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Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii

A specific signaling role for H2O2 in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H2O2 but also on light. In the dark, the induction of the reporter gene by H2O2 was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H2O2 from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H2O2 levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3′4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H2O2 in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H2O2 required to activate H2O2-dependent signaling pathways.


Introduction
Life in an oxygen-rich atmosphere has to cope with the danger of oxidative stress. Plants are particularly exposed to oxidative stress caused by photosynthetic processes. Reactive oxygen species (ROS) are produced during normal cell metabolism but their production is drastically enhanced when plants are exposed to stresses such as high light, low temperatures, or drought, or combinations thereof. Due to their high reactivity, ROS can damage macromolecules essential for the integrity of the cell such as lipids, proteins, and nucleic acids. Thus, the concentration and accumulation of ROS needs to be strictly controlled. For this purpose, cells have evolved a variety of very eYcient non-enzymatic (e.g., ascorbate, glutathione, tocopherols) and enzymatic (e.g., catalase, ascorbate peroxidase, superoxide dismutase, peroxiredoxins, thioredoxins, glutaredoxins) antioxidant systems (Apel and Hirt 2004;Foyer and Noctor 2005). The requirement for a control of ROS accumulation implicates that these reactive intermediates are sensed by cells in order to activate stress responses allowing adaptation to a new set of environmental conditions. It is now widely accepted that ROS, and especially H 2 O 2 , play a major role in cellular signaling pathways and regulation of gene expression in a wide variety of organisms including plants (Apel and Hirt 2004;Laloi et al. 2004). Several studies have used microarray analyses to study alterations in gene expression in response to ROS (Gadjev et al. 2006 and references therein). These studies have demonstrated that increased ROS levels alter the expression of a rather large set of genes (up to one-third of the genome). They also have shown that diVerent ROS have speciWc signaling properties; a result supported by the recent discovery that H 2 O 2 and singlet oxygen employ spe-ciWc targets for the activation of an ROS-responsive promoter (Shao et al. 2007). The mechanisms that allow transduction of ROS signals to the nucleus remain unknown. In plants, the signaling pathways mediated by ROS involve heterotrimeric G-proteins (Joo et al. 2005) and protein phosphorylation mediated by MAP kinases and protein phosphatases (Kovtun et al. 2000;Gupta and Luan 2003;Rentel et al. 2004).
Under conditions of intracellular H 2 O 2 production or treatment with exogenous H 2 O 2 , several plant H 2 O 2 detoxifying enzymes, such as catalases and ascorbate peroxidases, are activated at the transcript, protein, and/or activity levels (Gechev et al. 2002;Vandenabeele et al. 2004;Yabuta et al. 2004;Davletova et al. 2005). Several studies have also shown that some of these enzymes may be inactivated by H 2 O 2 (Miyake and Asada 1996;Shikanai et al. 1998;Hiner et al. 2000;Miyake et al. 2006).
Ascorbate peroxidases (APX) are the main H 2 O 2 detoxifying enzymes in plants. Several isoforms of APX exist in plants and are located in the chloroplasts, the cytosol and peroxisomes (Shigeoka et al. 2002;Panchuk et al. 2005;Teixeira et al. 2006). Incubation of tobacco cell cultures in the presence of an H 2 O 2 generating system (glucose + glucose oxidase) has been shown to result in inhibition of cytosolic APX activity (De Pinto et al. 2006). Also the endogenous generation of H 2 O 2 via photosynthetic electron transfer resulted in inhibition of chloroplastic APX activity in tobacco plants (Miyake et al. 2006). APX were recently found among new putative targets of thioredoxins (TRX) identiWed by proteomic studies (Marchand et al. 2004;Wong et al. 2004;Yamazaki et al. 2004). Moreover, puri-Wed recombinant cytosolic APX from poplar was shown to be inhibited by reduced cytosolic thioredoxin h, reduced glutathione, and dithiothreitol. The molecular mechanism of this inhibition though remains to be determined (Gelhaye et al. 2006).
