Probing the Influence of Novel Organometallic Copper(II) Complexes on Spinach PSII Photochemistry Using OJIP Fluorescence Transient Measurements.

Modern agricultural cultivation relies heavily on genetically modified plants that survive after exposure to herbicides that kill weeds. Despite this biotechnology, there is a growing need for new sustainable, environmentally friendly, and biodegradable herbicides. We developed a novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino[2,1-b]benzothiazole-2-amine,4-(2-imidazole) that is active on PSII by inhibiting photosynthetic oxygen evolution on the micromolar level. [CuL2]Br2 reduces the FV of PSII fluorescence. Artificial electron donors do not rescind the effect of [CuL2]Br2. The inhibitory mechanism of [CuL2]Br2 remains unclear. To explore this mechanism, we investigated the effect of [CuL2]Br2 in the presence/absence of the well-studied inhibitor DCMU on PSII-containing membranes by OJIP Chl fluorescence transient measurements. [CuL2]Br2 has two effects on Chl fluorescence transients: (1) a substantial decrease of the Chl fluorescence intensity throughout the entire kinetics, and (2) an auxiliary "diuron-like" effect. The initial decrease dominates and is observed both with and without DCMU. In contrast, the "diuron-like" effect is small and is observed only without DCMU. We propose that [CuL2]Br2 has two binding sites for PSII with different affinities. At the high-affinity site, [CuL2]Br2 produces effects similar to PSII reaction center inhibition, while at the low-affinity site, [CuL2]Br2 produces effects identical to those of DCMU. These results are compared with other PSII-specific classes of herbicides.

Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

The redox state of the plastoquinone (PQ) pool is connected to thylakoid lipid saturation in a marine diatom.

The redox state of the plastoquinone (PQ) pool is a known sensor for retrograde signaling. In this paper, we asked, "does the redox state of the PQ pool modulate the saturation state of thylakoid lipids?" Data from fatty acid composition and mRNA transcript abundance analyses suggest a strong connection between these two aspects in a model marine diatom. Fatty acid profiles of Phaeodactylum tricornutum exhibited specific changes when the redox state of the PQ pool was modulated by light and two chemical inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)]. Data from liquid chromatography with tandem mass spectrometry (LC-MS/MS) indicated a ca. 7-20% decrease in the saturation state of all four conserved thylakoid lipids in response to an oxidized PQ pool. The redox signals generated from an oxidized PQ pool in plastids also increased the mRNA transcript abundance of nuclear-encoded C16 fatty acid desaturases (FADs), with peak upregulation on a timescale of 6 to 12 h. The connection between the redox state of the PQ pool and thylakoid lipid saturation suggests a heretofore unrecognized retrograde signaling pathway that couples photosynthetic electron transport and the physical state of thylakoid membrane lipids.

Protective effect of the Spirulina platensis against toxicity induced by Diuron exposure in Mytilus galloprovincialis.

Diuron herbicide is widely used for weeds control in many kinds of cultivations. It reaches the waterbodies through various fate routes and can adversely threaten non-target organism. The current study was carried out to evaluate the antioxidant activity of Spirulina as feed additive against the toxicity of Diuron concentrations (40 and 80 µg/L) on the edible mollusk Mytilus galloprovincialis during seven days of exposure. Oxidative stress biomarkers were applied on mussel gills and digestive gland, investigating changes in enzymes activities such as catalase (CAT), Glutathione-S-transferase (GST) and Acetylcholinesterase (AChE) and the Malondialdehyde level (MDA). The obtained results show that diuron altered oxidative stress biomarkers in both organs, gills and digestive gland. Performed principle component analysis (PCA) highlighted relationship between biomarkers involved in functional response. Spirulina platensis supplemented diet (1 mg/L), completely ameliorated diuron-induced oxidative stress in mussel tissues. Thus, Spirulina seems to be a promising microalgae and eco-friendly tool helping the health recovery of aquatic animals subjected to environmental stressors.

This study provided recent and new data on the impact of Diuron in marine bivalve and the protective effect of Spirulina against Diuron-induced oxidative stress. The results of our study suggest that the antioxidant potential of Spirulina should be strongly candidate for the phytoremediation of Diuron-aquatic contaminated.

A new method for separate evaluation of PSII with inactive oxygen evolving complex and active D1 by the pulse-amplitude modulated chlorophyll fluorometry.

