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See detailInsertional mutagenesis to select mutants for modified hydrogen photoproduction in Chlamydomonas reinhardtii
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Cardol, Pierre ULg et al

Poster (2011, May 17)

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases ... [more ▼]

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases. This process occurs under anaerobic conditions and provides a biological basis for solar-driven hydrogen production. Nevertheless, the yield is a major limitation for an economic viability and fundamental knowledge is still needed in order to have a better understanding of the process. In 2000, Melis and co-worker defined a protocol allowing a sustainable hydrogen production in sulfur deprivation condition. By adjustment of an existent protocol called the Winkler test, we are trying to isolate mutants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). This kind of mutants could be able to reach anoxia needed for hydrogenases activity without the stressful impact of sulfur deprivation. An insertional mutagenesis of Chlamydomonas has been carried out with an hygromycin resistance cassette and about 2500 transformants have generated and screened by the adapted Winkler test. We have isolated several oxygen-consuming mutants and the most promising one is subject to functional, molecular and genetic characterization. To discover new genes involved in hydrogenases activity, we are also planning to screen the same insertional library for mutants with attenuated levels of hydrogen photoproduction, using sensitive chemochromic sensor films which turn in blue in presence of hydrogen. We are currently making the chemochromic sensor WO3 films by dip-coating which is on the brink of being useable. [less ▲]

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See detailA Chlamydomonas mutant locked in anaerobiosis
Ghysels, Bart ULg; Matagne, René-Fernand ULg; Franck, Fabrice ULg

Conference (2011, May)

The soil dwelling microalga Chlamydomonas reinhardtii most likely encounters transient periods of anaerobiosis in its natural environment, for instance at night time or when photosynthesis is turned down ... [more ▼]

The soil dwelling microalga Chlamydomonas reinhardtii most likely encounters transient periods of anaerobiosis in its natural environment, for instance at night time or when photosynthesis is turned down in response to macronutrient limitation. Anoxic conditions trigger state I to state II transition in C.r. and the induction of a chloroplast hydrogenase., which ability to accept electrons from reduced Fd results in a transient light driven H2 evolution. We present evidence that hydrogenase induction and state transitions are required for the induction of photosynthesis in anaerobiosis and therefore critical for this alga in order to survive transient anaerobic periods in the dark. In an anaerobic metabolic context the induction of photosynthesis is severely slowed down. The highly reduced state of the NAD(P) pools and the absence of O2 as electron sink hamper light driven reoxydation of the intersystem electron carriers while CO2 assimilation by the Calvin cycle is inhibited by ATP deficiency. We have seen that gradual increase of hydrogenase activity during anaerobiosis restores a PSI acceptor pool and leads to a reduction of the induction lag of oxygenic photosynthesis. A mutant HydEF devoid of hydrogenase maturation genes typically shows 3 to 4 times longer lag phases that the WT. State transitions provide another mechanism by which photosynthetic electron transport can be unlocked in anaerobic conditions. A state II conformation is known to stimulate photo-phosphorylation, and may therefore restore Calvin cycle activity in an ATP depleted metabolic context. We observed that an anaerobically adapted stt7 mutant locked in state I is only able to induce oxygenic photosynthesis upon hydrogenase expression. We therefore constructed a double mutant Stt7HydEF impaired of state transition ability and hydrogenase activity and found it to have lost the capacity of inducing photosynthesis in anaerobic conditions. [less ▲]

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See detailFunctional analysis of hydrogen photoproduction in respiratory-deficient mutants of Chlamydomonas reinhardtii
Lecler, Renaud ULg; Godaux, Damien ULg; Vigeolas, Hélène ULg et al

in International Journal of Hydrogen Energy (2011), 36

In this paper, mitochondrial mutants of Chlamydomonas reinhardtii defective for respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol cytochrome c oxidoreductase) and both ... [more ▼]

In this paper, mitochondrial mutants of Chlamydomonas reinhardtii defective for respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol cytochrome c oxidoreductase) and both complexes I and III were analyzed for H2 photoproduction. Several parameters were followed during the S-deficiency stage and the anaerobic stage leading to H2 photoproduction. At the early aerobic S-deficiency stage, starch and neutral lipids accumulated in all strains but their amount was significantly decreased in mutants compared to wild type. During the H2 photoproduction process, whereas starch content strongly decreased in all strains, neutral lipid amount remained nearly unchanged, suggesting that starch degraded by glycolysis is the preferential substrate for energy production during anaerobiosis. The mutants displayed a decrease in H2 photoproduction correlating to the number of active mitochondrial proton-pumping sites lost in the strains. Our results thus highlight the critical role of oxidative phosphorylation during the first (aerobic) stage of S-starvation when carbon resources are accumulated. [less ▲]

