Translatomics and physiological analyses of the detoxification mechanism of green alga Chlamydomonas reinhardtii to cadmium toxicity.

Cadmium (Cd) is one of the most toxic pollutants found in aquatic ecosystems. Although gene expression in algae exposed to Cd has been studied at the transcriptional level, little is known about Cd impacts at the translational level. Ribosome profiling is a novel translatomics method that can directly monitor RNA translation in vivo. Here, we analyzed the translatome of the green alga Chlamydomonas reinhardtii following treatment with Cd to identify the cellular and physiological responses to Cd stress. Interestingly, we found that the cell morphology and cell wall structure were altered, and starch and high-electron-density particles accumulated in the cytoplasm. Several ATP-binding cassette transporters that responded to Cd exposure were identified. Redox homeostasis was adjusted to adapt to Cd toxicity, and GDP-L-galactose phosphorylase (VTC2), glutathione peroxidase (GPX5), and ascorbate were found to play important roles in maintaining reactive oxygen species homeostasis. Moreover, we found that the key enzyme of flavonoid metabolism, i.e., hydroxyisoflavone reductase (IFR1), is also involved in the detoxification of Cd. Thus, in this study, translatome and physiological analyses provided a complete picture of the molecular mechanisms of green algae cell responses to Cd.

Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii.

Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]-ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms.

Novel atrazine-binding biomimetics inspired to the D1 protein from the photosystem II of Chlamydomonas reinhardtii.

Biomimetic design represents an emerging field for improving knowledge of natural molecules, as well as to project novel artificial tools with specific functions for biosensing. Effective strategies have been exploited to design artificial bioreceptors, taking inspiration from complex supramolecular assemblies. Among them, size-minimization strategy sounds promising to provide bioreceptors with tuned sensitivity, stability, and selectivity, through the ad hoc manipulation of chemical species at the molecular scale. Herein, a novel biomimetic peptide enabling herbicide binding was designed bioinspired to the D1 protein of the Photosystem II of the green alga Chlamydomonas reinhardtii. The D1 protein portion corresponding to the QB plastoquinone binding niche is capable of interacting with photosynthetic herbicides. A 50-mer peptide in the region of D1 protein from the residue 211 to 280 was designed in silico, and molecular dynamic simulations were performed alone and in complex with atrazine. An equilibrated structure was obtained with a stable pocked for atrazine binding by three H-bonds with SER222, ASN247, and HIS272 residues. Computational data were confirmed by fluorescence spectroscopy and circular dichroism on the peptide obtained by automated synthesis. Atrazine binding at nanomolar concentrations was followed by fluorescence spectroscopy, highlighting peptide suitability for optical sensing of herbicides at safety limits.

Lycorine and UV-C stimulate phenolic secondary metabolites production and miRNA expression in Chlamydomonas reinhardtii.

Studying stress pathways on the level of secondary metabolites that are found in very small concentration in the cells is complicated. In the algae, the role of individual metabolites (such as carotenoids, phenolic compounds, organic acids, and vitamins) and miRNAs that participate in plant's defence are very poorly understood during stressful conditions. Therefore, in the present experiment, the model organism Chlamydomonas reinhardtii was exposed to stress conditions (Lyc and UV-C irradiation) to detect these substances, even at very low concentrations. The purpose was to monitored changes at each response level with a future view to identifying their specific roles under different stress factors. In stress-treated cultures, numerous transcriptomic and metabolomic pathways were triggered in C. reinhardtii. Although Lyc significantly decreased the concentration of AA, suggesting that Lyc has a similar function in C. reinhardtii as in plants. The negative effect of UV-C radiation was based on the production of ROS and enhancement of antioxidant responses, resulting in increased levels of polyphenols and simple phenolic compounds. Both treatments did lead to extensive changes in transcript levels and miRNA expression patterns.

Whole-genome re-sequencing and transcriptome reveal cadmium tolerance related genes and pathways in Chlamydomonas reinhardtii.