In plants, also catalases and peroxiredoxins play an important role in detoxifying H 2 O 2 (for review see Feierabend 2005;Dietz et al. 2006). Light inactivation of catalase has been observed for enzymes of both plant and animal origin. DiVerent isoforms of plant catalases show diVerent sensitivities towards light (Grotjohann et al. 1997;Engel et al. 2006). The action spectrum of photoinactivation is identical to the absorption spectrum of catalase, indicating that the light inactivation of catalases is caused by direct light absorption by the heme moieties. However, when irradiation was performed in the presence of chloroplasts the in vitro inactivation of catalase was enhanced. Under these conditions red light which is not absorbed by catalase but by photosynthetic pigments is suYcient to inhibit catalase (Feierabend and Engel 1986).
Catalases from Chlamydomonas, wheat, spinach, pea and potato were found among the potential targets of thioredoxin (TRX) identiWed by proteomic studies Lemaire et al. 2004;Maeda et al. 2004;Wong et al. 2004;Michelet et al. 2006). TRX are small disulWde oxidoreductases which play a major role in light signaling and in the oxidative stress responses (Vieira Dos Santos and Rey 2006; Lemaire et al. 2007). We have previously shown that in total soluble extracts of Chlamydomonas, the activity of catalase could be modulated by cytosolic thioredoxin h: a 50% inhibition in catalase activity was observed after treatment with the cytosolic TRX system (NADPH, NADPH-thioredoxin reductase, TRX h) which could be mimicked by treatment with the strong reductant dithiothreitol (DTT) (Lemaire et al. 2004).
Hence, the question arises whether a down-regulation of the activity of ROS detoxifying enzymes, like APX and catalases, might be necessary to allow diVusion and perception of H 2 O 2 under stress conditions that would result in the activation of genes that allow acclimation to changes in the environment. In order answer this question, we investigated whether photosynthetic electron transfer may be involved in the regulation of H 2 O 2 detoxifying enzymes and thus the regulation of gene expression in response to H 2 O 2 . As a model system, we have chosen Chlamydomonas reinhardtii which was transformed with a construct containing a Renilla reniformis luciferase reporter gene under the control of an HSP70A promoter fragment that speciWcally responds to H 2 O 2 (Shao et al. 2007).

Nuclear transformation of Chlamydomonas
Plasmids used for Chlamydomonas transformation were puriWed by PEG precipitation. Prior to transformation, the plasmid containing the ARG7 gene (pCB412) was linearized by EcoRI digestion. Plasmids containing the reporter construct, P HSP70A-HSE -LUC (Shao et al. 2007) were linearized by ScaI digestion. Chlamydomonas reinhardtii strain 325 was cotransformed with pCB412 (selection plasmid) and the plasmid containing the P HSP70A-HSE -LUC reporter construct using the glass beads method (Kindle 1990). For transformation, cells were grown to 3-5 £ 10 6 cells ml ¡1 and concentrated to 3 £ 10 8 cells ml ¡1 . 1 £ 10 8 cells were mixed with 500 ng of linearized LUC reporter plasmid, 100 ng of linearized pCB412, 0.3 g of acid-washed glass beads, and 100 l of 10% PEG 6000. Immediately after vortexing for 20 s, cells were spread onto TAP-agar (1% agar) plates for the selection of arginine autotrophic clones. Transformants that harbored the LUC constructs were detected by luciferase assay.

Bioluminescence assay
Bioluminescence assays were performed essentially as described by Minko et al. (1999) at room temperature with a luminescence counter (MicroBeta TriLux) set in "Xash" mode with single auto-injection. After sampling, cells were spun down, resuspended in the same volume of sample buVer [1.5 mM Tris-HCI (pH 7.8), 1 mM EDTA], and frozen at ¡80°C for at least 20 min. After thawing, 20 l were transferred to 96-well microtiter plates and 125 l assay buVer [0.1 M K 2 HPO 4 (pH 7.6); 0.5 M NaCl; 1 mM EDTA] were added to each well. After incubation at room temperature for 15 min in the dark, bioluminescence was assayed using the MicroBeta TriLux. The substrate (0.01 mM Coelenterazine, Biosynth AG, Staad, Switzerland) was auto-injected into the wells, and luminescence was recorded over a 20 s period, following a 1 s delay window. The background was normalized using wells containing only buVer or buVer with cells lacking the LUC gene. Expression of the LUC reporter gene in transgenic C. reinhardtii cells was normalized for cell numbers. The induction factor was calculated by comparison with untreated cells.