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See detailLa réponse photosynthétique d'une algue verte à la carence en soufre
de Marchin, Thomas ULg; Ghysels, Bart ULg; Franck, Fabrice ULg

Book published by Editions universitaires europeennes (2010)

Chlamydomonas reinhardtii possède la capacité de produire de l'hydrogène à la lumière en absence d'oxygène. Cette condition peut être obtenue en cultivant les algues dans un milieu carencé en soufre. La ... [more ▼]

Chlamydomonas reinhardtii possède la capacité de produire de l'hydrogène à la lumière en absence d'oxygène. Cette condition peut être obtenue en cultivant les algues dans un milieu carencé en soufre. La carence en soufre entraîne une forte diminution de l'activité du photosystème II tout en maintenant une respiration élevée, ce qui provoque un passage de cultures fermées en anoxie et induit la production d'hydrogène. Dans cette étude, nous avons caractérisé la réponse photosynthétique à la carence en soufre chez la souche sauvage et la souche déficiente en oxydase alternative mictochondriale (AOX) dans des milieux contenant de l'ammonium ou du nitrate comme source d'azote. L'AOX, inductible par le nitrate, fait partie de la chaîne de transport d'électrons mitochondriale et catalyse l'oxydation de l'ubiquinol en transférant directement ses électrons à l'oxygène. Ainsi l'AOX entre en compétition avec le complexe III et est impliquée dans une voie de dissipation du pouvoir réducteur en excès. [less ▲]

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See detailAlternative photosynthetic electron pathways in different clades of Symbiodinium: the Mehler reaction
Roberty, Stéphane ULg; Poulicek, Mathieu ULg; Franck, Fabrice ULg

Poster (2010, December)

The high productivity of coral reef ecosystems is largely attributed to the mutualistic symbiosis between reef-building corals and their intracellular dinoflagellate in the genus Symbiodinium commonly ... [more ▼]

The high productivity of coral reef ecosystems is largely attributed to the mutualistic symbiosis between reef-building corals and their intracellular dinoflagellate in the genus Symbiodinium commonly referred to as zooxanthellae. These photosynthetic algae translocate a majority of their photosynthetically fixed carbon to the host and contribute greatly to their metabolic needs (Muscatine, 1990) and the calcification process (Gattuso, 1999). In the natural environment the holobiont have to cope with significant daily variations in light intensities that sometimes exceed Symbiodinium photosynthetic capacity. Fortunately, photosynthetic organisms possess regulatory features that help to ensure that high light intensities can be endured without the accumulation of photodamage. Thus, the regulation of photosynthesis can be viewed as a dynamic balance between photosynthetic efficiency (photochemical quenching) and photoprotection processes (i.e. non-photochemical quenching). Among them, the role of O2 as an alternative electron acceptor within the chloroplast could play a critical role (Ort & Baker, 2002). Under particular environmental conditions when sinks for photosynthetic electrons are scarce, the direct reduction of oxygen by the PSI could sustain significant levels of photosynthetic electron flux by initiating the ΔpH formation and of NPQ, regulating the ratio of ATP/NADPH to match the requirements of carbon reduction. However, this process leads to the formation of reactive oxygen species that are rapidly detoxified by superoxide dismutase and ascorbate peroxidase. An additional electron flux associated with this oxygen pathway is directed to the reduction of monodehydroascorbate (MDA), which is generated as a result of peroxide reduction by ascorbate (Asada, 2000). The present study aimed to highlight the existence of alternative photosynthetic electron pathways and more especially the Mehler ascorbate peroxidase pathway in different clades of Symbiodinium, cultivated at low and high light intensities. -Muscatine L (1990) The role of symbiotic algae in carbon and energy flux in reef corals. In: Dubinsky Z(ed) Ecosystems of the world: coral reefs. Elsevier, Amsterdam, pp 1-9. -Gattuso JP, Allemand D and M Frankignoulle (1999) Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: A review on interactions and control by carbonate chemistry. American Zoologist 39(1): 160-183. -Ort, D. R. and N. R. Baker (2002). A photoprotective role for O2 as an alternative electron sink in photosynthesis? Current Opinion in Plant Biology 5(3): 193-198. -Asada, K. (2000) The water-water cycle as alternative photon and electron sinks. Philosophical Transactions of the Royal Society B-Biological Sciences 355(1402): 1419–1431. [less ▲]

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See detail(Functionnal) analysis of hydrogen production in Chlamydomonas reinhardtii mitochondrial mutants
Lecler, Renaud ULg; Godaux, Damien ULg; Hamilton, Christopher ULg et al

Poster (2010, June 27)