Cadmium (Cd), a common environmental toxic contaminant, is easily accumulated in living organisms, leading to numerous harmful effects. Chlamydomonas reinhardtii, a unicellular eukaryotic green algae strain, is a very suitable candidate for bioremediation of Cd-contaminated water. However, for the poor resistance to Cd, application of C. reinhardtii was restricted and genes mediating Cd tolerance in C. reinhardtii remain unclear. In this paper, adaptive laboratory evolution was performed with algae constant exposure to Cd over 420-day at environmentally relevant concentrations to select C. reinhardtii strains with high tolerance to Cd. Physiological indicators, such as cell proliferation, photosynthetic pigment contents and photosynthetic activity of photosystem were detected to evaluate the Cd tolerance of selected algae strain ALE0.5. Then, whole-genome re-sequencing and transcriptome were applied to identify the genes related to Cd tolerance. Genes involved in photosynthesis (PSBP1), glutathione metabolism (CHLREDRAFT_167073, GPX5) and calcium transport (CHLREDRAFT_189266, CHLREDRAFT_191203, CHLREDRAFT_187187, CSE1) were related to Cd tolerance in C. reinhardtii. This study provides a basis for obtaining transgenic C. reinhardtii strains with high Cd tolerance used for bioremediation of Cd pollution in the future.

Toxicity of superparamagnetic iron oxide nanoparticles to the microalga Chlamydomonas reinhardtii.

Superparamagnetic iron oxide nanoparticles (SPION) have been widely studied for different biomedical and environmental applications. In this study we evaluated the toxicity and potential alterations of relevant physiological parameters caused to the microalga Chlamydomonas reinhardtii (C. reinhardtii) upon exposure to SPION. The results showed dose-dependent toxicity. A mechanistic study combining flow cytometry and physiological endpoints showed a toxic response consisting of a decrease in metabolic activity, increased oxidative stress and alterations in the mitochondrial membrane potential. Additionally, and due to the light absorption of SPION suspensions, we observed a significant shading effect, causing a marked decrease in photosynthetic activity. In this work, we demonstrated for the first time, the internalization of SPION by endocytosis in C. reinhardtii. These results demonstrated that SPION pose a potential risk for the environment if not managed properly.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

The role of size and protein shells in the toxicity to algal photosynthesis induced by ionic silver delivered from silver nanoparticles.

Because of their biocide properties, silver nanoparticles (AgNPs) are present in numerous consumer products. The biocidal properties of AgNPs are due to both the interactions between AgNP and cell membranes and the release of dissolved silver (Ag+). Recent studies emphasized the role of different nanoparticle coatings in complexing and storing Ag+. In this study, the availability of dissolved silver in the presence of algae was assessed for three AgNPs with different silver contents (59%, 34% and 7% of total Ag), silver core sizes and casein shell thicknesses. The impact of ionic silver on the photosynthetic yield of Chlamydomonas reinhardtii was used as a proxy to estimate the amount of ionic silver toxically active during in vivo assays. The results showed that cysteine, a strong silver ligand, mitigated the toxicity of AgNPs in all cases, demonstrating the key role of Ag+ in this toxicity. The results showed that the AgNPs presenting an intermediate level of silver (34%) were 10 times more effective in terms of total mass (EC50 ten times smaller) than those presenting more (59%) or less (7%) silver. The higher toxicity was due to the higher release of Ag+ under biotic conditions due to the high surface/mass ratio of the nanoparticle silver core. Protein shells played a minor role in altering the availability of Ag+, probably acting as intermediate reservoirs. This study highlighted the utility of a very sensitive biological endpoint (i.e., algal photosynthesis) for the optimization of ionic silver delivery by nanomaterials.

Sensitivity of Chlamydomonas reinhardtii to cadmium stress is associated with phototaxis.

Cadmium (Cd) is a common hazardous pollutant to aquatic environments and it easily accumulates in living organisms. The roles of phototactic behavior in Cd tolerance in motile organisms are poorly explored. In this study, two Chlamydomonas reinhardtii strains, a wild type with positive phototaxis (CC125) and a negatively phototactic mutant (agg1), were used to assess the effects of phototaxis on Cd-induced toxicity to algae. Exposure to Cd inhibited the cell growth and photosynthetic activities, reduced the photosynthetic pigment content, and enhanced the intracellular oxidative stress of algae. Well buffered by EDTA in algae medium, the concentrations of Cd causing 50% growth inhibition (EC50) of CC125 and agg1 for 72 h of exposure were 55.96 and 77.20 µM L-1, respectively. Photosystem II activities in CC125 were more sensitive to Cd than agg1 at 60 µM L-1 Cd. In addition, agg1 accumulated less intracellular Cd than CC125. The changes of extracellular polymeric substances and intracellular response to Cd stress might be related to the different tolerances of the two algae to Cd. Taken together, phototaxis was demonstrated to be associated with Cd-induced toxicity to C. reinhardtii.

PKA, PP1, and DC1 phosphorylation mediate alcohol-induced ciliary dysfunction in Chlamydomonas reinhardtii.

