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1.  Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii 
eLife  2014;3:e02286.
Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.
DOI: http://dx.doi.org/10.7554/eLife.02286.001
eLife digest
Plants, algae and some bacteria use photosynthesis to extract energy from sunlight and to convert carbon dioxide into the sugars needed for growth. One by-product of photosynthesis is a highly toxic molecule called singlet oxygen. Typically, organisms deal with stressful events such as the presence of toxic molecules by producing new proteins. However, protein production is generally initiated in the nucleus of the cell, and photosynthesis is carried out in structures called chloroplasts. Cells must therefore be able to alert the nucleus to the presence of toxic levels of singlet oxygen in the chloroplasts.
Like some plants that can withstand a gradual decrease in temperature, but not a sudden cold snap, the alga Chlamydomonas reinhardtii is capable of resisting high doses of singlet oxygen if it has previously been exposed to low doses of the molecule. Wakao et al. exploited this ability to hunt for algae that are unable to acclimate to singlet oxygen, and found that these cells are unable to produce a protein called SAK1.
Wakao et al. reveal that many factors involved in the algae's cellular response to singlet oxygen depend on the presence of SAK1. In addition, the response of the algae cells to singlet oxygen differs to the one seen in the model plant Arabidopsis thaliana, suggesting that the two organisms have found different ways to deal with the same problem.
The location of a protein in a cell can give clues to its function. SAK1 is present in the fluid surrounding cellular compartments—the cytosol—which is consistent with it acting as a signaling molecule between the chloroplast and the nucleus. Wakao et al. present further evidence for this hypothesis by demonstrating that the number of phosphate groups attached on SAK1 changes when exposed to singlet oxygen—a feature often seen in signaling proteins. In addition, part of SAK1 resembles proteins that can bind to DNA, which indicates that SAK1 may be directly involved in initiating protein production.
The discovery of SAK1 represents a starting point for understanding how the site of photosynthesis, the chloroplast, communicates with the nucleus. It also has implications for developing plants and algae that have a higher tolerance to environmental stress conditions for agriculture and biofuel production.
DOI: http://dx.doi.org/10.7554/eLife.02286.002
doi:10.7554/eLife.02286
PMCID: PMC4067076  PMID: 24859755
chlamydomonas; singlet oxygen; retrograde signaling; photosynthesis; photo-oxidative stress; other
2.  Acclimation to Singlet Oxygen Stress in Chlamydomonas reinhardtii▿  
Eukaryotic Cell  2007;6(6):919-930.
In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms.
doi:10.1128/EC.00207-06
PMCID: PMC1951523  PMID: 17435007
3.  Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts 
Journal of Experimental Botany  2014;65(12):3081-3095.
Summary
Arabidopsis cell suspension cultures are used as a biological tool to better understand the key role of functional chloroplasts in singlet oxygen-mediated programmed cell death in plants.
Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.
doi:10.1093/jxb/eru151
PMCID: PMC4071827  PMID: 24723397
Arabidopsis cell cultures; photosensitizers; programmed cell death; reactive oxygen species; Rose Bengal; singlet oxygen; transcriptional defence responses.
4.  Analysis of LhcSR3, a Protein Essential for Feedback De-Excitation in the Green Alga Chlamydomonas reinhardtii 
PLoS Biology  2011;9(1):e1000577.
To prevent photodamage by excess light, plants use different proteins to sense pH changes and to dissipate excited energy states. In green microalgae, however, the LhcSR3 gene product is able to perform both pH sensing and energy quenching functions.
In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O2. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b– and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.
Author Summary
Reactive oxygen species are formed during photosynthesis, particularly when electron transport is saturated in high light. The process of non-photochemical quenching (NPQ) helps protect plants against excess light by dissipating the excited states of chlorophyll into heat. By doing so, it prevents the formation of triplet excites that otherwise would react with molecular oxygen to form singlet oxygen, a damaging reactive oxygen species. In plants, NPQ is triggered by the PsbS protein, which senses pH changes caused by excess light and consequently triggers energy-quenching functions in other proteins. The green microalga C. reinhardtii lacks the PsbS proteins, and NPQ depends on the LhcSR3 protein. In this study, we show that, unlike PsbS, LhcSR3 not only binds pigments but is also a strong quencher for chlorophyll excited states. LhcSR3 carries protonatable residues that enable it to sense pH change. Its quenching activity is further enhanced by low pH, suggesting that this algal protein merges the functions of pH sensor and of excited state quencher into a single gene product.
doi:10.1371/journal.pbio.1000577
PMCID: PMC3022525  PMID: 21267060
5.  Does singlet oxygen activate cell death in Arabidopsis cell suspension cultures? 
Plant Signaling & Behavior  2011;6(12):1937-1942.
