Multiple stressor effects in Chlamydomonas reinhardtii--toward understanding mechanisms of interaction between effects of ultraviolet radiation and chemical pollutants.

The effects of chemical pollutants and environmental stressors, such as ultraviolet radiation (UVR), can interact when organisms are simultaneously exposed, resulting in higher (synergistic) or lower (antagonistic) multiple stressor effects than expected based on the effects of single stressors. Current understanding of interactive effects is limited due to a lack of mechanism-based multiple stressor studies. It has been hypothesized that effect interactions may generally occur if chemical and non-chemical stressors cause similar physiological effects in the organism. To test this hypothesis, we exposed the model green alga Chlamydomonas reinhardtii to combinations of UVR and single chemicals displaying modes of action (MOA) similar or dissimilar to the impact of UVR on photosynthesis. Stressor interactions were analyzed based on the independent action model. Effect interactions were found to depend on the MOA of the chemicals, and also on their concentrations, the exposure time and the measured endpoint. Indeed, only chemicals assumed to cause effects on photosynthesis similar to UVR showed interactions with UVR on photosynthetic yield: synergistic in case of Cd(II) and paraquat and antagonistic in case of diuron. No interaction on photosynthesis was observed for S-metolachlor, which acts dissimilarly to UVR. However, combined effects of S-metolachlor and UVR on algal reproduction were synergistic, highlighting the importance of considering additional MOA of UVR. Possible mechanisms of stressor effect interactions are discussed.

The toxicity of chemical pollutants in dynamic natural systems: the challenge of integrating environmental factors and biological complexity.

The dynamics of abiotic and biotic environmental factors, like temperature and predation, can strongly influence the effects of anthropogenic chemical pollutants in natural systems. Responses to toxicants and their interactions with environmental factors can occur at varying temporal scales and at different levels of biological complexity (from cells to organisms, populations, communities and ecosystems). Environmental factors may affect tolerance to toxic pollutants under non-stressful conditions, and cause adverse multiple stressor effects under stressful conditions. Adaptive processes, however, have the potential to either mitigate (by co-tolerance) or increase (due to associated costs) the sensitivity of individuals, populations, and communities to pollutants through selection and evolution of traits (at the individual and population levels) and changes in species composition (at the community level). Responses to such multiple stressor effects on different biological levels and temporal scales are not considered in current risk assessment practices. We suggest that these effects should and can be addressed by: (i) designing ecotoxicological experiments with temporal exposure patterns that accommodate adaptive processes, (ii) using trait-based approaches to assess biological responses and natural selection in an integrated manner, and (iii) using energy allocation models to link responses at different levels of biological organization.

Production, detection, and signaling of singlet oxygen in photosynthetic organisms.

SIGNIFICANCE: In photosynthetic organisms, excited chlorophylls (Chl) can stimulate the formation of singlet oxygen ((1)O(2)), a highly toxic molecule that acts in addition to its damaging nature as an important signaling molecule. Thus, due to this dual role of (1)O(2), its production and detoxification have to be strictly controlled. RECENT ADVANCES: Regulation of pigment synthesis is essential to control (1)O(2) production, and several components of the Chl synthesis and pigment insertion machineries to assemble and disassemble protein/pigment complexes have recently been identified. Once produced, (1)O(2) activates a signaling cascade from the chloroplast to the nucleus that can involve multiple mechanisms and stimulate a specific gene expression response. Further, (1)O(2) signaling was shown to interact with signal cascades of other reactive oxygen species, oxidized carotenoids, and lipid hydroperoxide-derived reactive electrophile species. CRITICAL ISSUES: Despite recent progresses, hardly anything is known about how and where the (1)O(2) signal is sensed and transmitted to the cytoplasm. One reason for that is the limitation of available detection methods challenging the reliable quantification and localization of (1)O(2) in plant cells. In addition, the process of Chl insertion into the reaction centers and antenna complexes is still unclear. FUTURE DIRECTIONS: Unraveling the mechanisms controlling (1)O(2) production and signaling would help clarifying the specific role of (1)O(2) in cellular stress responses. It would further enable to investigate the interaction and sensitivity to other abiotic and biotic stress signals and thus allow to better understand why some stressors activate an acclimation, while others provoke a programmed cell death response.

