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.