RNA isolation and RNA-blot analyses RNA extraction, electrophoretic separation of RNA and hybridizations were performed as described previously (von GromoV et al. 1989). The probe for LUC (accession number: AY004213) was a 0.9-kb BamHI/XhoI fragment from pCrLUC (Fuhrmann et al. 2004). The CBLP gene encoding a Chlamydomonas G -like polypeptide was employed as loading control gene (von Kampen et al. 1993).

Measurement of hydrogen peroxide
At each time point, 0.5-ml aliquots of the cultures were centrifuged at 13,000g in a microcentrifuge. The supernatants were mixed with an equal volume of 1 M KI. After 15 min at room temperature, the mixture was assayed for iodine formation by determining the OD at 390 nm. The absorbance at 390 nm was stable for at least 3 h (WaVenschmidt et al. 1993). Concentrations were determined using a standard calibration curve with known amounts of H 2 O 2 (Sigma).

Measurement of enzyme activities
Cultures of C. reinhardtii strain D66 were grown to 1-2 £ 10 6 cells ml ¡1 , transferred in the dark for 16 h, and subsequently exposed to light (70 mol photons m ¡2 s ¡1 ) or maintained in the dark. When indicated, DCMU (Wnal concentration 6 M) was added 45 min prior to illumination. Some cultures were supplemented with H 2 O 2 (2 mM Wnal concentration). Cells to be collected (1 £ 10 8 cells) were pelleted by centrifugation (microcentrifuge, maximal speed) and resuspended in 200 l 50 mM Hepes buVer (pH 8). After two consecutive freeze/thawing cycles in liquid nitrogen, the enzyme activities in crude extracts of Chlamydomonas cultures were measured.
Catalase activity was measured polarographically at 20°C with a Clark-type electrode in 50 mM Hepes buVer (pH 8) in the presence of 1 mM H 2 O 2 as substrate using a Wnal protein concentration of 5 g ml ¡1 . The protein concentration of the crude extracts was determined using the amido black assay.
Ascorbate peroxidase activity was measured photometrically at 290 nm in 20 mM phosphate buVer, pH 7.0, containing 0.5 mM ascorbate and 1 mM H 2 O 2 as substrates using crude extract with a Wnal protein concentration of 5 g ml ¡1 .
Initial NADP-malate dehydrogenase activity was measured photometrically at 340 nm in 100 mM Tris/HCl, pH 7.9, containing 0.75 mM oxalacetate and 0.27 mM NADPH as substrates using crude extract with a Wnal protein concentration of 10 g ml ¡1 . To activate MDH to its maximal activity, samples were incubated with 10 mM DTT, 20-M thioredoxin (cytosolic thioredoxin h1 from Chlamydomonas) in 100 mM Tris/HCl, pH 7.9 for 10 min prior to start of the assay by addition of substrates. The ratio between initial activity and total activity gives the percentage of activated MDH at the time point of harvest.

Statistics
Data represent means or representative examples from measurements repeated 3-6 times. Typical standard errors are shown, in Figs. 1b, 6 and 8; they are omitted for some data for clarity.

