Characterization of chloroplast ribulose-5-phosphate-3-epimerase from the microalga Chlamydomonas reinhardtii.

Carbon fixation relies on Rubisco and 10 additional enzymes in the Calvin-Benson-Bassham cycle. Epimerization of xylulose-5-phosphate (Xu5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco. Ribulose-5-phosphate-3-epimerase (RPE, EC 5.1.3.1) catalyzes the formation of Ru5P, but it can also operate in the pentose-phosphate pathway by catalyzing the reverse reaction. Here, we describe the structural and biochemical properties of the recombinant RPE isoform 1 from Chlamydomonas (Chlamydomonas reinhardtii) (CrRPE1). The enzyme is a homo-hexamer that contains a zinc ion in the active site and exposes a catalytic pocket on the top of an α8ß8 triose isomerase-type barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. By optimizing and developing enzyme assays to monitor the reversible epimerization of Ru5P to Xu5P and vice versa, we determined the catalytic parameters that differ from those of other plant paralogs. Despite being identified as a putative target of multiple thiol-based redox modifications, CrRPE1 activity is not affected by both reductive and oxidative treatments, indicating that enzyme catalysis is insensitive to possible redox alterations of cysteine residues. We mapped phosphorylation sites on the crystal structure, and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. This work provides an accurate description of the structural features of CrRPE1 and an in-depth examination of its catalytic and regulatory properties highlighting the physiological relevance of this enzyme in the context of photosynthetic carbon fixation.

How abiotic stress-induced socialization leads to the formation of massive aggregates in Chlamydomonas.

Multicellular organisms implement a set of reactions involving signaling and cooperation between different types of cells. Unicellular organisms, on the other hand, activate defense systems that involve collective behaviors between individual organisms. In the unicellular model alga Chlamydomonas (Chlamydomonas reinhardtii), the existence and the function of collective behaviors mechanisms in response to stress remain mostly at the level of the formation of small structures called palmelloids. Here, we report the characterization of a mechanism of abiotic stress response that Chlamydomonas can trigger to form massive multicellular structures. We showed that these aggregates constitute an effective bulwark within which the cells are efficiently protected from the toxic environment. We generated a family of mutants that aggregate spontaneously, the socializer (saz) mutants, of which saz1 is described here in detail. We took advantage of the saz mutants to implement a large-scale multiomics approach that allowed us to show that aggregation is not the result of passive agglutination, but rather genetic reprogramming and substantial modification of the secretome. The reverse genetic analysis we conducted allowed us to identify positive and negative regulators of aggregation and to make hypotheses on how this process is controlled in Chlamydomonas.

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii.

The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis.

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle.

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

Glutathionylation primes soluble glyceraldehyde-3-phosphate dehydrogenase for late collapse into insoluble aggregates.

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

The C-Terminal Acidic Tail Modulates the Anticancer Properties of HMGB1.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress.

Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.

The ATG4 protease integrates redox and stress signals to regulate autophagy.

Autophagy is a highly conserved degradative pathway that ensures cellular homeostasis through the removal of damaged or useless intracellular components including proteins, membranes, or even entire organelles. A main hallmark of autophagy is the biogenesis of autophagosomes, double-membrane vesicles that engulf and transport to the vacuole the material to be degraded and recycled. The formation of autophagosomes responds to integrated signals produced as a consequence of metabolic reactions or different types of stress and is mediated by the coordinated action of core autophagy-related (ATG) proteins. ATG4 is a key Cys-protease with a dual function in both ATG8 lipidation and free ATG8 recycling whose balance is crucial for proper biogenesis of the autophagosome. ATG4 is conserved in the green lineage, and its regulation by different post-translational modifications has been reported in the model systems Chlamydomonas reinhardtii and Arabidopsis. In this review, we discuss the major role of ATG4 in the integration of stress and redox signals that regulate autophagy in algae and plants.

Nitric Oxide Remodels the Photosynthetic Apparatus upon S-Starvation in Chlamydomonas reinhardtii.

Many photosynthetic autotrophs have evolved responses that adjust their metabolism to limitations in nutrient availability. Here we report a detailed characterization of the remodeling of photosynthesis upon sulfur starvation under heterotrophy and photo-autotrophy in the green alga (Chlamydomonas reinhardtii). Photosynthetic inactivation under low light and darkness is achieved through specific degradation of Rubisco and cytochrome b 6 f and occurs only in the presence of reduced carbon in the medium. The process is likely regulated by nitric oxide (NO), which is produced 24 h after the onset of starvation, as detected with NO-sensitive fluorescence probes visualized by fluorescence microscopy. We provide pharmacological evidence that intracellular NO levels govern this degradation pathway: the addition of a NO scavenger decreases the rate of cytochrome b 6 f and Rubisco degradation, whereas NO donors accelerate the degradation. Based on our analysis of the relative contribution of the different NO synthesis pathways, we conclude that the NO2-dependent nitrate reductase-independent pathway is crucial for NO production under sulfur starvation. Our data argue for an active role for NO in the remodeling of thylakoid protein complexes upon sulfur starvation.

Secondary Metabolites from the Culture of the Marine-derived Fungus Paradendryphiella salina PC 362H and Evaluation of the Anticancer Activity of Its Metabolite Hyalodendrin.