Mitochondrial Chlamydomonas mutants for respiratory complexes present a decreased dark respiration and apparent yield of photosynthetic linear electron flow. They accumulate reducing power such as NAD(P)H ... [more ▼]

Mitochondrial Chlamydomonas mutants for respiratory complexes present a decreased dark respiration and apparent yield of photosynthetic linear electron flow. They accumulate reducing power such as NAD(P)H and show lower levels of ATP. Under restrictive conditions, like sulfur depletion and anoxia, Chlamydomonas is able to produce hydrogen towards the activation of a chloroplatic O2-sensitive Fe-hydrogenase which catalyses the reduction of electrons to H2. In this study we used an adapted Melis protocol to analyse hydrogen evolution of mitochondrial mutants. For this aim a simple-flask system was built with gaz collecting tubes. A parallel flask was used for GC analyses. [less ▲]

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See detailEffect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009
Masset, Julien ULg; Hiligsmann, Serge ULg; Hamilton, Christopher ULg et al

in International Journal of Hydrogen Energy (2010), 35(8), 3371-3378

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production ... [more ▼]

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 l bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H2 mol-1 glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol-1 glucose and 0.6 mol butyrate mol-1 glucose. For starch a maximum yield of 2.0 mol H2 mol-1 hexose, and a maximum production rate of 15 mol H2 mol-1 hexose h-1 were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol-1 hexose and 0.67 mol butyrate mol-1 hexose. [less ▲]

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See detailLa production photosynthétique d'oxygène
Franck, Fabrice ULg

Conference given outside the academic context (2010)

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See detailProteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1
Mathy, Grégory ULg; Cardol, Pierre ULg; Dinant, Monique et al

in Journal of Proteome Research (2010), 9

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase ... [more ▼]

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase (AOX). The AOX-deficient strain displays a doubling of the cell volume and biomass without any alteration of the generation time, a significantly higher ROS production, no change in total respiration rate, and a slight decrease of the photosynthesis efficiency. In order to identify the molecular adaptation underlying these phenotypical effects, we carried out a comparative proteomic study at the level of the mitochondrial and cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways. [less ▲]

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See detailPlasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: the logic of Aox1 gene localization
Gérin, Stéphanie ULg; Mathy, Grégory ULg; Blomme, Arnaud ULg et al

in Biochimica et Biophysica Acta-Bioenergetics (2010), 1797

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one ... [more ▼]

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c1 together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster. [less ▲]

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See detailThe onset of NPQ and Deltamu(H)+ upon illumination of tobacco plants studied through the influence of mitochondrial electron transport.
Cardol, Pierre ULg; De Paepe, Rosine; Franck, Fabrice ULg et al

in Biochimica et Biophysica Acta (2010), 1797(2), 177-88

The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to ... [more ▼]

The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to assimilate CO(2) was studied in tobacco dark-adapted leaves at the onset of illumination with low light. These conditions induce the generation of a transient NPQ, which relaxes in the light in parallel with the activation of the Calvin cycle. Wild-type plants were compared with a CMSII mitochondrial mutant, which lacks the respiratory complex I and shows a delayed activation of photosynthesis. In the mutant, a slower onset of photosynthesis was mirrored by a decreased capacity to develop NPQ. This correlates with a reduced efficiency to reroute electrons at the PSI reducing side towards cyclic electron flow around PSI and/or other alternative acceptor pools, and with a smaller ability to generate a proton motive force in the light. Altogether, these data illustrate the tight relationship existing between the capacity to evacuate excess electrons accumulated in the intersystem carriers and the capacity to dissipate excess photons during a dark to light transition. These data also underline the essential role of respiration in modulating the photoprotective response in dark-adapted leaves, by poising the cellular redox state. [less ▲]

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See detailEukaryotic algae: where lies the diversity of oxygenic photosynthesis.
Cardol, Pierre ULg; Franck, Fabrice ULg

in Photosynthesis Research (2010), 106(1-2), 1-2

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See detailHydrogen photo-evolution upon S deprivation stepwise: An illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H2 production
Ghysels, Bart ULg; Franck, Fabrice ULg

in Photosynthesis Research (2010), 106

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails ... [more ▼]

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidise reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. 2000). This observation boosted research in algal H2 evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H2 generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H2 production procedures. Ten years later most of the critical steps and limitations of H2 production by this protocol have been studied from different angles particularly with the model organism C. reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review. [less ▲]

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See detailPhotoacclimation responses of a symbiotic sea anemone reveal an important host cellular plasticity
Roberty, Stéphane ULg; Fransolet, David ULg; Ladrière, Ophélie ULg et al

Poster (2010)

The high productivity of coral reef ecosystems is largely attributed to the mutualistic symbiosis between reef-building corals and their intracellular dinoflagellate in the genus Symbiodinium commonly ... [more ▼]

