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Proc Natl Acad Sci U S A. Dec 23, 2008; 105(51): 20546–20551.
Published online Dec 12, 2008. doi:  10.1073/pnas.0806896105
PMCID: PMC2629347
Plant Biology

A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas

Abstract

In photosynthetic eukaryotes, nonphotochemical plastoquinone (PQ) reduction is important for the regulation of photosynthetic electron flow. In green microalgae where this process has been demonstrated, the chloroplastic enzyme that catalyses nonphotochemical PQ reduction has not been identified yet. Here, we show by an RNA interference (RNAi) approach that the NDA2 gene, belonging to a type II NAD(P)H dehydrogenases family in the green microalga Chlamydomonas reinhardtii, encodes a chloroplastic dehydrogenase that functions to reduce PQ nonphotochemically in this alga. Using a specific antibody, we show that the Nda2 protein is localized in chloroplasts of wild-type cells and is absent in two Nda2-RNAi cell lines. In both mutant cell lines, nonphotochemical PQ reduction is severely affected, as indicated by altered chlorophyll fluorescence transients after saturating illumination. Compared with wild type, change in light excitation distribution between photosystems (‘state transition’) upon inhibition of mitochondrial electron transport is strongly impaired in transformed cells because of inefficient PQ reduction. Furthermore, the amount of hydrogen produced by Nda2-RNAi cells under sulfur deprivation is substantially decreased compared with wild type, which supports previous assumptions that endogenous substrates serve as source of electrons for hydrogen formation. These results demonstrate the importance of Nda2 for nonphotochemical PQ reduction and associated processes in C. reinhardtii.

Keywords: hydrogen, photosynthesis

Chloroplasts of algae and higher plants originate from a cyanobacterial endosymbiont, which was originally competent for both photosynthesis and respiration, whereas mitochondria have evolved from an endosymbiotic, nonphotosynthetic bacteria. With oxidative phosphorylation being efficiently operated in the mitochondrial inner membrane, the chloroplast thylakoid has specialized in photosynthetic NADP reduction and ADP photophosphorylation to drive reductive processes necessary for photoautotrophic growth. However, chloroplasts have not entirely lost competence for oxidizing NAD(P)H at the expense of oxygen. Twenty-five years ago, Bennoun first suggested that a chloroplast-localized respiratory chain, or chlororespiration, occurs in green microalgae such as Chlamydomonas reinhardtii and Chlorella vulgaris. This was primarily based on observations that the plastoquinones (PQs) of the electron transport chain could be reduced by NAD(P)H in open-cell preparations or in isolated chloroplasts (1, 2). Chlororespiration was defined as a thylakoid electron transport pathway involving NAD(P)H:plastoquinone oxidoreductase and plastoquinol oxidase activities (1, 3).

The identification of thylakoid-bound enzymes involved in light-independent PQ reduction and oxidation awaited the development of molecular biology approaches. As it turned out, the physiological functions of these enzymes were not, or not only, related to a respiratory activity per se. Genes encoding for several subunits of a NADPH-dehydrogenase (Ndh) complex homologous to mitochondrial complex I were found in the chloroplast genome of higher plants (4, 5). This type I, chloroplastic Ndh complex is involved in nonphotochemical (PSII-independent) PQ reduction, and is part of the PSI-driven cyclic electron transport pathways together with the Cyt b6/f complex (6, 7). The chloroplastic Ndh complex is suggested to be important for the adaptation of higher plant to conditions that lead to increased ATP demand or in stress conditions (8). In the plastoquinol (PQH2)-oxidizing side of the chlororespiratory pathway, different enzymes have been suspected. The best characterized of these was first discovered in Arabidopsis thaliana and shows homology to the mitochondrial alternative oxidase (AOX) (9). This plastid terminal oxidase (PTOX) was detected also in the green microalgae C. reinhardtii in which it was suggested to function as overflow device for excess PQH2 (10).

Although the function of type I Ndh in nonphotochemical PQ reduction is well-established in higher plants, Ndh1 genes are absent from the chloroplastic genome of many green algae (11). However, the ability of green microalgae to reduce PQ nonphotochemically is well-documented. Biochemical analysis of light-independent PQ reduction activity in isolated thylakoids of C. reinhardtii (12) points to its chloroplastic Ndh belonging to a family of type II dehydrogenases. Type II NAD(P)H dehydrogenases are present in a large variety of organisms (e.g., plants, fungi, and protists) but not in animals (13). These monomeric enzymes usually contain one noncovalently bound FAD as prosthetic group and have been located on the outer side or inner side of the mitochondrial inner membrane (13). They may form multiple gene families like in higher plants where seven type II NAD(P)H dehydrogenases are present in A. thaliana (14). Six of them are exclusively targeted to mitochondria (14, 15) whereas one of them would be dually targeted, to both mitochondria and chloroplasts (13).