Induction of an H 2 O 2 -responsive reporter gene by H 2 O 2 is strongly stimulated by light
In the present work, we investigated the regulation of an H 2 O 2 -responsive reporter gene by exogenously added H 2 O 2 . For these assays we employed transformants harboring an H 2 O 2 -responsive fragment of the HSP70A promoter fused to a Renilla-derived luciferase gene (LUC) (Shao et al. 2007). A schematic representation of the LUC Fig. 1 a-c Response of a P HSP70A -Luc reporter gene in Chlamydomonas transformants to the addition of H 2 O 2 in the light and in the dark. a A truncated version ( HSE) of the HSP70A promoter construct known to be inducible by H 2 O 2 (Shao et al. 2007) was fused to a luciferase reporter gene that at its upstream end harbored sequences of HSP70B. Dark shaded bars represent exons, light shaded bars indicate introns. The third exon of HSP70B was fused to a Renilla luciferase reporter gene with C. reinhardtii adapted codon usage (Fuhrmann et al. 2004) (LUC), indicated by a dark grey bar. The 3Ј UTR of the fusion gene is encoded by the corresponding region of RBCS2. b Induction of P HSP70A -LUC in Chlamydomonas transformants by exogenously applied hydrogen peroxide. Light grown C. reinhardtii cells were incubated in the presence of 2 mM H 2 O 2 with white light of an intensity of 70 mol photons m ¡2 s ¡1 (open circles) or in the dark (closed circles). H 2 O was added to light-incubated control cultures (open squares) and to control cultures transferred to the dark (closed squares). Samples for luciferase assays were taken at the time points indicated and processed as described in Sect. "Materials and methods". The activity was normalized with respect to cell numbers and the induction factors were calculated by comparison with untreated cells at time point 0 h. c Dependence of the degree of induction of the P HSP70A -LUC reporter construct in Chlamydomonas transformants in the light on the concentration of exogenous H 2 O 2 . Experiments were performed as described in b. Samples for luciferase assays were taken after incubation for 3 h (diamonds) and after 5 h (triangles) reporter construct is presented in Fig. 1a. Transformants with this construct did not respond to heat shock but expression of the reporter gene is induced by hydrogen peroxide, either produced inside the chloroplast or added exogenously to the culture medium (Shao et al. 2007). A strong increase in luciferase activity was observed in transformants with this construct after treatment by H 2 O 2 , but only when the cultures were incubated in the light (Fig. 1b). In the light, the activity increased about 200-fold within 5 h of incubation in the presence of 2 mM H 2 O 2 . In the dark, a distinctly weaker induction was observed. The luc activity was 11-fold higher after 3 h, and 19-fold higher after 5 h incubation. Light itself (70 mol photons m ¡2 s ¡1 ) had no eVect since no induction of the reporter gene could be observed in the light-grown control cultures supplemented with H 2 O. In the absence of H 2 O 2 , no diVerence was observed between light-grown cultures and cultures transferred from the light to the dark. When cultures were grown in the dark for 20 h and then shifted to light in the absence of H 2 O 2 , a weak increase in luciferase activity was observed (Shao et al. 2007). This increase was 2.5-to 3-fold smaller than the one observed in the presence of H 2 O 2 in the dark.
We next tested the level of expression in dependence on the concentration of H 2 O 2 added to the culture medium. Luciferase activity was shown to increase strongly with the concentration of H 2 O 2 as can be seen by plotting luciferase activity 3 and 5 h after H 2 O 2 addition as a function of the concentration of externally added H 2 O 2 (Fig. 1c). We have shown previously that much lower H 2 O 2 concentrations are suYcient to induce the reporter gene when H 2 O 2 is produced inside the chloroplast by the photosynthetic electron transfer chain in the presence of metronidazole as electron acceptor. In this case, a high level of induction of the reporter gene was observed already after 2 h, when the detectable H 2 O 2 concentration in the medium was 3 M (Shao et al. 2007).
Catalase activity contributes to a faster degradation of H 2 O 2 in the dark The strong induction of transformants with the P HSP70A-HSE -LUC reporter construct by externally added H 2 O 2 observed only in the light may possibly be linked to lower rate of H 2 O 2 degradation by Chlamydomonas cells incubated in the light than in the dark. This was tested by measuring the rate of H 2 O 2 disappearance from the culture medium kept in light and or darkness. Indeed, H 2 O 2 was more rapidly degraded by cultures in the dark than in the light (Fig. 2a). In the dark, H 2 O 2 almost completely disappeared from the culture medium within 4 h and more than 50% of the initial H 2 O 2 had disappeared already within 1 h. By contrast, in the light, about 75% of the H 2 O 2 was still present after 1 h and some H 2 O 2 could still be detected after 4 h. The slower disappearance of the H 2 O 2 from the culture medium in the light may possibly be explained by the continuous production of H 2 O 2 by the photosynthetic electron transport chain. However, the Mehler reaction as potential internal source of H 2 O 2 cannot explain the diVerence between H 2 O 2 degradation in the light and in the dark, because little H 2 O 2 was produced by cultures, even in the presence of metronidazole, an artiWcial electron acceptor of PSI (less than 2 M H 2 O 2 per hour of irradiation was detectable in the medium; Shao et al. 2007). This faster degradation of H 2 O 2 in the dark may be caused by a light-dependent inhibition of H 2 O 2 detoxifying enzymes. In plants, catalases and ascorbate peroxidases constitute the two major classes of enzymes involved in H 2 O 2 detoxiWcation. To test whether catalase is involved in the observed diVerences in H 2 O 2 degradation between light and dark conditions, we measured the rates of H 2 O 2 degradation in the dark in the presence or absence of the catalase inhibitor aminotriazole (AT; Fig. 2b). The concentration of H 2 O 2 in the culture medium decreased considerably slower in the presence of AT than in its absence. This indicates that, under the conditions tested, catalase appears to be one of the major enzymes involved in H 2 O 2 detoxiWcation in Chlamydomonas cells. This result also suggested that a light-dependent regulation of catalase activity may account for the diVerences in H 2 O 2 degradation observed between light and dark conditions (Fig. 2) and consequently for the light-dependence of the H 2 O 2 inducibility of the reporter gene (Fig. 1b).