Can Arabidopsis cell suspension cultures (ACSC) provide a useful working model to investigate genetically-controlled defense responses with signaling cascades starting in chloroplasts? In order to provide a convincing answer, we analyzed the early transcriptional profile of Arabidopsis cells at high light (HL). The results showed that ACSC respond to HL in a manner that resembles the singlet oxygen (1O2)-mediated defense responses described for the conditional fluorescent (flu) mutant of Arabidopsis thaliana. The flu mutant is characterized by the accumulation of free protochlorophyllide (Pchlide) in plastids when put into darkness and the subsequent production of 1O2 when the light is on. In ACSC, 1O2 is produced in chloroplasts at HL when excess excitation energy flows into photosystem II (PSII). Other reactive oxygen species are also produced in ACSC at HL, but to a lesser extent. When the HL stress ceases, ACSC recovers the initial rate of oxygen evolution and cell growth continues. We can conclude that chloroplasts of ACSC are both photosynthetically active and capable of initiating 1O2-mediated signaling cascades that activate a broad range of genetically-controlled defense responses. The upregulation of transcripts associated with the biosynthesis and signaling pathways of OPDA (12-oxophytodienoic acid) and ethylene (ET) suggests that the activated defense responses at HL are governed by these two hormones. In contrast to the flu mutant, the 1O2-mediated defense responses were independent of the upregulation of EDS1 (enhanced disease susceptibility) required for the accumulation of salicylic acid (SA) and genetically-controlled cell death. Interestingly, a high correlation in transcriptional expression was also observed between ACSC at HL, and the aba1 and max4 mutants of Arabidopsis, characterized by defects in the biosynthesis pathways of abscisic acid (ABA) and strigolactones, respectively.
doi:10.4161/psb.6.12.18264
PMCID: PMC3337182  PMID: 22112448
Acclimation; Arabidopsis cell culture; cell death; chloroplast; defence responses; high light; hormone stimulus; OPDA (12-oxophytodienoic acid); oxylipins; photosystem II; Reactive oxygen species; singlet oxygen; transcriptional profiling
6.  Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein 
BMC Plant Biology  2011;11:62.
Background
Reactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.
Results
To investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling.
Conclusions
We propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.
doi:10.1186/1471-2229-11-62
PMCID: PMC3083342  PMID: 21481232
7.  Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism 
A comprehensive genome-scale metabolic network of Chlamydomonas reinhardtii, including a detailed account of light-driven metabolism, is reconstructed and validated. The model provides a new resource for research of C. reinhardtii metabolism and in algal biotechnology.
The genome-scale metabolic network of Chlamydomonas reinhardtii (iRC1080) was reconstructed, accounting for >32% of the estimated metabolic genes encoded in the genome, and including extensive details of lipid metabolic pathways.This is the first metabolic network to explicitly account for stoichiometry and wavelengths of metabolic photon usage, providing a new resource for research of C. reinhardtii metabolism and developments in algal biotechnology.Metabolic functional annotation and the largest transcript verification of a metabolic network to date was performed, at least partially verifying >90% of the transcripts accounted for in iRC1080. Analysis of the network supports hypotheses concerning the evolution of latent lipid pathways in C. reinhardtii, including very long-chain polyunsaturated fatty acid and ceramide synthesis pathways.A novel approach for modeling light-driven metabolism was developed that accounts for both light source intensity and spectral quality of emitted light. The constructs resulting from this approach, termed prism reactions, were shown to significantly improve the accuracy of model predictions, and their use was demonstrated for evaluation of light source efficiency and design.
Algae have garnered significant interest in recent years, especially for their potential application in biofuel production. The hallmark, model eukaryotic microalgae Chlamydomonas reinhardtii has been widely used to study photosynthesis, cell motility and phototaxis, cell wall biogenesis, and other fundamental cellular processes (Harris, 2001). Characterizing algal metabolism is key to engineering production strains and understanding photobiological phenomena. Based on extensive literature on C. reinhardtii metabolism, its genome sequence (Merchant et al, 2007), and gene functional annotation, we have reconstructed and experimentally validated the genome-scale metabolic network for this alga, iRC1080, the first network to account for detailed photon absorption permitting growth simulations under different light sources. iRC1080 accounts for 1080 genes, associated with 2190 reactions and 1068 unique metabolites and encompasses 83 subsystems distributed across 10 cellular compartments (Figure 1A). Its >32% coverage of estimated metabolic genes is a tremendous expansion over previous algal reconstructions (Boyle and Morgan, 2009; Manichaikul et al, 2009). The lipid metabolic pathways of iRC1080 are considerably expanded relative to existing networks, and chemical properties of all metabolites in these pathways are accounted for explicitly, providing sufficient detail to completely specify all individual molecular species: backbone molecule and stereochemical numbering of acyl-chain positions; acyl-chain length; and number, position, and cis–trans stereoisomerism of carbon–carbon double bonds. Such detail in lipid metabolism will be critical for model-driven metabolic engineering efforts.