In green microalga, nonphotochemical PQ reduction is suggested to be involved in a variety of functionally important processes such as state transitions, cyclic electron flow and anaerobic hydrogen photoproduction.

In the process of state transitions (16), PQ reduction triggers activation of a thylakoid-associated kinase that catalyses phosphorylation of a mobile LHCII pool and of minor antenna polypeptides of PSII in appressed thylakoids (17). This weakens their association to PSII and allows their lateral diffusion to nonappressed regions of the thylakoids, where LHCII-PSI supercomplexes are then assembled. This defines the transition from state I to state II, which can be reversed in a phosphatase-dependent manner upon PQH2 reoxidation. By this process, the distribution of light-excitation energy between the two photosystems can be regulated. In green microalgae, the transition to state II is thought to favor photophosphorylation by PSI-driven cyclic electron flow at the expense of the generation of reducing power (NADPH) by PSII-dependent linear electron flow (16, 18). State transitions therefore represent a mechanism by which chloroplasts can respond to the energy status of the cell by modulating the ratio of ATP and NADPH produced in the light (19). In this regard, the putative type II chloroplastic NAD(P)H dehydrogenase represents the chloroplastic sensor of the cell energy status by its ability to modulate nonphotochemically the redox state of the PQ pool.

C. reinhardtii, like other green microalgae, evolves molecular hydrogen when illuminated under anaerobiosis (20). This is due to the anaerobiosis-induced expression of [FeFe]-hydrogenase, which catalyses proton reduction with electrons donated by ferredoxin in the chloroplast (21). In recent years, a protocol allowing sustained H2 evolution by sulfur-deprived C. reinhardtii has been developed (22). Sulfur deficiency causes a partial inhibition of PSII activity, which allows anaerobiosis to be maintained in the light because of continuous respiration. Rapid inhibition of hydrogenase by oxygen (23) is then avoided. In this condition, both photochemical (PSII-dependent) and nonphotochemical PQ reduction are thought to provide electrons for proton reduction via Cyt b6/f, PSI and hydrogenase (24, 25). Identification of the chloroplastic Ndh involved in this process is therefore important for further understanding and manipulation of the hydrogen metabolism of C. reinhardtii.

In the nuclear genome of Chlamydomonas, six sequences (NDA1, 2, 3, 5, 6, and 7) sharing similarity with type II NAD(P)H dehydrogenases have been identified (26). In this article, we identify Nda2 as a chloroplastic type II dehydrogenase responsible for nonphotochemical PQ reduction in this microalga. Furthermore, we show that inactivation of the NDA2 gene by RNA-interference leads to impairment of processes that were earlier suggested to depend on nonphotochemical PQ reduction.

Results

NDA2 Is Expressed in Mixotrophic Conditions and Knock-Down Transformants Can Be Isolated by RNAi.

We first looked at the expression of the six NAD(P)H dehydrogenase genes (NDA1, 2, 3, 5, 6, and 7). RNA blots from wild-type cells cultivated under mixotrophic conditions (light + acetate as organic carbon source) were hybridized with probes specific to each transcript. A strong signal was detected at 3.0 kb for NDA2 (Fig. 1A and Fig. S1). The size of the transcript is in good agreement with that predicted in GenBank (XM_001703591). For NDA6, a faint signal was detected at 3.8 kb, a size that is one kb larger than the size of the putative cDNA in GenBank (XM_001703003) (Fig. S1). No other signal could be seen. Because NDA2 seems well expressed in the conditions tested, we decided to suppress its expression by RNA interference to study its role. For that purpose, the cw15 mt- strain was cotransformed with pHyg3 conferring resistance to hygromycin b and pRNAi-NDA2 as described in Materials and Methods. Of 43 transformants resistant to the drug and bearing the pRNAi-NDA2 construct, two clones, Nda2-RNAi (1) and Nda2-RNAi (2), showed a complete lack of the NDA2 transcript (Fig. 1A) while the amount of COX3 transcript is shown as control of loading.

Fig. 1.
Expression and localization of NDA2 in wild-type and Nda2-RNAi transformants. (A) RNA blot analysis. Hybridization patterns were obtained with NDA2 and COX3 probes on RNA blots from wild type (WT) and Nda2-RNAi (1) and Nda2-RNAi (2) transformants. (B ...