Light has previously been shown to inactivate plant catalases (Grotjohann et al. 1997; for review see Feierabend 2005). Generally, this inactivation was observed under high light conditions (800 mol photons m ¡2 s ¡1 ). To test whether catalase activity was already aVected by the low light intensities used in the experiments shown in Figs. 1 and 2, cell extracts of Chlamydomonas were illuminated with white light of an intensity of 70 mol photons m -2 s ¡1 for up to 5 h. Under these conditions, no signiWcant photoinactivation of the enzyme could be observed within 5 h of illumination (Fig. 6). We therefore concluded that a direct photoinactivation of catalase is unlikely to play a role under the low light conditions that were employed for studying the induction of the reporter gene by H 2 O 2 .
The redox state of the photosynthetic electron transfer chain aVects H 2 O 2 detoxiWcation and catalase activity Recently, it was shown that the activity of Chlamydomonas catalase is inhibited by reduced TRX in vitro (Lemaire et al. 2004). In chloroplasts, TRXs are reduced by the photosynthetic electron transfer chain in the light. The reduction of TRXs may thus possibly be responsible for the inactivation of catalases; and therefore, the slower H 2 O 2 degradation observed in the light. Such a mechanism may account for the light dependence of the induction of the reporter gene by H 2 O 2 . In order to test whether photosynthetic electron transfer plays a role in this light dependence, we investigated the eVect of DCMU, an inhibitor of photosystem II, on the expression of the reporter gene, the degradation of H 2 O 2 , and the activity of H 2 O 2 detoxifying enzymes. In the presence of DCMU, linear photosynthetic electron transfer is blocked and, among other components which may play a role in redox regulation, chloroplastic TRXs remain oxidized. In the presence of DCMU, only a small increase in luciferase activity could be detected after treatment with H 2 O 2 in the light (Fig. 3), indicating only a weak induction of the reporter gene. The level of luciferase activity observed was similar to that seen after addition of H 2 O 2 in the dark. This suggests that linear photosynthetic electron Xow is required for an eYcient induction of the reporter gene by H 2 O 2 in the light.
If this requirement is linked to a light-dependent inactivation of catalase, implicating TRXs or other mediators, then DCMU was predicted to aVect the rate of H 2 O 2 degradation in the light and the activity of catalases. Therefore, the time course of H 2 O 2 consumption was measured in the light in the presence and absence of DCMU (Fig. 4). In the light, the H 2 O 2 concentration decreased signiWcantly faster in the presence of DCMU than in its absence. This suggests that photosynthetic electron Xow accounts for the eVect of light on the rate of H 2 O 2 degradation in the culture medium.