We experimentally verified transcripts accounted for in the network under permissive growth conditions, detecting >90% of tested transcript models (Figure 1B) and providing validating evidence for the contents of iRC1080. We also analyzed the extent of transcript verification by specific metabolic subsystems. Some subsystems stood out as more poorly verified, including chloroplast and mitochondrial transport systems and sphingolipid metabolism, all of which exhibited <80% of transcripts detected, reflecting incomplete characterization of compartmental transporters and supporting a hypothesis of latent pathway evolution for ceramide synthesis in C. reinhardtii. Additional lines of evidence from the reconstruction effort similarly support this hypothesis including lack of ceramide synthetase and other annotation gaps downstream in sphingolipid metabolism. A similar hypothesis of latent pathway evolution was established for very long-chain fatty acids (VLCFAs) and their polyunsaturated analogs (VLCPUFAs) (Figure 1C), owing to the absence of this class of lipids in previous experimental measurements, lack of a candidate VLCFA elongase in the functional annotation, and additional downstream annotation gaps in arachidonic acid metabolism.
The network provides a detailed account of metabolic photon absorption by light-driven reactions, including photosystems I and II, light-dependent protochlorophyllide oxidoreductase, provitamin D3 photoconversion to vitamin D3, and rhodopsin photoisomerase; this network accounting permits the precise modeling of light-dependent metabolism. iRC1080 accounts for effective light spectral ranges through analysis of biochemical activity spectra (Figure 3A), either reaction activity or absorbance at varying light wavelengths. Defining effective spectral ranges associated with each photon-utilizing reaction enabled our network to model growth under different light sources via stoichiometric representation of the spectral composition of emitted light, termed prism reactions. Coefficients for different photon wavelengths in a prism reaction correspond to the ratios of photon flux in the defined effective spectral ranges to the total emitted photon flux from a given light source (Figure 3B). This approach distinguishes the amount of emitted photons that drive different metabolic reactions. We created prism reactions for most light sources that have been used in published studies for algal and plant growth including solar light, various light bulbs, and LEDs. We also included regulatory effects, resulting from lighting conditions insofar as published studies enabled. Light and dark conditions have been shown to affect metabolic enzyme activity in C. reinhardtii on multiple levels: transcriptional regulation, chloroplast RNA degradation, translational regulation, and thioredoxin-mediated enzyme regulation. Through application of our light model and prism reactions, we were able to closely recapitulate experimental growth measurements under solar, incandescent, and red LED lights. Through unbiased sampling, we were able to establish the tremendous statistical significance of the accuracy of growth predictions achievable through implementation of prism reactions. Finally, application of the photosynthetic model was demonstrated prospectively to evaluate light utilization efficiency under different light sources. The results suggest that, of the existing light sources, red LEDs provide the greatest efficiency, about three times as efficient as sunlight. Extending this analysis, the model was applied to design a maximally efficient LED spectrum for algal growth. The result was a 677-nm peak LED spectrum with a total incident photon flux of 360 μE/m2/s, suggesting that for the simple objective of maximizing growth efficiency, LED technology has already reached an effective theoretical optimum.
In summary, the C. reinhardtii metabolic network iRC1080 that we have reconstructed offers insight into the basic biology of this species and may be employed prospectively for genetic engineering design and light source design relevant to algal biotechnology. iRC1080 was used to analyze lipid metabolism and generate novel hypotheses about the evolution of latent pathways. The predictive capacity of metabolic models developed from iRC1080 was demonstrated in simulating mutant phenotypes and in evaluation of light source efficiency. Our network provides a broad knowledgebase of the biochemistry and genomics underlying global metabolism of a photoautotroph, and our modeling approach for light-driven metabolism exemplifies how integration of largely unvisited data types, such as physicochemical environmental parameters, can expand the diversity of applications of metabolic networks.
Metabolic network reconstruction encompasses existing knowledge about an organism's metabolism and genome annotation, providing a platform for omics data analysis and phenotype prediction. The model alga Chlamydomonas reinhardtii is employed to study diverse biological processes from photosynthesis to phototaxis. Recent heightened interest in this species results from an international movement to develop algal biofuels. Integrating biological and optical data, we reconstructed a genome-scale metabolic network for this alga and devised a novel light-modeling approach that enables quantitative growth prediction for a given light source, resolving wavelength and photon flux. We experimentally verified transcripts accounted for in the network and physiologically validated model function through simulation and generation of new experimental growth data, providing high confidence in network contents and predictive applications. The network offers insight into algal metabolism and potential for genetic engineering and efficient light source design, a pioneering resource for studying light-driven metabolism and quantitative systems biology.
doi:10.1038/msb.2011.52
PMCID: PMC3202792  PMID: 21811229
Chlamydomonas reinhardtii; lipid metabolism; metabolic engineering; photobioreactor
8.  Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii  
Journal of Experimental Botany  2014;65(22):6563-6576.