Nda2 Protein Is Located in the Chloroplast of Wild Type and Its Amount Is Strongly Reduced or Absent in Nda2-RNAi Strains.

Immunodetection of the Nda2 protein in isolated mitochondria or chloroplast fractions was performed by using a specific antibody raised against the recombinant protein expressed in E. coli. The level of cross-contamination between the organellar fractions was assessed by using antibodies specific to each compartment (cytochrome f for chloroplast and alternative oxidase for mitochondria) and was shown to be null (Fig. 1B). In wild type, the Nda2 protein was only detected in chloroplasts. Immunodetection performed on chloroplastic fractions of Nda2-RNAi strains shows that the protein was absent or present in traces in the mutants.

Low levels or absence of detectable Nda2 protein in transformed strains had no significant effects on respiration rates in darkness. Cell pigmentation, photosystem II efficiency of dark-adapted samples, and O2 evolution in light-limited or light-saturated conditions were not affected in any significant way (Table 1). To determine the functional role of Nda2, a series of experiments were performed on the Nda2-RNAi strains comparatively to wild type.

Table 1.
Respiratory O2 uptake in the dark, chlorophyll content, and photosynthetic parameters in wild-type and Nda2-RNAi strains of C. reinhardtii grown in mixotrophic conditions

Nonphotochemical PQ Reduction After Pre-illumination Is Strongly Impaired in Nda2-RNAi Cells.

We have investigated nonphotochemical PQ reduction activity in Nda2-RNAi and wild-type strains by monitoring transient changes of PSII chlorophyll fluorescence yield under weak, modulated analytical light after actinic pre-illumination. This fluorescence yield is a sensitive indicator of the PQ redox state because of the quenching effect of their oxidized form. To lower the rate of O2-dependent PQH2 reoxidation, propyl-gallate (2 mM), an inhibitor of PTOX (27), was added before pre-illumination. In wild-type Chlamydomonas cells pre-illuminated for 5 min by actinic red light, primary quinone (Qa) reoxidation first caused an abrupt drop of the fluorescence yield at the offset of light (Fig. 2Inset). The fluorescence then showed a fluctuation with a maximum at ≈20 s, followed by a slow decline to a steady-state close to the minimal fluorescence (Fo) of the dark-adapted state (Fig. 2). The overall half-time of the fluorescence decline after the initial drop was ≈45 s. When the same experiment was performed with Nda2-RNAi strains, the slow fluorescence decline phase was completed within 1 min, lacked the transient maximum at 20 s, and had a half-time of only 10 s. In analogy to the interpretation of the postillumination fluorescence increase found in higher plants (7, 28), the shoulder observed here in wild-type cells can be interpreted as being due to a nonphotochemical PQ reduction process, which transiently overcomes PQH2 reoxidation. Absence of such fluctuation in transformed cells thus indicates that the Nda2 protein is involved in nonphotochemical PQ reduction after preillumination.

Fig. 2.
Changes in the chlorophyll fluorescence yield after a 5-min actinic preillumination (500 μmol/m2/s) in wild-type or Nda2-RNAi cells in the presence of 2 mM propyl-gallate (added in 0.5% DMSO 10 min before the experiment). Time 0 indicates the ...

NonPhotochemical PQ Reduction Mediated by Nda2 Is Essential for Efficient State Transition When Mitochondrial Oxidative Phosphorylation Is Inhibited.

Inhibitors of oxidative phosphorylation are known to induce a transition from state I to state II in C. reinhardtii (29). This effect is explained by the control of glycolysis by the ATP/ADP ratio. A decrease of this ratio stimulates glycolysis, which leads to increased NAD(P)H/NADH ratio (30). The PQ pool is then reduced via the putative dehydrogenase and a transition to state II follows. We have compared the ability of wild-type and Nda2-RNAi cells to undergo state II transition during a 30-min period of darkness after oxygen depletion by addition of the glucose oxidase/catalase system, or after addition of 10 μM myxothiazol (an inhibitor of mitochondrial complex III). For this purpose, excitation energy distribution between photosystems was evaluated by measuring low temperature (77 K) fluorescence emission spectra in different conditions. As Fig. 3A shows, the fluorescence spectra of control (aerated) samples were similar for wild-type and transformed cells, with a low fluorescence of PSI at 715 nm compared with the PSII fluorescence at 685 nm as expected for cells in state I. Under complete inhibition of oxidative phosphorylation by anaerobiosis, a strong increase of the relative PSI fluorescence intensity was found in wild-type cells because of the transition to state II. In contrast, only a modest increase was observed in transformed cells, thus indicating an inhibition of state II transition.