Excessive alcohol consumption impairs mucociliary clearance, in part, by compromising ciliary movement. Our previous study found alcohol reduces ciliary beat frequency in Chlamydomonas through a mechanism that involves the ß and γ heavy chains of the outer dynein arm (ODA). Moreover, we identified DC1, a subunit of the ODA-docking complex (ODA-DC), as the first ciliary target for alcohol. DC1 phosphorylation is alcohol sensitive and correlates with alcohol-induced ciliary dysfunction (AICD). Furthermore, DC1 phosphorylation is disrupted in the absence of the central pair and ODA. These results implicate a role for DC1 phosphorylation in regulating the ODA activity and mediating AICD. In our current study, we identified four alcohol-sensitive phosphosites in DC1: S33, T73, T351, and S628. Mutations of these sites rescue the assembly of the ODA-DC and ODA, resulting in wild-type swimming velocities. When cells were challenged with alcohol, we determined that three sites, S33, T351, and S628, are critical for mediating the ciliary slowing effects of alcohol. This result is consistent with our pharmacological studies, which reveal that both PP1 and PKA activities are required for AICD.

Cadmium accumulation and toxicity affect the extracytoplasmic polyphosphate level in Chlamydomonas reinhardtii.

Cadmium is a non-essential metal and highly toxic for biological functions. Depending on the dose of Cd2+, the biochemical response will differ. In this study, we investigated the level of extracytoplasmic polyphosphate (polyP) when Chlamydomonas reinhardtii was exposed to the effect of CdCl2. When compared to control cells, Cd2+-treated cells to 400-600 µM showed a decrease in the growth rate from 0.78 to 0.38 d-1 for the strain CC-125, and a decrease from 0.81 to 0.32-0.35 d-1 for CC-503. This indicated a strong toxicity effect on the population growth of cells during 72 h. In addition, the results demonstrated the decrease in the synthesis and/or the degradation of polyP that was correlated with the accumulation of Cd2+ in both algal strains. Furthermore, the level of polyP decreased in relation to the decrease of FV/FM value. The toxicity of Cd2+ induced a high level of cell necrosis for CC-503, and the level of polyP could not be recovered at 72 h. In response to the toxic effects of Cd2+, the observed formation of palmelloid colonies by CC-125 cells was correlated with the recovery of the polyP level. Nevertheless, both algal strains were able to accumulate significant amount of Cd2+ in their biomass. Therefore, our study demonstrated a distinct impact of Cd2+ on the metabolism of polyP (synthesis and/or degradation), which was dependent on the concentration of CdCl2 and the Chlamydomonas strain. Based on this study, the level of polyP can be used as a biomarker of Cd2+ toxicity at 24-48 h, even with the cell wall-deficient strain CC-503.

Interaction between 1,2-benzisothiazol-3(2H)-one and microalgae: Growth inhibition and detoxification mechanism.

Isothiazolinones, such as 1,2-benzisothiazol-3(2H)-one (BIT), are widely used as biocides for bacterial growth control in many domestic and industrial processes. Despite their advantages as biocides, they are highly toxic and pose a potential risk to the environment. This study investigated the inhibition process and detoxification mechanism involved in microalgal survival and growth recovery after BIT poisoning. BIT could seriously inhibit the growth of Scenedesmus sp. LX1, Chlorella sp. HQ, and Chlamydomonas reinhardtii with half maximal effective concentrations at 72 h (72h-EC50) of 1.70, 0.41, and 1.16 mg/L, respectively. The primary inhibition mechanism was the BIT-induced damage to microalgal photosynthetic systems. However, the inhibited strains could recover when their growth was not completely inhibited. The influence of this inhibiting effect on subsequent algal regrowth was negligible or weak. BIT consumption was the primary reason for their recovery. Notably, algae did not die even if their growth was completely inhibited. If the BIT concentration did not exceed a certain high level, then the inhibited algae could recover their growth relatively well. Microalgal generation of reduced glutathione (GSH) and the oxygen radical scavenging enzymes, superoxide dismutase (SOD) and catalase (CAT), played a key role in detoxification against BIT poisoning.

Physiological changes in Chlamydomonas reinhardtii after 1000 generations of selection of cadmium exposure at environmentally relevant concentrations.