The high productivity of coral reef ecosystems is largely attributed to the mutualistic symbiosis between reef-building corals and their intracellular dinoflagellate in the genus Symbiodinium commonly referred to as zooxanthellae. These photosynthetic algae translocate a majority of their photosynthetically fixed carbon to the host and contribute to their metabolic needs and the calcification process. <i>Symbiodinium</i> must maintain a balance between the energy derived from the light reactions in the chloroplast and the amount of energy used during dark reactions and other metabolic processes. Nevertheless, in the natural environment the holobiont have to cope with daily and seasonal changes in light intensity, upsetting that balance and creating a stress that induces a physiological response (photoacclimation) to optimize growth rates. After a ten day exposition to high and very low light intensity, morphological and photophysiological analysis conducted on the symbiotic sea anemone, Anemonia manjano, reveal significant modifications of the host tissues ultrastructure and the Symbiodinium metabolic processes (photosynthesis, respiration). Those results highlight particularly important gastrodermal and ectodermal plasticity in which symbiotic cnidarians acclimate to the Symbiodinium physiological status (mainly photosynthesis) by varying the density of particular cellular types (e.g.: cnidocytes, gastrodermal cells) contained in their tissues. [less ▲]

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See detailVitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress
Havaux, Michel; Ksas, Brigitte; Szewczyk, Agnieszka et al

in BMC Plant Biology (2009), 9

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See detailImpaired respiration discloses the physiological significance of state transitions in Chlamydomonas.
Cardol, Pierre ULg; Alric, Jean; Girard-Bascou, Jacqueline et al

in Proceedings of the National Academy of Sciences of the United States of America (2009), 106(37), 15979-84

State transitions correspond to a major regulation process for photosynthesis, whereby chlorophyll protein complexes responsible for light harvesting migrate between photosystem II and photosystem I in ... [more ▼]

State transitions correspond to a major regulation process for photosynthesis, whereby chlorophyll protein complexes responsible for light harvesting migrate between photosystem II and photosystem I in response to changes in the redox poise of the intersystem electron carriers. Here we disclose their physiological significance in Chlamydomonas reinhardtii using a genetic approach. Using single and double mutants defective for state transitions and/or mitochondrial respiration, we show that photosynthetic growth, and therefore biomass production, critically depends on state transitions in respiratory-defective conditions. When extra ATP cannot be provided by respiration, enhanced photosystem I turnover elicited by transition to state 2 is required for photosynthetic activity. Concomitant impairment of state transitions and respiration decreases the overall yield of photosynthesis, ultimately leading to reduced fitness. We thus provide experimental evidence that the combined energetic contributions of state transitions and respiration are required for efficient carbon assimilation in this alga. [less ▲]

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See detailS13.45 Chlamydomonas reinhardtii mitoproteome adaptation in response to inactivation of the energy-dissipating alternative oxidase 1 by RNA interference
Cloes, Marie ULg; Mathy, Grégory ULg; Cardol, Pierre ULg et al

in Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1777, Supplement 1, 19 July 2008, Page S99 (2008, July 18), 1777(Supplement 1), 99

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See detailImportance of the alternative pathway of respiration for avoidance of ROS production and for optimisation of photosynthesis in Chlamydomonas
Franck, Fabrice ULg; Dinant, M.; Cardol, Pierre ULg et al

Conference (2008, June)

The physiological function of the alternative pathway of respiration has been investigated by analysing two RNAi C.reinhardtii lines deprived of alternative oxidase protein (AOX1). Compared to wild-type ... [more ▼]

The physiological function of the alternative pathway of respiration has been investigated by analysing two RNAi C.reinhardtii lines deprived of alternative oxidase protein (AOX1). Compared to wild-type, AOX1- lines exhibited modified growth curves and reduced maximal cell density. These differences were more pronounced at high irradiance and in nitrate-containing medium (TAP NO3) rather than in ammonium-containing medium (TAP NH4). Although the alternative pathway was inactive, respiration was not significantly altered in transgenics. Light-saturation curves of O2-evolution were only slightly modified. However, non-photochemical quenching of fluorescence (NPQ) was strongly reduced. Further analysis showed that AOX1- transgenics present a reduced ability to promote the change in energy distribution between photosystems, known as state transition. This effect, which explains low NPQ in the light, was most pronounced in high-light cells cultivated in TAP NO3 medium. Moreover, AOX1- transgenics exhibited higher levels of intracellular peroxides, which suggests that inhibition of state transition might result from higher ROS production. In support of this hypothesis, addition of millimolar-range concentrations of H2O2 to wild-type inhibited the state transition promoted by the reduction of the plastoquinone pool in darkness. [less ▲]

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