A method that separately quantifies the PSII with inactive oxygen-evolving complex (OEC) and active D1 retaining the primary quinone acceptor (QA )-reducing activity from the PSII with damaged D1 in the leaf was developed using PAM fluorometry. It is necessary to fully reduce QA to obtain F m , the maximum fluorescence. However, QA in PSII with inactive OEC and active D1 would not be fully reduced by a saturating flash. We used the acceptor-side inhibitor DCMU to fully reduce QA . Leaves of cucumber (Cucumis sativus L.) were chilled at 4°C in dark or illuminated with UV-A to selectively inactivate OEC. After these treatments, F v /F m , the maximum quantum yield, in the leaves vacuum-infiltrated with DCMU were greater than those in water-infiltrated leaves. In contrast, when the leaves were illuminated by red light to photodamage D1, F v /F m did not differ between DCMU- and water-infiltrated leaves. These results indicate relevance of the present evaluation of the fraction of PSII with inactive OEC and active D1. Several examinations in the laboratory and glasshouse showed that PSII with inactive OEC and active D1 was only rarely observed. The present simple method would serve as a useful tool to clarify the details of the PSII photoinhibition.

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions.

Rate-limiting steps in the dark-to-light transition of Photosystem II (PSII) were discovered by measuring the variable chlorophyll-a fluorescence transients elicited by single-turnover saturating flashes (STSFs). It was shown that in diuron-treated samples: (i) the first STSF, despite fully reducing the QA quinone acceptor molecule, generated only an F1(

The hydrophobicity of mutations targeting D1:Val219 modifies formate and diuron binding in the quinone-Fe-acceptor complex of Photosystem II.

The D1:Val219 residue of Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 was mutated to alanine or isoleucine, creating the V219A and V219I mutants, respectively. Oxygen evolution was slowed in these mutants, while chlorophyll a fluorescence induction assays indicated slowed electron transfer. As previously observed [Erickson J.M., Rahire, M., Rochaix, J.-D. and Mets. L. (1985) Science, 228, 204-207], the V219I mutant was resistant to 3,4-dichloro-1,1-dimethyl urea (DCMU); however, the V219A strain displayed no DCMU resistance. Additionally, the V219A strain was less sensitive to the addition of formate than the control, while the V219I strain was more sensitive to formate. Both mutant strains were susceptible to photodamage and required protein synthesis for recovery. We hypothesize that the sensitivity to DCMU and the extent of bicarbonate-reversible formate-induced inhibition, as well as the capacity for recovery in cells following photodamage, are influenced by the hydrophobicity of the environment associated with the Val219 residue in D1.

Dark adaptation and ability of pulse-amplitude modulated (PAM) fluorometry to identify nutrient limitation in the bloom-forming cyanobacterium, Microcystis aeruginosa (Kützing).

Harmful algal blooms in inland waters are widely linked to excess phosphorus (P) loading, but increasing evidence shows that their growth and formation can also be influenced by nitrogen (N) and iron (Fe). Deficiency in N, P, and Fe differentially affects cellular photosystems and is manifested as changes in photosynthetic yield (Fv/Fm). While Fv/Fm has been increasingly used as a rapid and convenient in situ gauge of nutrient deficiency, there are few rigorous comparisons of instrument sensitivity and ability to resolve specific nutrient stresses. This study evaluated the application of Fv/Fm to cyanobacteria using controlled experiments on a single isolate and tested three hypotheses: i) single Fv/Fm measurements taken with different PAM fluorometers can distinguish among limitation by different nutrients, ii) measurements of Fv/Fm made by the addition of DCMU are comparable to PAM fluorometers, and iii) dark adaptation is not necessary for reliable Fv/Fm measurements. We compared Fv/Fm taken from the bloom-forming Microcystis aeruginosa (UTEX LB 3037) grown in nutrient-replete treatment (R) and N-, P-, and Fe-limited treatments (LN, LP, LFe, respectively), using three pulse-amplitude modulated (PAM) fluorometers and the chemical photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and evaluated the effects of dark adaptation prior to PAM measurement. There were significant differences in Fv/Fm estimates among PAM fluorometers for light- versus dark-adapted cell suspensions over the whole experiment (21 days), which were all significantly higher than the DCMU-based measurements. However, dark adaptation had no effect on Fv/Fm when comparing PAM-based values across a single nutrient treatment. All Fv/Fm methods could distinguish LN and LP from R and LFe treatments but none were able to resolve LFe from R, or LN from LP cultures. These results indicated that for most PAM applications, dark adaptation is not necessary, and furthermore that single measurements of Fv/Fm do not provide a robust measurement of nutrient limitation in Microcystis aeruginosa UTEX LB 3037, and potentially other, common freshwater cyanobacteria.

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

CIA2 and CIA2-LIKE are required for optimal photosynthesis and stress responses in Arabidopsis thaliana.