Fig. 3.
State II transition in Nda2-RNAi cells compared to wild-type cells. (A) Effect of 30-min anaerobiosis (glucose oxidase system) on 77 K fluorescence emission spectra of wild-type or Nda2-RNAi (1) cells. (B) Effect of myxothiazol (10 μM under aerobiosis) ...

The F715/F685 fluorescence ratio was measured to evaluate energy distribution between the two photosystems in different conditions (31). In wild type, the F715/F685 ratio increased from 0.7 to 1.2 after myxothiazol addition, and to 1.5 under anaerobiosis (Fig. 3B). In Nda2-RNAi cells this ratio only reached values of 0.8 and 1.1 in the respective conditions. These results show that Nda2 is required for efficient transition to state II when oxidative phosphorylation is either restricted (by myxothiazol) or completely inhibited (by anaerobiosis).

Measurements of fast chlorophyll fluorescence induction curves during a 1-s light pulse (1, 27) demonstrated that the inhibition of state transition in transformed cells was due to their inability to reduce PQ in the dark when oxidative phosphorylation was inhibited by myxothiazol (Fig. 3 C and D). In dark-adapted, untreated cells, the fluorescence rise shows a rapid phase because of primary QA reduction in PSII followed by a slower phase because of secondary reduction of the PQ pool as a result of multiple reaction center turnover. In wild-type cells, a 30-min incubation with 10 μM myxothiazol caused a marked increase of the relative amplitude of fast fluorescence rise phase (Fig. 3C). This shows that the PQ pool was largely reduced in the dark after myxothiazol addition. In Nda2-RNAi cells, this effect of myxothiazol was not observed: The relative amplitude of the fast phase of the fluorescence rise was only slightly increased compared with untreated cells (Fig. 3D). These results show that Nda2 is involved in nonphotochemical PQ reduction under restricted oxidative phosphorylation, which explains the inhibition of state transition in Nda2-RNAi cells in these conditions.

Sustained Hydrogen Photoevolution Under Sulfur Deficiency Is Largely Dependent on Nda2.

Sulfur-deprivation has been shown to limit PSII activity while keeping respiration unaffected in C. reinhardtii cultures. As a result, sealed cultures become anaerobic within approximately one day in the light and then start to generate hydrogen gas in a sustained manner (22). Under these conditions, electrons for proton reduction are thought to be supplied by a combination of two pathways: water photolysis because of the remaining PSII activity and nonphotochemical PQ reduction by a putative NAD(P)H dehydrogenase (24). Because Nda2-RNAi transformants showed decreased ability for nonphotochemical PQ reduction, it was of interest to check whether the transformed cells were also affected in their ability to generate hydrogen under standard sulfur-deprivation protocol. As Fig. 4 shows, sulfur-deprived Nda2-RNAi or wild-type cultures became anaerobic (O2 concentrations lower than the sensitivity limit of the electrode) after ≈12 h when placed in a closed photobioreactor. This was due to a strong decrease of PSII efficiency, which was observed in both cell lines [maximal PSII efficiency dropped to ≈0.3 (data not shown)]. In wild type, H2 gas evolution started shortly after anaerobiosis and maintained a high steady-state rate of 1.5–1.6 ml/l/h during ≈30 h, then the evolution rate progressively declined. In Nda2-RNAi 1 cultures, the rate of H2 evolution showed maximal values similar to that of wild type shortly after anaerobiosis, but declined much faster than in wild type. As a result, the total amount of H2 gas produced was in average 50% lower in transformed cells. Similar results were obtained with Nda2-RNAi 2 cultures (data not shown).

Fig. 4.
Hydrogen evolution by sulfur-deprived cells.(A) Typical time-courses of dissolved oxygen concentration (triangles) and of the amount of gas (circles) evolved during incubation of sulfur-deprived wild-type (open symbols) and Nda-RNAi (1) cells (closed ...

Discussion

Previous studies on C. reinhardtii and other green microalgae have strongly suggested the occurrence of a thylakoid-bound NAD(P)H:PQ oxidoreductase in these organisms. Yet, while such enzyme was discovered in higher plants, the microalgal enzyme remained unidentified up to now. In this work, we have shown that C. reinhardtii transformants with undetectable expression of the nuclear NDA2 gene by Northern blot are strongly impaired in their ability to reduce PQ nonphotochemically.