Summary
Impaired carbon precursor supply through cpPDC is detrimental for TAG biosynthesis in Chlamydomonas reinhardtii under conditions of photoautotrophy and nitrogen starvation.
The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.
doi:10.1093/jxb/eru374
PMCID: PMC4246187  PMID: 25210079
Acetyl-CoA; chlorophyll fluorescence transients; fatty acid synthesis; lipid; microalgae; photosystem II; pyruvate dehydrogenase.
9.  Key players of singlet oxygen-induced cell death in plants 
The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis. Singlet oxygen (1O2) is a highly reactive species to which has been attributed a major destructive role during the execution of ROS-induced cell death in photosynthetic tissues exposed to excess light. The study of the specific biological activity of 1O2 in plants has been hindered by its high reactivity and short lifetime, the concurrent production of other ROS under photooxidative stress, and limited in vivo detection methods. However, during the last 15 years, the isolation and characterization of two 1O2-overproducing mutants in Arabidopsis thaliana, flu and ch1, has allowed the identification of genetically controlled 1O2 cell death pathways and a 1O2 acclimation pathway that are triggered at sub-cytotoxic concentrations of 1O2. The study of flu has revealed the control of cell death by the plastid proteins EXECUTER (EX)1 and EX2. In ch1, oxidized derivatives of β-carotene, such as β-cyclocitral and dihydroactinidiolide, have been identified as important upstream messengers in the 1O2 signaling pathway that leads to stress acclimation. In both the flu and ch1 mutants, phytohormones act as important promoters or inhibitors of cell death. In particular, jasmonate has emerged as a key player in the decision between acclimation and cell death in response to 1O2. Although the flu and ch1 mutants show many similarities, especially regarding their gene expression profiles, key differences, such as EXECUTER-independent cell death in ch1, have also been observed and will need further investigation to be fully understood.
doi:10.3389/fpls.2015.00039
PMCID: PMC4316694
singlet oxygen; oxidative stress; cell death; acclimation; EXECUTER; β-cyclocitral; phytohormones; oxylipins
10.  Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS 
BMC Plant Biology  2014;14(1):242.
Background
PsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ.
Results
In studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that.
Conclusion
These results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0242-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0242-2
PMCID: PMC4219129  PMID: 25342550
Photoprotection; PsbS; ROS; Superoxide; Photosynthesis; NPQ; Rice
11.  A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains 
BMC Plant Biology  2008;8:107.
Background
Sealed Chlamydomonas reinhardtii cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating C. reinhardtii transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation.
Results
To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify C. reinhardtii DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain apr1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of apr1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted apr1 cells in the light led to a 2-3-fold higher in vivo hydrogen production than previously reported for the sulfur-deprived C. reinhardtii wild type.
Conclusion
Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high in vivo hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.
doi:10.1186/1471-2229-8-107
PMCID: PMC2576467  PMID: 18928519
12.  Increased production of mitochondrial reactive oxygen species and reduced adult life span in an insecticide-resistant strain of Anopheles gambiae 
Bulletin of entomological research  2014;104(3):323-333.
Control of the malaria vector An. gambiae is still largely obtained through chemical intervention using pyrethroids, such as permethrin. However, strains of An. gambiae that are resistant to the toxic effects of pyrethroids have become widespread in several endemic areas over the last decade. The objective of this study was to assess differences in five life-history traits (larval developmental time and the body weight, fecundity, hatch rate, and longevity of adult females) and energy metabolism between a strain of An. gambiae that is resistant to permethrin (RSP), due to knockdown resistance and enhanced metabolic detoxification, and a permethrin susceptible strain reared under laboratory conditions. We also quantified the expression levels of five antioxidant enzyme genes: GSTe3, CAT, GPXH1, SOD1, and SOD2. We found that the RSP strain had a longer developmental time than the susceptible strain. Additionally, RSP adult females had higher wet body weight and increased water and glycogen levels. Compared to permethrin susceptible females, RSP females displayed reduced metabolic rate and mitochondrial coupling efficiency and higher mitochondrial ROS production. Furthermore, despite higher levels of GSTe3 and CAT transcripts, RSP females had a shorter adult life span than susceptible females. Collectively, these results suggest that permethrin resistance alleles might affect energy metabolism, oxidative stress, and adult survival of An. gambiae. However, because the strains used in this study differ in their genetic backgrounds, the results need to be interpreted with caution and replicated in other strains in order to have significant implications for malaria transmission and vector control.
doi:10.1017/S0007485314000091
PMCID: PMC4008687  PMID: 24555527
Anopheles gambiae; permethrin; ROS; mitochondria; insecticide resistance
13.  Light-Induced Changes within Photosystem II Protects Microcoleus sp. in Biological Desert Sand Crusts against Excess Light 
PLoS ONE  2010;5(6):e11000.