Down-regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii.

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H2 O2 in the 10 µm range. This concentration range seems to be necessary to activate H2 O2 -dependent signalling pathways stimulating the expression of H2 O2 responsive genes, such as the heat shock protein HSP22C. Catalase knock-down mutants had lost the transient accumulation of H2 O2 , suggesting that a decrease in catalase activity was the key element for establishing a transient H2 O2 burst. Catalase was inactivated by a one-electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H2 O2 , inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast-directed protection enzymes.

Multiple stressor effects of predation by rotifers and herbicide pollution on different Chlamydomonas strains and potential impacts on population dynamics.

Environmental factors can interact with the effects of chemical pollutants on natural systems by inducing multiple stressor effects in individual organisms as well as by altering selection pressure on tolerant strains in heterogeneous populations. Predation is a stressful environmental factor relevant for many species. Therefore, the impact of predation by the rotifer Brachionus calyciflorus on tolerance of eight genetically different strains of the green alga Chlamydomonas reinhardtii to simultaneous exposure to each of the three herbicides (diuron, paraquat, and S-metolachlor) was tested. Interactions of combined stressors were analyzed based on the independent action model; additive, synergistic, and antagonistic effects of the combined exposure could be detected depending on the herbicide and strain tested. If cultures were acclimated (pre-exposed) to one stressor, tolerance to the second stressor could be increased. This indicates that physiological changes can induce cotolerance of predation-exposed algae to herbicides and of herbicide-treated algae to predation depending on the combination of stressors. The strain-specific differences in multiple stressor effects also changed the correlation of strains' tolerances to individual stressors determined during combined and single-stressor exposure. Changes in cotolerance to stressors affect selection pressure and population dynamics during long-term exposure. This shows that predation stress can have adverse effects on the toxicity of chemical pollutants to microalgae on the organism and population levels.

SINGLET OXYGEN RESISTANT 1 links reactive electrophile signaling to singlet oxygen acclimation in Chlamydomonas reinhardtii.

Acclimation of Chlamydomonas reinhardtii cells to low levels of singlet oxygen, produced either by photoreactive chemicals or high light treatment, induces a specific genetic response that strongly increases the tolerance of the algae to subsequent exposure to normally lethal singlet oxygen-producing conditions. The genetic response includes the increased expression of various oxidative stress response and detoxification genes, like the glutathione peroxidase homologous gene GPXH/GPX5 and the σ-class glutathione-S-transferase gene GSTS1. To identify components involved in the signal transduction and activation of the singlet oxygen-mediated response, a mutant selection was performed. This selection led to the isolation of the singlet oxygen resistant 1 (sor1) mutant, which is more tolerant to singlet oxygen-producing chemicals and shows a constitutively higher expression of GPXH and GSTS1. Map-based cloning revealed that the SOR1 gene encodes a basic leucine zipper transcription factor, which controls its own expression and the expression of a large number of oxidative stress response and detoxification genes. In the promoter region of many of these genes, a highly conserved 8-bp palindromic sequence element was found to be enriched. This element was essential for GSTS1 induction by increased levels of lipophilic reactive electrophile species (RES), suggesting that it functions as an electrophile response element (ERE). Furthermore, GSTS1 overexpression in sor1 requires the ERE, although it is unknown whether it occurs through direct binding of SOR1 to the ERE. RES can be formed after singlet oxygen-induced lipid peroxidation, indicating that RES-stimulated and SOR1-mediated responses of detoxification genes are part of the singlet oxygen-induced acclimation process in C. reinhardtii.

Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in Chlamydomonas reinhardtii.

