentrations of 0.005-0.04 g L(-1). Bare and polymer-coated CuO NPs induced a decrease of Photosystem II activity and the formation of reactive oxygen species. Polymer-coated CuO NP was found to be more toxic than the uncoated CuO NP. The higher toxicity of CS-CuO NP was mainly associated with the increased capacity of polymer-coated CuO NP to penetrate the cell compared to bare CuO NPs. These results indicates that the high toxicity of polymer-coated CuO NPs in algal cells results of intracellular interactions between NPs and the cellular system.

Evaluation of toxicity and oxidative stress induced by copper oxide nanoparticles in the green alga Chlamydomonas reinhardtii.

Copper oxide nanoparticles (CuO NP) are frequently employed for their antimicrobial properties in antifouling paints. Their extensive use can contaminate aquatic ecosystems. However, the toxicological effects of this NP in the environment are poorly known. In this study, we evaluated the toxicity and oxidative stress induced by CuO NP on Chlamydomonas reinhardtii using several toxicological assays. CuO NP was found to induce growth inhibition and a significant decrease in carotenoids levels. From data on cells density after 72 h of CuO NP exposure in light, the EC50 value was calculated to be 150.45±1.17 mg L(-1) and the NOEC≤100 mg L(-1). Evaluation of esterase activity demonstrates a decrease in cell metabolism activity with the increase of CuO NP concentration. The CuO NP induced an increase of reactive species level (190±0.45% at 1000 mg L(-1) after 72 h of exposition, compared to control) and lipid peroxidation of cellular membranes (73±2% at 1000 mg L(-1) of CuO NP in 72 h of exposition, compared to control). Investigation of CuO NP uptake showed the presence of NP into C. reinhardtii cells in different sites of the cell and, biomarkers of enzymatic antioxidants showed a change of activity after CuO NP exposition. In conclusion, C. reinhardtii was shown to be sensitive to the presence of CuO NP in their environment and CuO NP treatments induced a toxic response from 0.1 mg L(-1) after 72 h of treatment.

Mercury inhibition at the donor side of photosystem II is reversed by chloride

Mercury is an environmental contaminant that strongly inhibits photosynthetic electron transport, photosystem II being the most sensitive target. We investigated in greater detail the effect of mercury using photosystem II submembrane fractions of higher plants. Oxygen evolution was strongly inhibited and variable chlorophyll fluorescence was severely quenched by mercury. Chloride, an inorganic cofactor known to be essential for the optimal function of photosystem II, significantly reversed the inhibitory effect of mercury. However, calcium, another essential cofactor, showed no reversal capacity. It is concluded that on the donor side of PSII, mercury exerts its action by perturbing chloride binding and/or function. Considering the exceptional affinity of mercury for sulfhydryl groups of proteins, the results suggest the implication of cystein residue(s) in maintaining structural and functional integrity of photosystem II.

Evidence for the Rapid Phytotoxicity and Environmental Stress Evaluation Using the PAM Fluorometric Method: Importance and Future Application

Modulated fluorometry has opened new possibilities for the interpretation of photosynthetic parameters concerning the physiological state of plants. By exposing plant to continuous actinic light and to pulses of saturating light, it is possible to calculate from the Kautsky fluorescence transient four important values: ΦM, the maximum quantum yield as a measure of the maximum photosystem II (PSII) photochemistry; Φ′M, the steady state quantum yield as a real PSII photochemistry at the equilibrium state of electron transport; QP, the photochemical quenching as a measure of light-energy dissipation via photosynthetic electron transport; and QN, the non-photochemical quenching as a measure of dissipation of the absorbed light energy via non-photochemical processes. The change of these PAM-fluorescence parameters is related to the physiological state of the plant and therefore they can be used as reliable indicators of different environmental stress effects. However, these parameters have never been seen as useful standard tools for toxicological monitoring of ecosystems. Highly sensitive modulated fluorometers are today available which allow to measure the fluorescence parameters either in the lab or in the field. In this report we evaluate the use of these parameters in assessing the rapid environmental impact of mercury toxicity on Selenastrum capricornutum and of temperature stress on tomato plants. In interpreting our results, we suggest the utility of some of these parameters as technologically advanced tools in future bioassays.

Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures.

Copper oxide nanoparticles (CuO NPs) are used for their biocide potential however they were also shown to be highly toxic to mammalian cells. Therefore, the effects of CuO NPs should be carefully investigated to determine the most sensitive processes for CuO NP toxicity. In this study, the genotoxicity of CuO NPs was investigated in vitro, using the mouse neuroblastoma cell line Neuro-2A. Genotoxic effects related to DNA fragmentation, DNA methylation and chromosomal damage, as well as lipid peroxidation, were investigated and compared to cytotoxic effects, measured by the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. Based on mitochondrial activity, CuO NPs were found to be cytotoxic. At the highest concentration tested (400 mg l⁻¹), 63% of cell viability was found in Neuro-2A cells after 24 h of treatment to CuO NPs. CuO NPs were also found to induce DNA fragmentation, lipid peroxidation and micronucleus formation. The micronucleus assay was the most sensitive to evaluate CuO NP genotoxicity and micronucleus frequency was increased significantly at 12.5 mg l⁻¹ CuO NPs after 24h of treatment. At this concentration, no significant change of cell viability was found using the mitochondrial activity assay. These results highlight the important risk of genotoxic effects of CuO NPs and show that genotoxicity assays are a sensitive approach to evaluate the risk of CuO NP toxicity.