Cadmium (Cd) is a nonessential and toxic trace element widely existing in waters through various anthropogenic activities such as mining and waste disposal. The physiological responses of aquatic organisms to long-term Cd exposure at environmentally relevant concentrations are still not well explored. In the present study, two strains of unicellular green algae Chlamydomonas reinhardtii, a walled strain CC125 and a wall-less strain CC406 were selected to investigate the physiological changes of aquatic organisms after long-term Cd exposure at environmentally relevant concentrations (4.92 and 49.2 µg L-1). After about 1000 generations of selection, all of the two strains showed higher intracellular lipid peroxidation and lower photosynthetic activities, and failed to evolve specific adaptation to high levels of Cd (4.92 mg L-1) compared to the control. However, short-term low dose Cd exposure exerted hormetic effects on C. reinhardtii and the hormetic stimulation of growth rate, chlorophyll contents and photochemical activities at the lower concentration of Cd (4.92 µg L-1) groups were more pronounced than those at higher ones (49.2 µg L-1). Taken together, this study confirmed that long-term exposure to Cd at environmentally relevant concentrations which were regarded as nontoxic in acute experiments would produce toxic effects on C. reinhardtii and should be paid more attention.

The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae.

Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyper-accumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and "omics" approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (-N) in the KD. RNA-seq and network analyses showed that under -N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.

Effects of TiO2, SiO2, Ag and CdTe/CdS quantum dots nanoparticles on toxicity of cadmium towards Chlamydomonas reinhardtii.

Nanoparticles (NPs) are inevitably released into the aquatic environment for being widely used and may affect the toxicity of other contaminants already present in the environment, such as trace metals. However, the effects of NPs on the ecotoxicity of cadmium (Cd), a common environmental trace metal pollutant, are not well explored. In this study, effects of four widely used NPs TiO2 (n-TiO2), SiO2 (n-SiO2), Ag (n-Ag) and CdTe/CdS core/shell quantum dots (QD) on the toxicity of Cd to the freshwater algae Chlamydomonas reinhardtii were assessed respectively. Cd reduced the algae biomass, impaired the photosynthetic activities, and led to intracellular oxidative stress of algae. At non-toxic concentrations, both n-TiO2 (100 mg L-1) and n-SiO2 (400 mg L-1) attenuated the toxicity of Cd towards the algae for reducing the intracellular Cd contents, and the former was more pronounced. QD (0.5 mg L-1) increased the toxicity of Cd to algae, but n-Ag (0.2 mg L-1) had no significant influence on the Cd toxicity to algae. The microscopic observations on the ultrastructure of algae cells presented the same phenomena and n-TiO2, n-SiO2 aggregations were clearly observed outside the cell wall. Furthermore, the regulation of NPs to the Cd toxicity towards algae was related to the intracellular nitric oxide (NO), an important signaling molecule, rather than the phototaxis of algae. Above all, this study provided a basic understanding about the difference in joint toxicity of different kinds of NPs and Cd to aquatic organisms.

Effect of cadmium accumulation on green algae Chlamydomonas reinhardtii and acid-tolerant Chlamydomonas CPCC 121.

Cadmium is one of the most dangerous metals found in wastewater since exposure to low concentrations are highly toxic for cellular functions. In this study, the effect of cadmium accumulation on Chlamydomonas reinhardtii and acid-tolerant strain CPCC 121 was investigated during 48 h under 100-600 µM of Cd and two pH conditions (4 and 7). The toxicity of accumulated Cd was determined by the change of cellular and photosynthetic parameters. Obtained results showed that the maximum capacity of Cd accumulation in algal biomass was reached for both strains at 24 h of exposure to 600 µM of Cd. Under this condition, C. reinhardtii showed a higher uptake of Cd compared to the strain CPCC 121, inducing a stronger cellular toxic impact. Chlamydomonas CPCC 121 showed a tolerance for Cd due to the exclusion of Cd at the cell wall surface, which was higher at pH 4 than pH 7. TEM images and EDX spectrum of Cd distribution within the cell confirmed the role of the cell wall as a barrier for Cd uptake. Although Cd2+ concentration was the highest in the medium, CPCC 121 was the most tolerant at pH 4, but was not enough efficient to be considered for the phycoremediation of Cd. At neutral pH, the efficiency of C. reinhardtii for the removal of Cd was limited by its toxicity, which was dependent to the concentration of Cd in the medium and the time of exposure.

Remediation of Cadmium Toxicity by Sulfidized Nano-Iron: The Importance of Organic Material.