BACKGROUND: When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed. RESULTS: Mutants with altered GPXH response were isolated from UV-mutagenized cells containing a GPXH-arylsulfatase reporter gene construct. Out of 5500 clones tested, no mutant deficient in GPXH induction was isolated, whereas several clones showed constitutive high GPXH expression under normal light conditions. Many of these GPXH overexpressor (gox) mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. Interestingly, most gox mutants produced increased singlet oxygen levels correlating with high GPXH expression. Furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants. CONCLUSIONS: Screening for mutants with altered GPXH expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ROS in photosynthetic organisms. Phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. Furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and GPXH expression, which could either result from mutations in multiple loci or in a single gene encoding for a global regulator of cellular photoprotection mechanisms.

Multiple stressor effects of high light irradiance and photosynthetic herbicides on growth and survival of the green alga Chlamydomonas reinhardtii.

Exposure of the green alga Chlamydomonas reinhardtii Dangeard to a combination of environmental stress by high light irradiance and chemical stress by each of the three herbicides paraquat, atrazine, and norflurazon resulted in diverse multiple stressor effects on growth and survival of the cells. Under low light conditions, growth analyzed by cell numbers was generally more sensitive to herbicide treatment than optical density-based growth rates or colony-forming unit endpoints, which both also analyzed the viability of the cells. However, growth analyzed by optical density and colony-forming units in herbicide-treated cultures was affected much more strongly by high light irradiance, as shown by reduced 50% effective concentrations, indicating extensive multiple stressor effects of the combined treatment on the viability of the cells. None of the currently used concepts for mixture toxicity (concentration addition, independent action, or effect summation) could accurately describe the effects measured by the two stressors in combination. Both synergistic and antagonistic interactions seem to occur depending on the light conditions and the parameter analyzed. The strong stimulation of toxicity by the combined stresses can be explained by the similar mode of toxic action of the treatments, all increasing the production of reactive oxygen species. Antagonistic effects, conversely, are probably attributable to the various protection mechanisms of photosynthetic organisms to increased light irradiance, which help the cells acclimate to specific light conditions and defend against the deleterious effects of excess light. These protection mechanisms can affect growth and viability under increased light conditions and also might influence the toxicity of the photosynthetic herbicides.

Transcriptomics in ecotoxicology.

The emergence of analytical tools for high-throughput screening of biomolecules has revolutionized the way in which toxicologists explore the impact of chemicals or other stressors on organisms. One of the most developed and routinely applied high-throughput analysis approaches is transcriptomics, also often referred to as gene expression profiling. The transcriptome represents all RNA molecules, including the messenger RNA (mRNA), which constitutes the building blocks for translating DNA into amino acids to form proteins. The entirety of mRNA is a mirror of the genes that are actively expressed in a cell or an organism at a given time. This in turn allows one to deduce how organisms respond to changes in the external environment. In this article we explore how transcriptomics is currently applied in ecotoxicology and highlight challenges and trends.

Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii.

When exposed to strong sunlight, photosynthetic organisms encounter photooxidative stress by the increased production of reactive oxygen species causing harmful damages to proteins and membranes. Consequently, a fast and specific induction of defense mechanisms is required to protect the organism from cell death. In Chlamydomonas reinhardtii, the glutathione peroxidase homologous gene GPXH/GPX5 was shown to be specifically upregulated by singlet oxygen formed during high light conditions presumably to prevent the accumulation of lipid hydroperoxides and membrane damage. We now showed that the GPXH protein is a thioredoxin-dependent peroxidase catalyzing the reduction of hydrogen peroxide and organic hydroperoxides.Furthermore, the GPXH gene seems to encode a dual-targeted protein, predicted to be localized both in the chloroplast and the cytoplasm, which is active with either plastidic TRXy or cytosolic TRXh1. Putative dual-targeting is achieved by alternative transcription and translation start sites expressed independently from either a TATA-box or an Initiator core promoter. Expression of both transcripts was upregulated by photooxidative stress even though with different strengths. The induction required the presence of the core promoter sequences and multiple upstream regulatory elements including a Sp1-like element and an earlier identified CRE/AP-1 homologous sequence. This element was further characterized by mutation analysis but could not be confirmed to be a consensus CRE or AP1 element. Instead, it rather seems to be another member of the large group of TGAC-transcription factor binding sites found to be involved in the response of different genes to oxidative stress.