To investigate the eVect of the redox state of photosynthetic electron Xow further, the degradation of H 2 O 2 was tested using mutants lacking either the cytochrome b 6 f complex ( petD) or deWcient in ribulose-1,5-bisphosphate carboxylase (Rubisco) (rbcL). As shown in Fig. 5, only a small diVerence in the kinetics of H 2 O 2 degradation was observed between dark-treated and light-treated cultures of the petD mutant lacking the cytochrome b 6 f complex. The diVerences were statistically not signiWcant according to the Student's t test. In this mutant, forward electron transport to PSI is completely abolished, the plastoquinone pool stays Relative LUC activity reduced and components downstream from PSI remain oxidized. In the mutant lacking the large subunit of Rubisco, the diVerence in H 2 O 2 degradation between light and dark is comparable to that observed in the wild-type strain. In this mutant, upon irradiation, the electron transport chain, including electron acceptors like NADP + and thioredoxins stays highly reduced. These data suggest that photosynthetic electron Xow regulates the activity of H 2 O 2 detoxifying enzymes, possibly involving TRXs. In order to test this possibility, we measured catalase activity in cells opened up by freeze/thawing. The activity could be inhibited by 75% by addition of 5-mM DTT (Fig. 6). This indicates that the catalase can undergo redox dependent inactivation. Figure 6 shows that incubation of the cells in the light clearly inhibited the activity of catalase. Indeed, about 40% of the activity was lost within 2 h and only 25% remained after 5 h of incubation in the light. The remaining activity after 5 h illumination corresponded to the activity of the catalase in the presence of DTT. By contrast, in cells maintained in the dark, the activity remained above 85% of the initial activity throughout the experiment. Presence of DCMU during the light incubation protected against the loss of activity (Fig. 6), supporting the idea that the activity of H 2 O 2 -consuming enzymes is regulated via the redox state of the electron transfer chain. The kinetics of inactivation were not dependent on the presence of H 2 O 2 in the culture medium, since the same degree of inhibition was observed when no H 2 O 2 was added prior to the shift of the cultures from dark to light (data not shown).
The activity of ascorbate peroxidases did not show any inhibition during the time course of illumination of the cultures in the presence of 2 mM H 2 O 2 (Fig. 7), indicating that indeed the redox-dependent inhibition of catalase is the important factor leading to a loss of H 2 O 2 degradation and subsequent expression of the reporter gene. To show a correlation between the state of reduction of thioredoxins and the activity of catalase, we measured the activity of the NADP-dependent malate dehydrogenase (MDH), one of the classical enzymes activated by reduction of its disul-Wde bonds by reduced thioredoxin (e.g., Lemaire et al. 2005Lemaire et al. , 2007. MDH activity increased upon incubation in the light while catalase activity decreased (Fig. 7). These data support the hypothesis that the activity of catalase is linked to the redox state of thioredoxin in the chloroplast. DCMU inhibited the activation of MDH as expected (Fig. 7).
It has to be mentioned that H 2 O 2 inhibits CO 2 Wxation, since the transketolase reactions of the Calvin cycle are very sensitive to inhibition by H 2 O 2 (Kaiser 1976). Under our conditions, photosynthetic oxygen evolution measured in the presence of 1 mM NaHCO 3 was inhibited by 50% upon addition of 2 mM H 2 O 2 (data not shown) in accordance with data reported for a diVerent green algal species (Drabokova et al. 2007). This eVect, however, seems not to interfere with our observations, neither with the induction of the reporter gene nor with the light-dependent inactivation of catalase.
Aminotriazole and DCMU aVect the expression of the reporter gene at the mRNA level We have shown above that a down-regulation of catalase activity mediated either by photosynthetic electron Xow or the addition of AT appears to be responsible for a slower degradation of H 2 O 2 in the light. This suggested that a down-regulation of catalase activity in the light also accounts for the light dependence of the LUC reporter gene induction by H 2 O 2 . In order to conWrm that the activity of H 2 O 2 degrading enzymes and photosynthetic electron Xow indeed are involved in the regulation of the reporter gene and to rule out the possibility that DCMU and AT interfere with luciferase activity directly, we measured the eVect of AT and DCMU on H 2 O 2 induction of the reporter at the mRNA level (Fig. 8). In the light, an increase of LUC mRNA level was observed after H 2 O 2 treatment. This induction was abolished by the presence of DCMU, conWrming that photosynthetic electron Xow is required for induction of the reporter gene, as already observed at the activity level (Fig. 3). In the dark, a small increase in LUC mRNA levels was observed upon H 2 O 2 addition, consistent with the low level increase of LUC activity observed under these conditions (Fig. 1b). Addition of AT in the dark allowed a stronger increase in LUC mRNA levels after H 2 O 2 treatment, consistent with a lower degradation of this ROS (Fig. 2b). These results are in agreement with those observed at the LUC activity level and indicate that down-regulation of the activity of H 2 O 2 degrading enzymes is a prerequisite for an eYcient induction of the reporter gene by H 2 O 2 . consumption by the crude extracts was measured polarographically. 100% activity corresponds to an activity of 10 mol O 2 mg protein ¡1 min ¡1 produced when 1 mM H 2 O 2 was added as substrate. The catalase activity in the crude extract of cells incubated in the light was strongly inhibited when 5 mM DTT was added (stars). Also shown is the catalase activity of crude extract of dark-grown cells (diamonds) that were illuminated (white light, 70 mol photons m ¡2 s ¡1 ) for the time indicated

Discussion
The data presented here show that the expression of the H 2 O 2 -responsive reporter construct not only depended on the H 2 O 2 concentration in the media (Fig. 1c) but also on the activity state of H 2 O 2 degrading enzymes (Figs. 2,3,8).