The filamentous cyanobacterium Microcoleus vaginatus, a major primary producer in desert biological sand crusts, is exposed to frequent hydration (by early morning dew) followed by desiccation during potentially damaging excess light conditions. Nevertheless, its photosynthetic machinery is hardly affected by high light, unlike “model” organisms whereby light-induced oxidative stress leads to photoinactivation of the oxygen-evolving photosystem II (PSII). Field experiments showed a dramatic decline in the fluorescence yield with rising light intensity in both drying and artificially maintained wet plots. Laboratory experiments showed that, contrary to “model” organisms, photosynthesis persists in Microcoleus sp. even at light intensities 2–3 times higher than required to saturate oxygen evolution. This is despite an extensive loss (85–90%) of variable fluorescence and thermoluminescence, representing radiative PSII charge recombination that promotes the generation of damaging singlet oxygen. Light induced loss of variable fluorescence is not inhibited by the electron transfer inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), nor the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that reduction of plastoquinone or O2, or lumen acidification essential for non-photochemical quenching (NPQ) are not involved. The rate of QA− re-oxidation in the presence of DCMU is enhanced with time and intensity of illumination. The difference in temperatures required for maximal thermoluminescence emissions from S2/QA− (Q band, 22°C) and S2,3/QB− (B band, 25°C) charge recombinations is considerably smaller in Microcoleus as compared to “model” photosynthetic organisms, thus indicating a significant alteration of the S2/QA− redox potential. We propose that enhancement of non-radiative charge recombination with rising light intensity may reduce harmful radiative recombination events thereby lowering 1O2 generation and oxidative photodamage under excess illumination. This effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacteria to the higher plant chloroplast.
doi:10.1371/journal.pone.0011000
PMCID: PMC2882322  PMID: 20544016
14.  Integrative “Omics”-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses 
PLoS Genetics  2013;9(6):e1003576.
Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level (“expressome”). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.
Author Summary
Bacteria are frequently exposed to disadvantageous conditions, like elevated temperatures or nutrient depletion. The ability to maintain viable populations is based on cellular stress responses, which are regulated in a complex manner with different outputs on different regulatory levels. For example, mRNA levels do not ultimately determine protein amounts since translation of mRNAs can be influenced irrespective of mRNA levels. To appreciate nature and frequency of these regulatory events, multi-layered experimental approaches are required on a global scale. The photo-oxidative stress response of the purple bacterium Rhodobacter sphaeroides was chosen as a model. Changes of total mRNAs (transcriptome) and ribosomal-bound mRNAs (translatome) were monitored by microarrays. The proteome was assessed by mass spectrometry, applying a “bacterial SILAC standard” for indirect quantification, an approach which additionally identified new open reading frames. Integration of the three expression levels provided a comprehensive insight into regulatory events and identified new stress-responsive genes, including genes for transcriptional regulators and for quorum sensing. We found that translational control exceeds simple regulation on the transcriptional level. Furthermore, polar expression patterns within inducible operons point at the possibility of expression fine-tuning by gene positioning.
doi:10.1371/journal.pgen.1003576
PMCID: PMC3688512  PMID: 23818867
15.  Leaf Variegation in the Rice zebra2 Mutant Is Caused by Photoperiodic Accumulation of Tetra-Cis-Lycopene and Singlet Oxygen 
Molecules and Cells  2011;33(1):87-97.
In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O2−], hydrogen peroxide [H2O2], and singlet oxygen [1O2]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during the light period, which consequently altered the expression of 1O2-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive 1O2 under natural day-night conditions.
doi:10.1007/s10059-012-2218-0
PMCID: PMC3887748  PMID: 22134723
carotenoid isomerase; rice; singlet oxygen; tetra-cis-lycopene; zebra2
16.  Proteins Needed to Activate a Transcriptional Response to the Reactive Oxygen Species Singlet Oxygen 
mBio  2013;4(1):e00541-12.