Sensing and responding to excess light.

Plants and algae often absorb too much light-more than they can actually use in photosynthesis. To prevent photo-oxidative damage and to acclimate to changes in their environment, photosynthetic organisms have evolved direct and indirect mechanisms for sensing and responding to excess light. Photoreceptors such as phototropin, neochrome, and cryptochrome can sense excess light directly and relay signals for chloroplast movement and gene expression responses. Indirect sensing of excess light through biochemical and metabolic signals can be transduced into local responses within chloroplasts, into changes in nuclear gene expression via retrograde signaling pathways, or even into systemic responses, all of which are associated with photoacclimation.

The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii.

Thiol/selenol peroxidases are ubiquitous nonheme peroxidases. They are divided into two major subfamilies: peroxiredoxins (PRXs) and glutathione peroxidases (GPXs). PRXs are present in diverse subcellular compartments and divided into four types: 2-cys PRX, 1-cys PRX, PRX-Q, and type II PRX (PRXII). In mammals, most GPXs are selenoenzymes containing a highly reactive selenocysteine in their active site while yeast and land plants are devoid of selenoproteins but contain nonselenium GPXs. The presence of a chloroplastic 2-cys PRX, a nonselenium GPX, and two selenium-dependent GPXs has been reported in the unicellular green alga Chlamydomonas reinhardtii. The availability of the Chlamydomonas genome sequence offers the opportunity to complete our knowledge on thiol/selenol peroxidases in this organism. In this article, Chlamydomonas PRX and GPX families are presented and compared to their counterparts in Arabidopsis, human, yeast, and Synechocystis sp. A summary of the current knowledge on each family of peroxidases, especially in photosynthetic organisms, phylogenetic analyses, and investigations of the putative subcellular localization of each protein and its relative expression level, on the basis of EST data, are presented. We show that Chlamydomonas PRX and GPX families share some similarities with other photosynthetic organisms but also with human cells. The data are discussed in view of recent results suggesting that these enzymes are important scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) but also play a role in ROS signaling.

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii.

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear-encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.

The role of Yap1p and Skn7p-mediated oxidative stress response in the defence of Saccharomyces cerevisiae against singlet oxygen.

The production of the reactive oxygen species superoxide and hydrogen peroxide in Saccharomyces cerevisiae induces the expression of various defence genes involved in an oxidative stress response. Expression of many of these genes has been shown to be coordinated by two transcriptional regulators, Yap1p and Skn7p, either alone or in concert. Here, we investigated the role of the Yap1p and Skn7p-mediated stress response in the defence against singlet oxygen, a non-radical reactive oxygen species produced mainly by photosensitized reactions in illuminated cells. Both, a yap1 and skn7 mutant were highly sensitive to Rose Bengal, an exogenous photosensitizer producing singlet oxygen in the light. The expression of a Yap1p-dependent reporter gene was induced by increased singlet oxygen production, showing that singlet oxygen activates general oxidative stress response mechanisms required for the resistance against Rose Bengal treatment. This response was also slightly stimulated by light in the absence of the photosensitizer, possibly due to singlet oxygen production by endogenous photosensitizers. The expression pattern of four oxidative stress genes in a yap1, skn7 and wild-type strain and the sensitivity of the corresponding mutants exposed to different oxidative stress conditions proved a role of Yap1p and Skn7p in the defence against singlet oxygen. Similarities in the genetic responses against singlet oxygen and hydroperoxides suggest an overlap in the oxidative stress response against these reactive oxygen species.

Growth condition-dependent sensitivity, photodamage and stress response of Chlamydomonas reinhardtii exposed to high light conditions.