Chloroplast-to-nucleus retrograde signaling is essential for cell function, acclimation to fluctuating environmental conditions, plant growth and development. The vast majority of chloroplast proteins are nuclear-encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses that influence nuclear gene expression. Signaling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual-localized TFs involved in chloroplast retrograde signaling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homolog CIA2-like (CIL) are involved in the regulation of Arabidopsis responses to UV-AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signaling events in the chloroplast-nucleus cross-talk.

Comparative effect of tenuazonic acid, diuron, bentazone, dibromothymoquinone and methyl viologen on the kinetics of Chl a fluorescence rise OJIP and the MR820 signal.

In this study, the comparative effect of TeA, DCMU, bentazone, DBMIB and MV on prompt fluorescence and the MR820 signal was simultaneously analyzed to provide an insight into how to elucidate their precise influence on Ageratina adenophora photosystems. The herbicides that interrupt electron transport beyond QA, such as TeA, DCMU and bentazone, mainly increased the J-step level of fluorescence rise kinetics as a result of accumulation of QA-, but showed differences in detail. The IP phase disappeared in the presence of DCMU and bentazone with a significant increase in FO value. TeA treatment retained the IP phase with lowering FM. As an inhibitor of plastoquinone re-oxidation, DBMIB increased the I-step (IP phase almost unnoticable) without changing FO and FM values. MV blocking PSI electron transfer through intercepting electrons from the FeS clusters suppressed the IP phase by decreasing the P level. Considering the WIP kinetics, TeA and DBMIB also affected PSI activity. After DCMU and MV treatment, the major change in the MR820 kinetics was the loss of the slow phase due to the complete prevention of electron movement from PSII to re-reduce PC+ and P700+. TeA, bentazone and DBMIB clearly suppressed the MR820 slow phase and decreased the re-reduction rate of PC+ and P700+ (Vred), significantly. However, there were still parts of electrons being donated to PC+ and P700+, showing a smaller slow phase and PC+ and P700+ re-reduction rate. Additionally, TeA and DBMIB also somewhat declined the fast phase and PC and P700 oxidation rate (Vox).

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2 under illumination. One factor limiting the H2 photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2 production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H2 at three to five times the rate of untreated cells, when exposed to light. The highest H2 photoproduction rates, 2.26 ±â€Š0.24 and 3.63 ±â€Š0.26 µmol H2 g-1 dry weight h-1, were found in cells treated with 0.5 µM CCCP and 50 µM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H2 production compared with cells that were incubated in the light. Only CCCP treatment increased H2 production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2 production rate of 39.50 ±â€Š2.13 µmol H2 g-1 dry weight h-1 was found in cells treated with 0.5 µM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O2 level. In contrast, DCMU reduced the respiration rate in A. halophytica.

The function of PROTOPORPHYRINOGEN IX OXIDASE in chlorophyll biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii.

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b6f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii. The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis.

Influence of Chemically Disrupted Photosynthesis on Cyanobacterial Thylakoid Dynamics in Synechocystis sp. PCC 6803.

The photosynthetic machinery of the cyanobacterium Synechocystis sp. PCC 6803 resides in flattened membrane sheets called thylakoids, situated in the peripheral part of the cellular cytoplasm. Under photosynthetic conditions these thylakoid membranes undergo various dynamical processes that could be coupled to their energetic functions. Using Neutron Spin Echo Spectroscopy (NSE), we have investigated the undulation dynamics of Synechocystis sp. PCC 6803 thylakoids under normal photosynthetic conditions and under chemical treatment with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), an herbicide that disrupts photosynthetic electron transfer. Our measurements show that DCMU treatment has a similar effect as dark conditions, with differences in the undulation modes of the untreated cells compared to the chemically inhibited cells. We found that the disrupted membranes are 1.5-fold more rigid than the native membranes during the dark cycle, while in light they relax approximately 1.7-fold faster than native and they are 1.87-fold more flexible. The strength of the herbicide disruption effect is characterized further by the damping frequency of the relaxation mode and the decay rate of the local shape fluctuations. In the dark, local thicknesses and shape fluctuations relax twice as fast in native membranes, at 17% smaller mode amplitude, while in light the decay rate of local fluctuations is 1.2-fold faster in inhibited membranes than in native membranes, at 56% higher amplitude. The disrupted electron transfer chain and the decreased proton motive force within the lumenal space partially explain the variations observed in the mechanical properties of the Synechocystis membranes, and further support the hypothesis that the photosynthetic process is tied to thylakoid rigidity in this type of cyanobacterial cell.

Change in the antimicrobial resistance profile of Pseudomonas aeruginosa from soil after exposure to herbicides.