The decrease in the activity of H 2 O 2 degrading enzymes observed upon illumination was prevented by DCMU ( Fig. 4) and by mutations perturbing photosynthetic electron Xow (Fig. 5), indicating that catalase activity was correlated with the redox state of the photosynthetic electron transport chain (Fig. 6). These data illustrate that Chlamydomonas cells have evolved mechanisms that ensure maintenance of a certain level of H 2 O 2 in order to allow activation of H 2 O 2 -responsive genes. In photosynthetic organisms, ROS are mainly formed in the light as a consequence of electron transport reactions under conditions where the electron Xow system is saturated and the electron acceptors are reduced. In the so-called Mehler reaction, the electron acceptors of photosystem I react with oxygen when the ferredoxin-NADP system and alternative electron sinks are already reduced. Superoxide anions and H 2 O 2 are formed. These ROS are released into the stroma where they encounter an eYcient antioxidant system and detoxifying enzymes (for review see Asada 1999;Foyer and Noctor 2005). In general, it is assumed that detoxifying enzymes have to be present in an active state during oxidative stress conditions. However, it was recently observed that higher plant ascorbate peroxidase is inhibited in the light under conditions where both, the Calvin cycle and photorespiration, are inhibited (Miyake et al. 2006). This paradoxical situation makes sense when the function of ROS in gene regulation is taken into account. Maintenance of a certain H 2 O 2 concentration inside the chloroplast may be achieved in higher plants via control of the activity of ascorbate peroxidases, the main H 2 O 2 -detoxifying enzyme in this organelle. The H 2 O 2 concentration in the cell will depend on the activity of the whole set of detoxifying enzymes; also on those located in diVerent organelles because H 2 O 2 can easily diVuse across biological membranes (Bienert et al. 2007). As shown for diVerent organisms, the expression of a number of genes, encoding, e.g., heat shock proteins, catalases, and ascorbate peroxidases involved in the abrogation of oxidative stress, is regulated via H 2 O 2 signaling. Thus, it can be postulated that a down-regulation of the activity of detoxifying enzymes under stress conditions is a necessary prerequisite for the activation of those genes that allow acclimation of the organism to oxidative stress conditions. In Chlamydomonas, this may be achieved via inactivation of catalase; the activity of ascorbate peroxidase under these conditions being constant (Fig. 7). Inactivation of plant catalases by high intensity irradiation (Feierabend and Engel 1986;Grotjohann et al. 1997) needs to be distinguished from low light induced inactivation as studied here by exposing Chlamydomonas cells to light of an intensity of 70 mol photons m ¡2 s ¡1 or less. It has also been reported previously that the catalase activity in Chlamydomonas is higher in cells grown in the dark than in the light (Kato et al. 1997).