Different substrate conditions, such as varying CO(2) concentrations or the presence of acetate, strongly influence the efficiency of photosynthesis in Chlamydomonas reinhardtii. Altered photosynthetic efficiencies affect the susceptibility of algae to the deleterious effects of high light stress, such as the production of reactive oxygen species (ROS) and PSII photodamage. In this study, we investigated the effect of high light on C. reinhardtii grown under photomixotrophy, i.e. in the presence of acetate, as well as under photoautotrophic growth conditions with either low or high CO(2) concentrations. Different parameters such as growth rate, chlorophyll bleaching, singlet oxygen generation, PSII photodamage and the total genomic stress response were analyzed. Although showing a similar degree of PSII photodamage, a much stronger singlet oxygen-specific response and a broader general stress response was observed in acetate and high CO(2)-supplemented cells compared with CO(2)-limited cells. These different photooxidative stress responses were correlated with the individual cellular PSII content and probably directly influenced the ROS production during exposure to high light. In addition, growth of high CO(2)-supplemented cells was more susceptible to high light stress compared with cells grown under CO(2) limitation. The growth of acetate-supplemented cultures, on the other hand, was less affected by high light treatment than cultures grown under high CO(2) concentrations, despite the similar cellular stress. This suggests that the production of ATP by mitochondrial acetate respiration protects the cells from the deleterious effects of high light stress, presumably by providing energy for an effective defense.

The glutathione peroxidase homologous gene Gpxh in Chlamydomonas reinhardtii is upregulated by singlet oxygen produced in photosystem II.

The expression of the glutathione peroxidase homologous gene Gpxh, known to be specifically induced by the formation of singlet oxygen (1O2), was analyzed in cells of Chlamydomonas reinhardtii exposed to environmental conditions causing photoinhibition. Illumination with high light intensities, leading to an increased formation of 1O2 in photosystem II, continuously induced the expression of Gpxh in cell for at least 2 h. Phenolic herbicides like dinoterb, raise the rate of 1O2 formation by increasing the probability of charge recombination in photosystem II via the formation of the primary radical pair and thereby 3P680 formation (Fufezan C et al. 2002, FEBS Letters 532, 407-410). In the presence of dinoterb the light-induced loss of the D1 protein in C. reinhardtii was increased and the high light-induced Gpxh expression was further stimulated. DCMU, a urea-type herbicide, causing reduced 1O2 generation in photosystem II, protected the D1 protein slightly against degradation and downregulated the expression of the Gpxh gene compared to untreated cells exposed to high light intensities. This indicates that the Gpxh expression is induced by 1O2 under environment conditions causing photoinhibition.

Photosensitizers neutral red (type I) and rose bengal (type II) cause light-dependent toxicity in Chlamydomonas reinhardtii and induce the Gpxh gene via increased singlet oxygen formation.

The connection between the mode of toxic action and the genetic response caused by the type I photosensitizer and photosynthesis inhibitor neutral red (NR) and the type II photosensitizer rose bengal (RB) was investigated in the green alga Chlamydomonas reinhardtii. For both photosensitizers, a light intensity-dependent increase in toxicity and expression of the glutathione peroxidase homologous gene (Gpxh) was found. The toxicity of RB was reduced by the singlet oxygen (1O2) quenchers 1,4-diazabicyclo[2.2.2]octane and L-histidine, and the RB-induced Gpxh expression was stimulated in deuterium oxide-supplemented growth medium. These observations clearly indicate the involvement of 1O2 in both toxicity and the genetic response caused by RB. NR up-regulated the expression of typical oxidative and general stress response genes, probably by a type I mechanism, and also strongly induced the Gpxh expression. The stimulating effect of deuterium oxide in the growth medium suggested the involvement of 1O2 also in the NR-induced response. Indeed, an increased 1O2 formation was detected with EPR-spin trapping in NR-treated spinach thylakoids. However, none of the 102 quenchers could reduce the light-dependent toxicity of NR in C. reinhardtii, indicating that NR has a different mode of toxic action than RB.