Doctoral thesis (Dissertations and theses)
Function of the chloroplastic hydrogenase in the microalga Chlamydomonas reinhardtii: A trvel from dark to light
Godaux, Damien
2014
 

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Keywords :
Chlamydomonas; Hydrogenase; Photosynthesis induction
Abstract :
[en] The decreasing availability of fossil energy stocks and the eventuality of tragic climate changes caused by greenhouse gases lead to search for alternative renewable energy sources. Biological hydrogen might be one promising renewable energy carrier. A specific and restricted group of microalgae developed the ability to produce hydrogen based on an oxygen-sensitive hydrogenase enzyme coupled to the photosynthetic pathway, acting as a putative valve for excess electrons in conditions where other electron acceptors are scarce. The unicellular green alga Chlamydomonas reinhardtii is widely regarded as a model organism for various biological processes, especially for photosynthesis. Moreover, the capacity of Chlamydomonas hydrogenase is claimed as the highest recorded in literature. Less than twenty years ago, a group of American scientists designed a new approach for sustained photobiological production of hydrogen, based on a two-stage protocol that temporally separates photosynthetic O2 evolution from the H2 production phase (Melis et al., 2000). The transition occurs upon sulfur deprivation of the culture and leads to an operating continuous production for several days, opening new possibilities in the aim of an economically rentable bioproduction. For these reasons, hydrogen photoproduction in Chlamydomonas reinhardtii has been extensively examined in the last decade as extension of photosynthesis research entailing the understanding of hydrogen metabolism in microalgae (for reviews, see Hankamer et al., 2007; Ghirardi et al., 2009; Ghysels and Franck, 2010). Despite the attractive trait of generating a renewable fuel from nature’s most plentiful resources, i.e. light and water, the physiological significance of such oxygen-sensitive enzyme coupled to oxygenic photosynthesis has been poorly investigated with the exception of some old studies (Kessler, 1973; Schreiber and Vidaver, 1974). In this work, hydrogenase implication in photosynthetic reactivation from dark and anoxic environment is investigated. In the first part of the work, by analyzing several strains affected in hydrogen metabolism (e.g. nda2-RNAi (Jans et al., 2008), pfl1 (Philipps et al., 2011), dum11 (Dorthu et al., 1992)), we show that the PSII–dependent photosynthetic electron flow upon dark to light shift is linearly related to the activity of hydrogenase, both for short and long-terms adaptation (Publication I). In agreement with this conclusion, a hydrogenase-deficient strain for the HydEF maturation factor (hydef, Posewitz et al., 2004) shows peculiar chlorophyll fluorescence induction kinetics after adaptation to dark and anoxia. Based on these findings, a novel imaging screening method is developed, allowing rapid identification of strains impaired in hydrogen metabolism. Compared to existing screens (for review, see Hemschemeier et al., 2009), our protocol is remarkably fast, sensitive and non-invasive. At this stage, application of this new screening method allowed us to isolate several hydrogenase-deficient strains, among which one was impaired for the hydrogenase maturation protein HydG (hydg-2 mutant). Chlamydomonas reinhardtii might frequently encounter period of dark and anoxia in its natural habitat, especially during the night when the microbial community respires the available oxygen. In the second part of my work, the physiological importance of hydrogenase is investigated in the context of photosynthesis induction at the onset of light upon anoxia. In such conditions, the plastoquinone pool is known as being overreduced. This triggers the process of state transitions which is described as allowing the redistribution of light capture between both photosystems to manage the redox poise of the photosynthetic pathway (for review, see Lemeille and Rochaix, 2010). We therefore revisit the impact of both state transitions and hydrogenase activity on the reactivation of photosynthetic electron flow (Publication II). Here we show that, in presence of hydrogenase, photosynthesis reactivation is slightly faster in stt7 mutant locked in state 1 (Depege et al., 2003) compared to wild type which is in state 2. However, photosynthesis reactivation is delayed in hydef stt7-9 double mutant compared to hydef mutant. This indicates that, in a hydrogenase-deficient context, state 2 promotes photosynthesis reactivation. Considered for a long time as being tightly interconnected (Finazzi et al., 1999; Finazzi et al., 2002; Finazzi and Forti, 2004), state transitions and PSI-CEF have recently been revealed as unrelated to each other (Takahashi et al., 2013). Nonetheless, the increasing of PSI antenna size in state 2 could even though enhance the PSI-CEF rate, in an indirect way, by enhancing PSI energy capture (Cardol et al., 2009; Alric, 2014). This reasonably raises the question of a possible involvement of PSI-CEF in photosynthesis induction. This possibility is further studied in the third and last part of the work. Thanks to mutants devoid of PSI-CEF (i.e. pgrl1 mutant (Tolleter et al., 2011)) and hydrogenase activity (i.e. hydg-2 mutant (Publication I)), we investigate the role played by PSI-CEF along with hydrogenase during photosynthesis reactivation during a shift from dark anoxia to light (Publication III). Herein, we demonstrate that Calvin cycle reactivation is proton gradient-dependent, most likely due to ATP requirement for carbon dioxide fixation. By measuring the PSI/PSII efficiency ratio during the re- illumination period, we point out the physiological occurrence of PSI-CEF within the first minutes of ilumination. We therefore propose a schematic model that assesses the electron flow through hydrogenase, PSI-CEF and Calvin cycle in function of the illumination period in all studied strains. Although lack of PSI-CEF does not appear to be essential for cell survival, photosynthesis reactivation is delayed in pgrl1 mutants. We also isolate a pgrl1 hydg-2 double mutant and demonstrate that the combination of both defects prevents any photosynthetic activity and strongly impairs growth. This highlights the importance for algae to keep both pathways in the course of evolution, being critical for the survival of Chlamydomonas reinhardtii in its natural environment.
Research center :
Laboratoire de génétique et physiologie des microalgues
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Godaux, Damien ;  Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Language :
English
Title :
Function of the chloroplastic hydrogenase in the microalga Chlamydomonas reinhardtii: A trvel from dark to light
Alternative titles :
[fr] Fonction de l'hydrogénase chloroplastique de la microalgue Chlamydomonas reinhardtii: De l'ombre à la lumière
Defense date :
03 October 2014
Number of pages :
146
Institution :
ULiège - Université de Liège
Degree :
Docteur en Sciences
Promotor :
Cardol, Pierre  ;  Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
Remacle, Claire  ;  Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
President :
Galleni, Moreno ;  Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
Secretary :
Franck, Fabrice ;  Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Jury member :
Dommes, Jacques ;  Université de Liège - ULiège > Département des sciences de la vie > Biologie végétale translationnelle
Morosinotto, Tomas
Rappaport, Fabrice
Funders :
FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
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since 09 February 2015

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