High-throughput screening assays have been designed to identify compounds capable of inhibiting phenotypes involved in cancer aggressiveness. However, most studies used commercially available chemical libraries. This prompted us to explore natural products isolated from marine-derived fungi as a new source of molecules. In this study, we established a chemical library from 99 strains corresponding to 45 molecular operational taxonomic units and evaluated their anticancer activity against the MCF7 epithelial cancer cell line and its invasive stem cell-like MCF7-Sh-WISP2 counterpart. We identified the marine fungal Paradendryphiella salina PC 362H strain, isolated from the brown alga Pelvetia caniculata (PC), as one of the most promising fungi which produce active compounds. Further chemical and biological characterizations of the culture of the Paradendryphiella salina PC 362H strain identified (-)-hyalodendrin as the active secondary metabolite responsible for the cytotoxic activity of the crude extract. The antitumor activity of (-)-hyalodendrin was not only limited to the MCF7 cell lines, but also prominent on cancer cells with invasive phenotypes including colorectal cancer cells resistant to chemotherapy. Further investigations showed that treatment of MCF7-Sh-WISP2 cells with (-)-hyalodendrin induced changes in the phosphorylation status of p53 and altered expression of HSP60, HSP70 and PRAS40 proteins. Altogether, our study reveals that this uninvestigated marine fungal crude extract possesses a strong therapeutic potential against tumor cells with aggressive phenotypes and confirms that members of the epidithiodioxopiperazines are interesting fungal toxins with anticancer activities.

High-Resolution Crystal Structure of Chloroplastic Ribose-5-Phosphate Isomerase from Chlamydomonas reinhardtii-An Enzyme Involved in the Photosynthetic Calvin-Benson Cycle.

The Calvin-Benson cycle is the key metabolic pathway of photosynthesis responsible for carbon fixation and relies on eleven conserved enzymes. Ribose-5-phosphate isomerase (RPI) isomerizes ribose-5-phosphate into ribulose-5-phosphate and contributes to the regeneration of the Rubisco substrate. Plant RPI is the target of diverse post-translational modifications including phosphorylation and thiol-based modifications to presumably adjust its activity to the photosynthetic electron flow. Here, we describe the first experimental structure of a photosynthetic RPI at 1.4 Å resolution. Our structure confirms the composition of the catalytic pocket of the enzyme. We describe the homo-dimeric state of the protein that we observed in the crystal and in solution. We also map the positions of previously reported post-translational modifications and propose mechanisms by which they may impact the catalytic parameters. The structural data will inform the biochemical modeling of photosynthesis.

Phenomenal, bodily and brain correlates of fictional reappraisal as an implicit emotion regulation strategy.

The ability to modulate our emotional experience, depending on our current goal and context, is of critical importance for adaptive behavior. This ability encompasses various emotion regulation strategies, such as fictional reappraisal, at stake whenever one engages in fictional works (e.g., movies, books, video games, virtual environments). Neuroscientific studies investigating the distinction between the processing of real and fictional entities have reported the involvement of brain structures related to self-relevance and emotion regulation, suggesting a threefold interaction between the appraisal of reality, aspects of the Self, and emotions. The main aim of this study is to investigate the effect of implicit fictional reappraisal on different components of emotion, as well as on the modulatory role of autobiographical and conceptual self-relevance. While recording electrodermal, cardiac, and brain activity (EEG), we presented negative and neutral pictures to 33 participants, describing them as either real or fictional. After each stimulus, the participants reported their subjective emotional experience, self-relevance of the stimuli, as well as their agreement with their description. Using the Bayesian mixed-modeling framework, we showed that stimuli presented as fictional, compared with real, were subjectively appraised as less intense and less negative, and elicited lower skin conductance response, stronger heart-rate deceleration, and lower late positive potential amplitudes. Finally, these phenomenal and physiological changes did, to a moderate extent, rely on variations of specific aspects of self-relevance. Implications for the neuroscientific study of implicit emotion regulation are discussed.

Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma.

BALF0/1 is a putative Epstein-Barr virus (EBV) protein that has been described as a modulator of apoptosis. So far, the lack of specific immunological reagents impaired the detection of native BALF0/1 in EBV-infected cells. This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0) using a heterologous bacterial expression system. tBALF0 was further used as an antigen in an indirect Enzyme-linked Immunosorbent Assay (ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of nasopharyngeal carcinoma (NPC) patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis.

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories.

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

Modulation of the specific glutathionylation of mitochondrial proteins in the yeast Saccharomyces cerevisiae under basal and stress conditions.

The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H2O2, or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell.

Control of Autophagy in Chlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease.

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii Our results indicate that the activity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can be efficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon, an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes the oxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redox state, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii.

A Light Switch Based on Protein S-Nitrosylation Fine-Tunes Photosynthetic Light Harvesting in Chlamydomonas.

Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light-harvesting proteins and efficient light capture. In contrast, elevated light supply causes its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting.

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii.

BACKGROUND: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2+. METHODS: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography. RESULTS: CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2+. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2+-dependent and proceeds with a slow kinetics. The 3D-structure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2+ participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation. CONCLUSION: These evidences indicate that Mg2+ is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2+ is involved in the control of redox sensitivity of CrTK. GENERAL SIGNIFICANCE: The importance of Mg2+ in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2+ in chloroplasts.

Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes.

Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. Here, we employ the reference unicellular green alga Chlamydomonas reinhardtii to identify the effect of H2O2 on gene expression by monitoring the changes in the transcriptome in a time-course experiment. Comparison of transcriptomes from cells sampled immediately prior to the addition of H2O2 and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts that increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that addition of H2O2 is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O2 output. Additionally, we analyze our transcriptomes in the context of changes in transcript abundance in response to singlet O2 (O2*), and relate our H2O2 -induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H2O2 -induced transcripts early in the light phase, late in the light phase and 2 h prior to light. On this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses.