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The unicellular green alga Chlamydomonas reinhardtii (referred to here as Chlamydomonas throughout) has been used for years as a model organism to study a variety of cell processes, including photosynthesis, cell division and flagellar function (Rochaix, 1995; Harris, 2001a).
Hydrogen production in Chlamydomonas was reported nearly 70 yr ago, (Gaffron & Rubin, 1942) although it has only been the subject of significant attention by scientists for the past decade. (Melis et al., 2000). Its ability to synthesize hydrogen, if a way were found to harness it, could potentially revolutionize the renewable energies market (Lee et al., 2010). An advantage of hydrogen over other alternatives to fossil fuels is that the final product of its combustion is water rather than CO2, and therefore it does not negatively contribute to climate change (Lee et al., 2010).
Chlamydomonas is an aerobic organism, although it may experience hypoxia on a daily basis depending on the environment. Chlamydomonas contains two iron (Fe)-hydrogenase encoding genes, usually found with strict anaerobes, allowing hydrogen production under anoxic conditions (Happe & Kaminski, 2002; Forestier et al., 2003; Mus et al., 2007). Anoxia is required because the hydrogenase enzyme catalytic site is inactivated by oxygen and so does not function in an oxygenic environment (Ghirardi et al., 2007; Stripp et al., 2009).
Chlamydomonas must therefore be in anoxia as a precondition to activate hydrogen production (Happe et al., 1994; Ghirardi et al., 2007), but it is likely that it can survive anoxia only for limited periods. Cells may be grown in anoxia in sealed containers either in the dark, to avoid photosynthetic oxygen generation (dark anoxia), or in the light, provided that photosynthesis somehow be reduced to a level low enough so that all the oxygen it produces is consumed by cellular respiration (light anoxia) (Melis, 2007). This latter approach may be carried out by starving cells of sulfur (Melis et al., 2000). In dark anoxia all electrons for H2 synthesis come from the fermentation of organic substrates accumulated in the cell.
Under anoxia, cells potentially confront an energy crisis, as O2, their favored final electron acceptor, is not available. In such conditions, NAD(P)H deriving from respiration cannot be reoxidized and the whole process is blocked (Greenway & Gibbs, 2003). It has been proposed that H2 synthesis may act as a back-up mechanism to sequester electrons permitting NAD(P)H re-oxidation and the generation of ATP, which is essential for cell maintenance and repair functions, and ultimately for survival (Melis et al., 2000; Happe & Kaminski, 2002).
In other systems, such as Arabidopsis, acclimatization to anoxia comprises whole array of metabolic adaptations, including the induction of alcohol dehydrogenase (ADH) (Ellis et al., 1999) whose role is to reoxidize NADH produced in the glycolytic pathway (Perata & Alpi, 1993). Together, they determine the outcome: survival or death (Licausi & Perata, 2009).
The Chlamydomonas genome sequence (available online at http://genome.jgi-psf.org/Chlre4/Chlre4.home.html) contains a significant number of genes encoding proteins involved in anaerobic metabolism (Grossman et al., 2007; Merchant et al., 2007). Networks of pathways that ferment pyruvate derived from starch terminate in the production of a range of metabolites (heterofermentation) including acetate, ethanol, formate and small amounts of malate, CO2 and H2 (Kreuzberg, 1984; Mus et al., 2007). The ratio of fermentation products may change with culture conditions and the interruption of one of these main pathways may activate additional alternative pathways (Gfeller & Gibbs, 1984; Kosourov et al., 2003). A mutant (hydEF-1) defective in hydrogenase activity activates an otherwise non functioning pathway terminating in the production of succinate, seemingly to compensate for the loss of the ability to reduce protons to H2 as final electron acceptor (Posewitz et al., 2004; Dubini et al., 2009). While these fermentative pathways have been identified, some questions remain regarding the individual role and contribution of each pathway as well as factors involved in their regulation.
The ADH gene, encoding alcohol dehydrogenase, has been largely associated with the hypoxia/anoxia response in the plant kingdom, although it may also play a role in other environmental stress, including cold, dehydration and salinity (Dolferus et al., 1994; Dennis et al., 2000; Ismond et al., 2003; Senthil-Kumar et al., 2010). Most plant species upregulate ADH in response to low oxygen, although exceptions have been reported (Kennedy et al., 1992). ADH is necessary to survive flooding in Arabidopsis thaliana, as highlighted by the decreased tolerance in an ADH-null mutant, although over-expressing ADH does not increase tolerance (Ismond et al., 2003).
In Chlamydomonas ADH1 catalyses the conversion of acetyl-CoA into ethanol, and the presence of an ADH able to convert acetaldehyde into ethanol, thus resembling plant ADH, has been hypothesized (Mus et al., 2007).
In Chlamydomonas the expression of the hydrogenase genes (HYD) has similarly been studied. Two hydrogenase genes were characterized in Chlamydomonas: HYD1 and HYD2 (Happe & Kaminski, 2002; Forestier et al., 2003). They show 68% identity in their amino acid sequence (Forestier et al., 2003). Both HYD1 and HYD2 are upregulated in response to dark anoxia (Mus et al., 2007), and to light anoxia in sulfur starvation (Forestier et al., 2003). In addition, Chlamydomonas possesses two hydrogenase maturation genes, HYDEF and HYDG, whose proteins are essential for constructing the metal core present in the active site of the hydrogenase themselves (Posewitz et al., 2004).
Chlamydomonas, like most other organisms (Moore-Ede et al., 1982; Johnson & Golden, 1999) shows temporal organization of its behavioral, physiological and biochemical processes to adapt them to the 24-h cycle of its environment (Takahashi, 1991; Dunlap, 1999). For example, its division has been observed to coincide with the night period, and the survival of Chlamydomonas cells after irradiation by UV light depended heavily on the time of day the treatment was carried out (Spudich & Sager, 1980; Nikaido & Johnson, 2000).
Evolution wise, an organism may posses a competitive advantage if its cellular physiology were organized such that oxygen sensitive reactions could be restricted to times when photosynthesis does not occur (e.g. the night). In this way energy would not be wasted in synthesizing proteins that cannot be active when oxygen or light is present. (Johnson & Golden, 1999; Nikaido & Johnson, 2000). In this context, it would be logical to expect that some phases of the daily cycle are more favorable to hydrogen production than others. During the day, Chlamydomonas photosynthesizes and produces its own oxygen but at night photosynthetic oxygen production ceases and whatever oxygen is dissolved in the water may quickly be consumed. This, together with the slow rate of diffusion of oxygen (from the atmosphere in this case) in water, makes it possible that Chlamydomonas experiences a few hours of hypoxia, or even anoxia on a daily basis. An experiment performed on a synchronous culture of wild-type strain CC124 following anoxic induction by sulfur starvation demonstrated that the amount of hydrogen produced varies according to the time of day the treatment starts (Tsygankov et al., 2002).
Industrial-scale H2 production, will likely involve growing Chlamydomonas outdoors and subject to day–night rhythms. In this context it is important to take cell cycle factors into consideration in scientific studies. In this article, we demonstrate that ability to survive dark anoxia depends on the time of day treatment starts. We further show that Chlamydomonas ADH1, HYD1, HYD2, HYDEF and HYDG expression displays a day–night fluctuation pattern, which can be only partly explained by oxygen availability in the medium.
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Chlamydomonas possesses a variety of fermentative pathways, which are activated when oxygen is not available (Mus et al., 2007). The hydrogenase genes themselves, encoding the proteins responsible for hydrogen production, are part of anaerobic pathways (Mus et al., 2007).
In this work, we demonstrated that the expression of anaerobic genes fluctuates during the day–night cycles, in line with the different tolerance to low oxygen displayed by cultures exposed to anoxia at different times of the day. The most obvious explanation for a day–night fluctuation of genes reported to be induced by low oxygen (Mus et al., 2007) is related to oxygen availability. Photosynthetic O2 generation varies over the 24-h period and photosynthesis itself is reported to be under circadian regulation in higher plants (Dodd et al., 2005; Fukushima et al., 2009).
The O2 levels in the medium of synchronous cultures of Chlamydomonas were, as expected, high during the day and low at night (Fig. 2a). However, counter-intuitively, ADH1 expression was high during the day when oxygen is high (Fig. 2b, top panel), while its expression was actually lower in continuous darkness (Fig. 2b, middle panel), when no photosynthesis occurred and dissolved O2 levels were quite low (Fig. 2a).
One explanation could be that ADH1 is not hypoxia responsive in Chlamydomonas. Indeed, we did not observe ADH1 induction in response to dark anoxia (Fig. 8), and increasing oxygen availability did not inhibit ADH1 expression (Fig. 9). Upregulation of ADH1 by anoxia was observed in Chlamydomonas by Mus et al. (2007), but the different experimental set-up may explain the different conclusions reached in our work.
The function of ADH1 in Chlamydomonas, when uncoupled from its otherwise obvious role in the hypoxic metabolism, remains obscure. Ethanol production through the action of ADH recycles NADH and allows ATP production through glycolysis to continue in absence of oxygen. For this reason ADH is important for survival under anoxia (Perata & Alpi, 1993; Gibbs & Greenway, 2003). In Chlamydomonas the picture might be different, as ethanol and H2 production pathways may compete for reductants during anoxia. Following anoxia induction by sulfur starvation in the light, ethanol production is inhibited when H2 production is maximized, possibly because reductants from starch may be preferentially used by NAD(P)H-PQ oxidoreductase to fuel H2 production (Kosourov et al., 2003). The expression of ADH1 might therefore be downregulated during the night (Fig. 2b) to avoid the competition for reducing agents required for the action of hydrogenases.
Overall, the hydrogenases (HYD1 and HYD2) and hydrogenase maturation genes (HYDEF, and HYDG) (Fig. 8) displayed a pattern of expression that is consistent with hydrogen production during dark-induced anaerobiosis as reported by Mus et al. (2007).
ADH1 and HYD2 deregulation in continuous light showed that the dark phase is essential to maintain their daily pattern (Fig. 2b, bottom panel).
Deregulation of ADH1 and HYD2 in continuous light could be a direct response to light conditions, or an indirect response to a light-dependent disruption of the cell cycle. Under continuous light the circadian rhythm seemed to be maintained, as demonstrated by the tufA gene (Fig. 4), leaving the cell cycle hypothesis viable.
The green alga Ostreococcus was reported to modify its cell division rhythms in response to light conditions (Moulager et al., 2007). Interrupted cell synchrony in continuous light was also observed in Chlamydomonas (Hwang et al., 1996). Interestingly, CDKB1, a cell cycle marker gene (Bisova et al., 2005), was deregulated in continuous light, whereas it maintained a regular oscillation in continuous darkness (Fig. 6b,c). These results suggested that the regulation of ADH1 and of HYD2, could be linked, at least in part, to the cell cycle. This hypothesis certainly deserves further investigation.
In conclusion, our results demonstrated that fermentative genes are expressed following precise day–night fluctuations. The regulation of the anaerobic metabolism of Chlamydomonas can only be partly explained by responses to anoxia, but the cell cycle and light–dark cycles are equally important elements in the regulatory network modulating the anaerobic response in Chlamydomonas. Intriguingly, the regulation and metabolic role of ADH1 in Chlamydomonas is apparently not explained by our knowledge of the fermentative metabolism in higher plants and further work is warranted on this topic.