Nanozerovalent iron (nZVI) is widely used for its ability to remove or degrade environmental contaminants. However, the effect of nZVI-pollutant complexes on organisms has not been tested. We demonstrate the ability of a sulfidized derivative of nZVI (FeSSi) to sorb cadmium (Cd) from aqueous media and alleviate Cd toxicity to a freshwater alga for 32 days. FeSSi particles removed over 80% of the aqueous Cd in the first hour and nearly the same concentration of free Cd remained unbound at the end of the experiment. We found that FeSSi particles with Cd sorbed onto them are an order of magnitude more toxic than FeSSi alone. Further, algal-produced organic material facilitates safer remediation of Cd by FeSSi by decreasing the toxicity of FeSSi itself. We developed a dynamic model to predict the maximum Cd concentration FeSSi can remediate without replacing Cd toxicity with its own. FeSSi can remediate four times as much Cd to phytoplankton populations when organic material is present compared to the absence of organic material. We demonstrate the effectiveness of FeSSi as an environmental remediator and the strength of our quantitative model of the mitigation of nanoparticle toxicity by algal-produced organic material.

Toxicity of emerging antifouling biocides to non-target freshwater organisms from three trophic levels.

Antifouling (AF) systems provide the most cost-effective protection against biofouling. Several AF biocides have, however, caused deleterious effects in the environment. Subsequently, new compounds have emerged that claim to be more environment-friendly, but studies on their toxicity and environmental risk are necessary in order to ensure safety. This work aimed to assess the toxicity of three emerging AF biocides, tralopyril, triphenylborane pyridine (TPBP) and capsaicin, towards non-target freshwater organisms representing three trophic levels: algae (Chlamydomonas reinhardtii), crustacean (Daphnia magna) and fish (Danio rerio). From the three tested biocides, tralopyril had the strongest inhibitory effect on C. reinhardtii growth, effective quantum yield and adenosine triphosphate (ATP) content. TPBP caused sub-lethal effects at high concentrations (100 and 250µgL-1), and capsaicin had no significant effects on algae. In the D. magna acute immobilisation test, the most toxic compound was TPBP. However, tralopyril has a short half-life and quickly degrades in water. With exposure solution renewals, tralopyril's toxicity was similar to TPBP. Capsaicin did not cause any effects on daphnids. In the zebrafish embryo toxicity test (zFET) the most toxic compound was tralopyril with a 120h - LC50 of 5µgL-1. TPBP's 120h - LC50 was 447.5µgL-1. Capsaicin did not cause mortality in zebrafish up to 1mgL-1. Sub-lethal effects on the proteome of zebrafish embryos were analysed for tralopyril and TPBP. Both general stress-related and compound-specific protein changes were observed. Five proteins involved in energy metabolism, eye structure and cell differentiation were commonly regulated by both compounds. Tralopyril specifically induced the upregulation of 6 proteins implicated in energy metabolism, cytoskeleton, cell division and mRNA splicing whilst TPBP lead to the upregulation of 3 proteins involved in cytoskeleton, cell growth and protein folding. An ecological risk characterization was performed for a hypothetical freshwater marina. This analysis identified capsaicin as an environment-friendly compound while tralopyril and TPBP seem to pose a risk to freshwater ecosystems. Noneless, more studies on the characterization of the toxicity, behaviour and fate of these AF biocides in the environment are necessary since this information directly affects the outcome of the risk assessment.

Oxidative stress in the algae Chlamydomonas reinhardtii exposed to biocides.

The toxicity of biocides can be associated with the formation of reactive oxygen species (ROS) and subsequent oxidative damage, interfering with the normal function of photosynthetic organisms. This study investigated the formation and effects of ROS in the unicellular green algae Chlamydomonas reinhardtii exposed to three environmentally relevant biocides, aclonifen, dichlofluanid and triclosan. After a first screening to identify which biocides induced ROS, a 24h multi-endpoint analysis was used to verify the possible consequences. A battery of high-throughput methods was applied in algae for measuring ROS formation, reduced glutathione (GSH), lipid peroxidation (LPO), photosystem (PS) II performance and pigments (chlorophylls a, b and carotenoids). Results show that only aclonifen induced ROS after the first 6h exposure, with the other two biocides not showing any ROS formation. Aclonifen, a Protox and carotenoid inhibitor, induced a concentration-dependent ROS formation, LPO and interfered with algae pigments content, while no alterations were detected in GSH content. A significant effect was also seen in the photosynthetic process, especially a reduction in the maximum and effective quantum yields, accompanied by alterations in energy dissipation in PSII reaction centers and the impairment of the electron transport rate. This study demonstrated the successful use of a battery of high-throughput methods for quickly screening biocides capacity to induce the formation of ROS and the subsequent effects in C. reinhardtii, thus revealing their mode of action (MoA) at concentrations before an impact on growth can become effective.

Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii.

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO4 at 100 µM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO4. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO4 was reduced by AO. After cultivation with CuSO4 at 100 µM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO4 stress.