The inactivation of H 2 O 2 degrading enzymes by light of moderate intensity as observed in Chlamydomonas could be linked to photosynthetic electron transfer since this inactivation was prevented by DCMU known to block the photosynthetic apparatus at PSII (Fig. 4). In vitro measurements revealed that catalase is the target for this inactivation (Fig. 6). The redox sensor which mediates the inactivation of H 2 O 2 degrading enzymes appears to be located downstream from PSI, since a mutation leading to an inactivation of the cytochrome b 6 f complex ( petD) resulted in a protection against inhibition of the activity of H 2 O 2 degrading enzymes by light (Fig. 5). The reduction state of the plastoquinone pool (Pfannschmidt and Liere 2005) can thus be ruled out to be of importance in the regulation of catalase activities, because mutations leading to an inactivation of the cytochrome b 6 f complex are characterized by a high reduction state of the plastoquinone pool while in the presence of DCMU, the plastoquinone pool stays oxidized. Since a reduction of TRX by the photosyn-   (Lemaire et al. 2004;Gelhaye et al. 2006). Proteomic studies revealed that also several ROS scavenging enzymes which do not use TRXs as substrates such as catalase, superoxide dismutase, or APX, are putative TRX targets since they are bound to TRX aYnity columns Lemaire et al. 2004;Maeda et al. 2004;Wong et al. 2004;Michelet et al. 2006).
In the genomic sequence of Chlamydomonas (http:// genome.jgi-psf.org/Chlre3/Chlre3.home.html), only a single gene encoding a typical catalase (gene model Chlre3/ scaVold_30:1312932-1317626) was observed. A gene (gene model Chlre3/scaVold_1:4550894-4558523) encoding a putative catalase-peroxidase homologous to those found in prokaryotes (Jakopitsch et al. 2005) was also found. However, as judged on the basis of the number of EST sequences, expression of the latter gene appears to be very weak as compared to the expression of the typical catalase gene, even under oxidative stress conditions. Consequently, the catalase activities reported here are likely to correspond to the activity of the typical catalase, although it cannot be excluded that the putative catalase-peroxidase also contributes to H 2 O 2 degrading activity measured in extracts.
In higher plants, catalases were localized to peroxisomes (Heazlewood et al. 2005), while in Chlamydomonas the enzyme is most likely located in mitochondria (Kato et al. 1997;Lemaire et al. 2004). This implies that a signal about the redox state of the photosynthetic electron transfer chain has to be transmitted to targets outside of the chloroplast. This signal could be initiated by a component whose redox state depends on the redox state of a functional photosynthetic electron transfer chain such as TRXs. The signal is possibly transmitted from the chloroplast by redox mediators such as glutathione or NADPH. Indeed, reducing equivalents of chloroplastic NADPH have been reported to be transferred outside of the chloroplast in the form of malate (Scheibe 2004). This malate is formed by reduction of oxalacetate using NADPH as electron donor. Interestingly, the NADP-malate dehydrogenase of the chloroplast is activated by light by a mechanism dependent on the redox state of plastidic TRXs (Lemaire et al. 2005(Lemaire et al. , 2007. Indeed, a reverse correlation between the activities of catalase and NADP-malate dehydrogenase was observed (Fig. 7). Once outside of the chloroplast, the signal may be transduced to a redox regulator of the catalase via diVerent redox mediators. Since the activity of Chlamydomonas catalase has been shown to be regulated by TRXs in vitro (Lemaire et al. 2004), the redox regulator of catalase is likely to be a TRX or involves TRX-related proteins like glutaredoxins. There is some evidence that thioredoxins belonging to the TRX o and TRX h subtypes are present in the mitochondria of Chlamydomonas (Lemaire et al. 2003).
In conclusion, the data presented provide support for the idea that Chlamydomonas has evolved mechanisms that allow maintenance of a certain level of H 2 O 2 . We assume that these mechanisms ensure a regulation of gene expression in response to ROS, allowing for an adaptation of the organisms to stress conditions. A fast inactivation of catalase activity is seen when cells are exposed to a 10fold higher light intensity (data not shown). Regulation of the activity of ROS detoxifying enzymes appears to play an important role. A regulation via TRX or other components of the regulatory redox network like glutaredoxins, peroxiredoxins or glutathione provides an attractive strategy for the regulation of such enzyme activities. These compounds may ensure a fast and reversible inactivation of the enzymes, thereby allowing for a rapid sensing of changes in environmental conditions. Most importantly, through thioredoxins or other redox compounds, the regulation of the activity of H 2 O 2 -consuming enzymes may directly be coupled to photosynthetic electron Xow, and thus to the main source for ROS production in photosynthetic organisms.