The extensive use of pesticides represents a risk to human health and to the environment. This study aimed to investigate if the exposure to atrazine and diuron, two herbicides widely used in Brazil, could induce changes in the susceptibility profile to aztreonam, colistin and polymyxin B antimicrobials in isolates of P. aeruginosa obtained from soil samples by using the determination of minimum inhibitory concentration (MIC) test. Three isolates had an increase of MIC to aztreonam after exposure to both herbicides and one isolate did not show any MIC change. The MexAB-OprM efflux pump has already been upregulated in these isolates and the herbicides atrazine and diuron did not increase MexAB-OprM overexpression. Therefore, the decrease in aztreonam susceptibility was not directly related to this pump, suggesting that probably other mechanisms should be involved.

A novel psbA mutation (Phe274-Val) confers resistance to PSII herbicides in wild radish (Raphanus raphanistrum).

BACKGROUND: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides. RESULTS: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides. CONCLUSION: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides. © 2018 Society of Chemical Industry.

Evaluation of Alkaloids Isolated from Ruta graveolens as Photosynthesis Inhibitors.

Eight alkaloids (1⁻8) were isolated from Ruta graveolens, and their herbicide activities were evaluated through in vitro, semivivo, and in vivo assays. The most relevant results were observed for Compounds 5 and 6⁻8 at 150 µM, which decreased dry biomass by 20% and 23%, respectively. These are significant results since they presented similar values with the positive control, commercial herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Based on the performed assays, Compound 5 (graveoline) is classified as an electron-transport inhibitor during the light phase of photosynthesis, as well as a plant-growth regulator. On the other hand, Compounds 6⁻8 inhibited electron and energy transfers, and are also plant-growth inhibitors. These phytotoxic behaviors based on acridone and quinolone alkaloids may serve as a valuable tool in the further development of a new class of herbicides since natural products represent an interesting alternative to replace commercial herbicides, potentially due their low toxicity.

Herbicides diuron and fluazifop-p-butyl affect avoidance response and multixenobiotic resistance activity in earthworm Eisenia andrei.

The usage of pesticides has been steadily increasing over the last decades, and among them herbicides are the most commonly used ones. Despite their main mode of action targeting plant organisms, they can also have adverse effects on non-target animal organisms. In soil ecosystems, earthworms play an important role due to their positive impacts on the soil functioning and they represent good model organisms in soil ecotoxicology. The aim of the present study was to assess the effects of two herbicides on several endpoints at different levels of biological organization in the earthworm Eisenia andrei. Diuron and fluazifop-p-butyl were selected for the investigation and their lethal concentrations were determined: LC50 48 h: 89.087 µg/cm2 for diuron and 6.167 µg/cm2 for fluazifop-p-butyl. Furthermore, measurements of enzymatic biomarkers (catalase (CAT), acetylcholinesterase (AChE), carboxylesterase (CES) and glutathione S-transferase (GST)), multixenobiotic resistance (MXR) activity and gene expression of antioxidative enzymes (only for fluazifop-p-butyl) were conducted. Enzymatic biomarker responses showed no significant differences compared to the control after the exposure to the investigated herbicides, whereas the MXR activity was significantly inhibited. The gene expression level of superoxide dismutase (sod) and glutathione S-transferase (gst) after fluazifop-p-butyl exposure showed a significant increase. Finally, avoidance behavior in soil was assessed and it was determined that both herbicides caused significant avoidance response. The obtained results show that both investigated herbicides significantly affect earthworms on different levels of biological organization. This emphasizes the importance of comprehensive ecotoxicological assessment of herbicide effects on non-target organisms at all organizational levels.

Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum.

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N'-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.

Thylakoid Containing Artificial Cells for the Inhibition Investigation of Light-Driven Electron Transfer during Photosynthesis.

The fabrication of artificial cells containing nature components is challenging. Herein we construct a thylakoid containing artificial cell (TA-cell) by forming multicompartmental structure inside giant unilamellar vesicles (GUVs) using osmotic stress. The thylakoids are selectively loaded inside each compartment in GUVs to mimic "chloroplast". The TA-cells are able to carry out photosynthesis upon light on. The TA-cells keep their 50% functionality of electron transfer for 12 days, which is twice of those of free thylakoids. Using TA-cells the inhibition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and heavy metal ions (Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+) on the electron transfer process in TA-cells is systematically investigated. Their half maximal inhibitory concentration (IC50) values are 36.23 ± 1.87, 0.02 ± 0.01, 0.42 ± 0.08, 0.82 ± 0.12, 1.97 ± 0.21, and 4.08 ± 0.18 µM, respectively. Hg2+ is the most toxic ion for the photosynthesis process among these five heavy metal ions. This biomimetic system can be expanded to study other processes during the photosynthesis. The TA-cells pave a way to fabricate more complicated nature component containing artificial cells.