ABSTRACT
Singlet oxygen (1O2) is a reactive oxygen species generated by energy transfer from one or more excited donors to molecular oxygen. Many biomolecules are prone to oxidation by 1O2, and cells have evolved systems to protect themselves from damage caused by this compound. One way that the photosynthetic bacterium Rhodobacter sphaeroides protects itself from 1O2 is by inducing a transcriptional response controlled by ChrR, an anti-σ factor which releases an alternative sigma factor, σE, in the presence of 1O2. Here we report that induction of σE-dependent gene transcription is decreased in the presence of 1O2 when two conserved genes in the σE regulon are deleted, including one encoding a cyclopropane fatty acid synthase homologue (RSP2144) or one encoding a protein of unknown function (RSP1091). Thus, we conclude that RSP2144 and RSP1091 are each necessary to increase σE activity in the presence of 1O2. In addition, we found that unlike in wild-type cells, where ChrR is rapidly degraded when 1O2 is generated, turnover of this anti-σ factor is slowed when cells lacking RSP2144, RSP1091, or both of these proteins are exposed to 1O2. Further, we demonstrate that the organic hydroperoxide tert-butyl hydroperoxide promotes ChrR turnover in both wild-type cells and mutants lacking RSP2144 or RSP1091, suggesting differences in the ways different types of oxidants increase σE activity.
IMPORTANCE
Oxygen serves many crucial functions on Earth; it is produced during photosynthesis and needed for other pathways. While oxygen is relatively inert, it can be converted to reactive oxygen species (ROS) that destroy biomolecules, cause disease, or kill cells. When energy is transferred to oxygen, the ROS singlet oxygen is generated. To understand how singlet oxygen impacts cells, we study the stress response to this ROS in Rhodobacter sphaeroides, a bacterium that, like plants, generates this compound as a consequence of photosynthesis. This paper identifies proteins that activate a stress response to singlet oxygen and shows that they act in a specific response to this ROS. The identified proteins are found in many free-living, symbiotic, or pathogenic bacteria that can encounter singlet oxygen in nature. Thus, our findings provide new information about a stress response to a ROS of broad biological, agricultural, and biomedical importance.
doi:10.1128/mBio.00541-12
PMCID: PMC3546557  PMID: 23300250
17.  Singlet oxygen-mediated signaling in plants: moving from flu to wild type reveals an increasing complexity 
Photosynthesis research  2013;116(0):10.1007/s11120-013-9876-4.
Singlet oxygen (1O2)-mediated signaling has been established in the conditional fluorescent (flu) mutant of Arabidopsis. In the dark, the flu mutant accumulates free protochlorophyllide (Pchlide), a photosensitizer that in the light generates 1O2. The release of 1O2 leads to growth inhibition of mature plants and bleaching of seedlings. These 1O2-mediated responses depend on two plastid proteins, EXECUTER (EX) 1 and 2. An ex1/ex2/flu mutant accumulates in the dark Pchlide and upon illumination generates similar amounts of 1O2 as flu, but 1O2-mediated responses are abrogated in the triple mutant. The 1O2- and EX-dependent signaling pathway operates also in wild type placed under light stress. However, it does not act alone as in flu, but interacts with other signaling pathways that modulate 1O2-mediated responses. Depending on how severe the light stress is, 1O2- and EX-dependent signaling may be superimposed by 1O2-mediated signaling that does not depend on EX and is associated with photo-oxidative damage. Because of its high reactivity and short half-life, 1O2 is unlikely to be a signal that is translocated across the chloroplast envelope, but is likely to interact with other plastid components close to its site of production and to generate more stable signaling molecules during this interaction. Depending on the site of 1O2 production and the severity of stress, different signaling molecules may be expected that give rise to different 1O2-mediated responses.
doi:10.1007/s11120-013-9876-4
PMCID: PMC3833438  PMID: 23832611
Singlet oxygen-mediated signaling; Executer; Light stress; flu mutant; Arabidopsis thaliana
18.  Singlet Oxygen Scavenging Activity and Cytotoxicity of Essential Oils from Rutaceae 
Since we have been exposed to excessive amounts of stressors, aromatherapy for the relaxation has recently become very popular recently. However, there is a problem which responds to light with the essential oil used by aromatherapy. It is generally believed that singlet oxygen is implicated in the pathogenesis of various diseases such as light-induced skin disorders and inflammatory responses. Here we studied whether essential oils can effectively scavenge singlet oxygen upon irradiation, using the electron spin resonance (ESR) method. Green light was used to irradiate twelve essential oils from rutaceae. Among these twelve essential oils, eight were prepared by the expression (or the compression) method (referred to as E oil), and four samples were prepared by the steam distillation method (referred to as SD oil). Five E oils enhanced singlet oxygen production. As these essential oils may be phototoxic, it should be used for their use whit light. Two E oils and three SD oils showed singlet oxygen scavenging activity. These results may suggest that the antioxidant activity of essential oils are judged from their radical scavenging activity. Essential oils, which enhance the singlet oxygen production and show higher cytotoxicity, may contain much of limonene. These results suggest that limonene is involved not only in the enhancement of singlet oxygen production but also in the expression of cytotoxic activity, and that attention has to be necessary for use of blended essential oils.
