Beat B. Fischer

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 glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen

The glutathione peroxidase homologous gene (Gpxh gene) in Chlamydomonas reinhardtii is up-regulated under oxidative stress conditions. The Gpxh gene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The Gpxh mRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the Gpxh mRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the Gpxh mRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxh gene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxh than for the other genes investigated. Using Gpxh promoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxh was transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxh promoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2 but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome.

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.

Transcriptomics in ecotoxicology

AbstractThe 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. FigureThe transcriptome (RNA) is a mirror of the genes that are actively expressed in a cell or organism at a given time, providing information on how organisms respond to chemicals or other stressors in the environment

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.

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. Copyright

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.

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.

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.

Phenotypic plasticity influences the eco-evolutionary dynamics of a predator-prey system

There is increasing evidence that rapid phenotypic evolution can strongly influence population dynamics, but how are such eco-evolutionary dynamics influenced by the source of trait variation (i.e., genetic variation or phenotypic plasticity)? To investigate this, we used rotifer–algae microcosm experiments to test how the phenotypic and genetic composition of prey populations affect predator–prey population dynamics. We chose four genetically distinct strains of the green alga Chlamydomonas reinhardtii that varied in their growth rate, standing levels of defense, and inducible defense. To additionally test for strain specificity of plasticity responses, we quantified protein expression of each strain in the presence and absence of rotifer predators (Brachionus calyciflorus). We then tested how different strain combinations influenced the outcome of pairwise competition trials with and without rotifer predation. We tracked individual strain frequencies using quantitative polymerase chain reaction (qPCR), ...