Protective role of CaCl2 against Pb2+ inhibition in photosystem II.

The Pb2+ treatment of Photosystem II (PS II) membrane fragments, either intact or depleted in 17 and 23 kDa extrinsic polypeptides, inhibits PS II activity. When CaCl2 was present in the assay, the Pb2+ inhibition was significantly reduced in both types of PS II membranes, suggesting a protective role of CaCl2 against Pb2+ inhibition. However, in either case, the degree of PS II inhibition by Pb2+ was higher in the protein depleted than in intact PS II. It showed that the loss of endogenous Ca2+ induced by polypeptide depletion causes the PS II to be more susceptible to Pb2+. The interaction of Pb2+ with CaCl2 in protein‐depleted PS II was competitive. Our results suggest that Pb2+ competes for binding to the Ca2+ and Cl− active sites in the water‐splitting complex. Since Pb2+ inhibition of PS II activity cannot be reversed by CaCl2 but can be reversed by diphenylcarbazide, we conclude that Pb2+ induced inhibition of PS II activity was mediated via the water‐splitting system.

Applications of Chlorophyll Fluorescence in Ecotoxicology: Heavy Metals, Herbicides, and Air Pollutants

Environmental stress factors, such as herbicides, heavy metals, and air pollutants, may produce deleterious effects on photosynthesis and consequently damage higher plant or algal growth. The inhibition of photosynthesis or biochemical processes linked to photosynthesis can represent the physiological state of the plant and therefore measurement of photosynthesis can be used as an indicator of environmental stress effects (Krause and Weis, 1984; Lichtenthaler and Rinderle, 1988; Bolhar-Nordenkampf et al., 1989). Very early in environmental studies in photosynthesis it was concluded that measuring an induced change of the photosynthetic process could be useful to monitor the presence of environmental pollutants (Neubauer and Schreiber, 1987). It has been established that measurements of variable chlorophyll a fluorescence from intact plants offer several parameter values that are very useful for understanding the functioning of specific processes of photosynthesis (Govindjee, 1995). As a consequence, some effects of pollutants detected by the change of fluorescence parameters were found to be directly associated with the photosynthetic process (Van Coillie et al., 1983; Wong and Couture, 1986). Different algal species are used in studying pollutant effects or for routine bioassays based on growth rate or fluorescence emission (Blanck et al., 1984; Juneau et al., 2002).

Induction to oxidative stress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells and Chlamydomonas reinhardtii alga

Saxitoxin (STX) is a cyanotoxin, which can cause neurotoxic effects and induce ecological changes in aquatic environments, a potential risk to public and environmental health. Many studies of cytotoxicity on animal cells and algae have been performed, although few compare the toxic effects between the two models. In this sense, we investigated the oxidative stress induced by STX (0.4-3.0 nM) in two different cellular models: Neuro-2A (N2A) cells and Chlamydomonas reinhardtii alga by quantification of malondialdehyde (MDA) levels as indicative of lipid peroxidation (LPO). Also was evaluated the antioxidant defense of these cells systems after exposure to STX by the addition of antioxidants in N2A cells culture, and by the measure of antioxidants enzymes activity in C. reinhardtii cells. The MDA levels of N2A cells increased from 15% to 113% for 0.4 and 3.0 nM of STX, respectively, as compared to control. Superoxide-dismutase and catalase did not appear to protect the cell from STX effect while, in cells treated with vitamin E, the rates of MDA production decreased significantly, except for higher concentrations of STX. No MDA productions were observed in algal cells however some effects on antioxidant enzymes activity were observed when algae were exposed to 3.0 nM STX. Our results indicate that the concentrations of STX that may induce oxidative stress through LPO are different in animal and phytoplankton communities. A combination of algal and animal bioassays should be conducted for reliable assessment of oxidative stress induced by STX.

Studies on the O-J-I-P transient of chlorophyll fluorescence in relation to photosystem II assembly and heterogeneity in plastids of greening barley

Abstract The polyphasic variable fluorescence in saturating light (O-J-I-P transient, Strasser et al. (1995) Photochem. Photobiol. 61: 21–42) has been investigated in etiochloroplasts during the greening of etiolated leaves of Hordeum vulgare . The initial photochemical phase (O-J) due to reduction of the primary quinone acceptor Q A was found to represent a constant proportion (65–70%) of total variable fluorescence independent of greening time. The partial fluorescence quenching in the Q A -reduced state seems, therefore, to represent a basic property of PSII electron transport. The biphasic character of the slower J-I-P transient due to reduction of the plastoquinone pool developped progressively during the first hours of greening. In the same period of time the proportion and rate constant of rapid PSIIα sub-population increased, as calculated from the induction curve in the presence of DCMU. Etiochloroplasts or chloroplasts resuspended in low salt medium showed a low I level, which was restored upon readdition of 5 MM MgCl 2 and NaCl. Salts also increased the apparent proportion of PSIIα. These results suggest that the J-I and I-P phases of the induction curve are related to different rates of plastoquinone photoreduction by two distinct PSII populations. The effects of DMQ and of DCBQ on the O-J-I-P transient were also studied in (etio-)chloroplasts. In addition to the already reported quenching of the initial ( F O ) and variable fluorescence by DCBQ, a slow fluorescence increase phase was found to appear upon the addition of DCBQ but not of DMQ. The latter observations confirm that DCBQ differs from DMQ by its higher efficiency as PSII electron acceptor.