doi:10.3164/jcbn.2008037
PMCID: PMC2459252  PMID: 18648659
aromatherapy; essential oil; rutaceae; ESR; singlet oxygen
19.  Studies on the Singlet Oxygen Scavenging Mechanism of Human Macular Pigment 
It is thought that direct quenching of singlet oxygen and scavenging free radicals by macular pigment carotenoids is a major mechanism for their beneficial effects against light-induced oxidative stress. Corresponding data from human tissue remains unavailable, however. In the studies reported here, electron paramagnetic resonance (EPR) spectroscopy was used to measure light-induced singlet oxygen generation in postmortem human macula and retinal pigment epithelium/choroid (RPE/choroid). Under white-light illumination, production of singlet oxygen was detected in RPE/choroid but not in macular tissue, and we show that exogenously added macular carotenoids can quench RPE/choroid singlet oxygen. When the singlet oxygen quenching ability of the macular carotenoids was investigated in solution, it was shown that a mixture of meso-zeaxanthin, zeaxanthin, and lutein in a ratio of 1:1:1 can quench more singlet oxygen than the individual carotenoids at the same total concentration.
doi:10.1016/j.abb.2010.07.024
PMCID: PMC2957523  PMID: 20678467
Human macula; singlet oxygen; electron paramagnetic resonance (EPR)
20.  Single Cell Responses to Spatially-Controlled Photosensitized Production of Extracellular Singlet Oxygen 
Photochemistry and photobiology  2011;87(5):1077-1091.
The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed post-irradiation “waiting period” before necrosis became apparent depended on (a) the distance between the cell membrane and the domain irradiated, (b) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced, and (c) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach of using extracellularly-generated singlet oxygen to induce cell death can provide a solution to a problem that often limits mechanistic studies of intracellularly photosensitized cell death: it can be difficult to quantify the effective light dose, and hence singlet oxygen concentration, when using an intracellular photosensitizer.
doi:10.1111/j.1751-1097.2011.00951.x
PMCID: PMC3166418  PMID: 21668871
21.  Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen. 
Journal of Bacteriology  1989;171(4):2188-2194.
Gram-negative and gram-positive bacteria were found to display different sensitivities to pure singlet oxygen generated outside of cells. Killing curves for Salmonella typhimurium and Escherichia coli strains were indicative of multihit killing, whereas curves for Sarcina lutea, Staphylococcus aureus, Streptococcus lactis, and Streptococcus faecalis exhibited single-hit kinetics. The S. typhimurium deep rough strain TA1975, which lacks nearly all of the cell wall lipopolysaccharide coat and manifests concomitant enhancement of penetration by some exogenous substances, responded to singlet oxygen with initially faster inactivation than did the S. typhimurium wild-type strain, although the maximum rates of killing appeared to be quite similar. The structure of the cell wall thus plays an important role in susceptibility to singlet oxygen. The outer membrane-lipopolysaccharide portion of the gram-negative cell wall initially protects the bacteria from extracellular singlet oxygen, although it may also serve as a source for secondary reaction products which accentuate the rates of cell killing. S. typhimurium and E. coli strains lacking the cellular antioxidant, glutathione, showed no difference from strains containing glutathione in response to the toxic effects of singlet oxygen. Strains of Sarcina lutea and Staphylococcus aureus that contained carotenoids, however, were far more resistant to singlet oxygen lethality than were both carotenoidless mutants of the same species and other gram-positive species lacking high levels of protective carotenoids.
PMCID: PMC209876  PMID: 2703469
22.  Convergence of the Transcriptional Responses to Heat Shock and Singlet Oxygen Stresses 
PLoS Genetics  2012;8(9):e1002929.
Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoHI and RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the −35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several bases in the predicted −10 element were important for activity with either RpoHII or both RpoH homologs. Genes that are transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.
Author Summary
An important property of living systems is their ability to survive under conditions of stress such as increased temperature or the presence of reactive oxygen species. Central to the function of these stress responses are transcription factors that activate specific sets of genes needed for this response. Despite the central role of stress responses across all forms of life, the processes driving their organization and evolution across organisms are poorly understood. This paper uses genomic, computational, and mutational analyses to dissect stress responses controlled by two proteins that are each members of the RpoH family of alternative σ factors. RpoH family members usually control gene expression during a heat shock response. However, the photosynthetic bacterium Rhodobacter sphaeroides and several other α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. Our findings predict that a gene duplication event followed by changes in DNA recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria. Our approach and findings should interest those studying the evolution of transcription factors or the signal transduction pathways that control stress responses.
doi:10.1371/journal.pgen.1002929
PMCID: PMC3441632  PMID: 23028346
23.  The Transcription Factor BcLTF1 Regulates Virulence and Light Responses in the Necrotrophic Plant Pathogen Botrytis cinerea 
PLoS Genetics  2014;10(1):e1004040.
Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious, but infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB-1). By deletion and over-expression of bcltf1, we confirmed the predicted role of the transcription factor in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 light-responsive genes, and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. In addition, the deletion of bcltf1 affects the expression of 1,539 genes irrespective of the light conditions, including the overexpression of known and so far uncharacterized secondary metabolism-related genes. Increased expression of genes encoding alternative respiration enzymes, such as the alternative oxidase (AOX), suggest a mitochondrial dysfunction in the absence of bcltf1. The hypersensitivity of Δbctlf1 mutants to exogenously applied oxidative stress - even in the absence of light - and the restoration of virulence and growth rates in continuous light by antioxidants, indicate that BcLTF1 is required to cope with oxidative stress that is caused either by exposure to light or arising during host infection.
Author Summary
Both fungal pathogens and their host plants respond to light, which represents an important environmental cue. Unlike plants using light for energy generation, filamentous fungi use light, or its absence, as a general signal for orientation (night/day, underground/on the surface). Therefore, dependent on the ecological niche of the fungus, light may control the development of reproductive structures (photomorphogenesis), the dispersal of propagules (phototropism of reproductive structures) and the circadian rhythm. As in other organisms, fungi have to protect themselves against the detrimental effects of light, i.e. the damage to macromolecules by emerging singlet oxygen. Adaptive responses are the accumulation of pigments, especially in the reproductive and survival structures such as spores, sclerotia and fruiting bodies. Light is sensed by fungal photoreceptors leading to quick responses on the transcriptional level, and is furthermore considered to result in the accumulation of reactive oxygen species (ROS). In this study, we provide evidence that an unbalanced ROS homoeostasis (generation outweighs detoxification) caused by the deletion of the light-responsive transcription factor BcLTF1 impairs the ability of the necrotrophic pathogen Botrytis cinerea to grow in the presence of additional oxidative stress arising during illumination or during infection of the host.
doi:10.1371/journal.pgen.1004040
PMCID: PMC3886904  PMID: 24415947
24.  The aniline-to-azobenzene oxidation reaction on monolayer graphene or graphene oxide surfaces fabricated by benzoic acid 
Nanoscale Research Letters  2013;8(1):372.
The oxidation of aniline to azobenzene was conducted in the presence of either monolayer graphene (EG) or graphene-oxide-like surface, such as GOx, under ultra-high vacuum conditions maintaining a 365-nm UV light exposure to enhance the oxidation reaction. The surface-bound products were investigated using micro Raman spectroscopy, high-resolution photoemission spectroscopy, and work function measurements. The oxygen carriers present on the GOx surfaces, but not on the EG surfaces, acted as reaction reagents to facilitate the oxidation reaction from aniline to azobenzene. Increasing the aniline concentration at 300 K confirmed that the exchange ratio from the aniline to the azobenzene was enhanced, as determined by the intensity ratio between the aniline- and azobenzene-induced N 1 s core-level spectra. The work function changed dramatically as the aniline concentration increased, indicating that the aniline on the GOx surface conveyed n-type doping characteristics at a low coverage level. A higher aniline concentration increased the p-type doping character by increasing the azobenzene concentration on the GOx surface. A comparison of the oxidation reactivity of aniline molecules on the EG or GOx surfaces revealed the role of the oxygen carriers on the GOx surfaces in the context of catalytic oxidation.
doi:10.1186/1556-276X-8-372
PMCID: PMC3766250  PMID: 24229051
Oxidation reaction; GO surface; Raman spectroscopy; HRPES; Reaction reagent
25.  Bacterial responses to photo-oxidative stress 
Nature reviews. Microbiology  2009;7(12):856-863.
Singlet oxygen is one of several reactive oxygen species that can destroy biomolecules, microorganisms and other cells. Traditionally, the response to singlet oxygen has been termed photo-oxidative stress, as light-dependent processes in photosynthetic cells are major biological sources of singlet oxygen. Recent work identifying a core set of singlet oxygen stress response genes across various bacterial species highlights the importance of this response for survival by both photosynthetic and non-photosynthetic cells. Here, we review how bacterial cells mount a transcriptional response to photo-oxidative stress in the context of what is known about bacterial stress responses to other reactive oxygen species.
doi:10.1038/nrmicro2237
PMCID: PMC2793278  PMID: 19881522

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