NDA genes code for membrane-bound, flavin-containing type II dehydrogenases, which in higher plants have been localized in mitochondria where they act as NAD(P)H:ubiquinone oxidoreductases. The NDA2 gene is predicted to have a size of 12,347 bp with 17 exons and 16 introns (26). Alignments of Nda2 from Chlamydomonas with type II NAD(P)H dehydrogenases of fungi and higher plants reveal conserved domains, for example, dual motifs for dinucleotide binding (32) (Fig. S2). These regions may form the binding for the noncovalently attached FAD cofactor or the substrate [NAD(P)H] (33). In addition, a region that could correspond to an EF-hand motif involved in calcium binding is present (32) (Fig. S2). Plant type II NAD(P)H dehydrogenases can be grouped into three classes upon phylogenetic analyses: The NDA and NDB families are closely related to fungal homologues, the NDB family possessing an inserted domain with more or less degenerate EF-hand motif and the NDC family, which clusters together with cyanobacterial proteins (14). The Nda2 protein is clearly related to fungal homologues and groups into the NDB family because of the presence of the EF-hand motif (Fig. S3). The NDA2 gene is therefore suggested to have entered the cell through the mitochondrial progenitor and later moved to the nucleus, by intracellular gene transfer (34) where it acquired a presequence specifying chloroplastic targeting. The C-terminal part of the sequence is also rather well conserved between the organisms (Fig. S2) and could form a membrane attachment domain (33).

The consequences of low Nda2 activity in RNAi mutants are in line with previous studies in which nonphotochemical PQ reduction was suggested to be part of various processes. The fluorescence transient after strong illumination is interpreted as an after-effect of cyclic electron flow, as excess NADPH generated in the light at the reducing side of PSI is used to reduce the PQ pool. Therefore, the acceleration of the postillumination fluorescence decrease in Nda2-RNAi cells suggests that the Nda2 protein is involved in cyclic electron flow.

State transitions are initiated by changes in the PQ pool redox state (16, 17). In C. reinhardtii, the latter strongly varies depending on the energy status of the cell (29). In agreement with earlier findings (18, 29) wild-type cells were found here to undergo partial state II transition after myxothiazol addition, and a complete transition under anaerobiosis. In both cases, this transition was strongly inhibited in Nda2-RNAi cells. Fluorescence induction analysis demonstrated that the failure of Nda2-RNAi cells to perform complete state transition was due to their decreased ability for nonphotochemical PQ reduction. This study shows therefore that Nda2 is essential for the PQ pool to function as sensor of the cell energy status.

Recent work on anaerobic hydrogen photoproduction by sulfur-deprived C. reinhardtii cultures has suggested that sustained H2 production results partly from the degradation of starch, which accumulates under sulfur deficiency. Under anaerobiosis, reducing equivalents arising from starch catabolism would be redirected to the photosynthetic electron transport chain where they would be used for ATP generation coupled to H2-producing electron transport (24). Consistent with this idea, mutants with a disrupted isoamylase gene display attenuated H2 photoproduction (35). Electron donation from NAD(P)H to PQ is regarded as the link between carbohydrate catabolism through glycolysis and the electron transport chain leading to H2 formation in the chloroplast. In this context, our results provide the first direct evidence that nonphotochemical PQ reduction, catalyzed by the NDA2-gene product, is important for H2 generation in sulfur-deprived C. reinhardtii cultures. Over-expressing the NDA2 gene might be promising in efforts aimed at increasing H2-photoproduction yields by microalgae in view of using this process to produce renewable fuel (36).

In conclusion, our study on Nda2-RNAi cells of C. reinhardtii demonstrates that several processes, suspected earlier to depend on nonphotochemical PQ reduction, rely strongly on the expression of the sole NDA2 gene. We therefore propose that the Nda2 protein is the chloroplastic type II dehydrogenase that catalyses electron donation from NAD(P)H to plastoquinone in green microalgae, which lack the chloroplastic type I dehydrogenase.

Materials and Methods

Strains and Growth Conditions.

Strains are derived from strain 137c of C. reinhardtii. Reference strains are cw15 mt+ (reference number 25 in our stock collection) and cw15 mt- (reference number 84), both deficient for the cell wall. Cells were routinely grown in liquid or solid agar medium under mixotrophic conditions, that is, under moderate light (70 μE/m2/s) on Tris-acetate phosphate (TAP) medium (37). When required, cells were grown under phototrophic conditions (light + minimal medium) (37). For transformation experiments, selection was performed on TAP agar medium containing 10 mg/l hygromycin B.

Construction of Plasmid for RNAi (pRNAi-NDA2).

The pPN10 plasmid (4,422 bp) used to express double-stranded RNA contains the 1.1-kb promoter of nitrate reductase (38) cloned in the ApaI and SalI restriction sites of pBCKS(+). The two cDNA fragments of NDA2 were cloned in opposite orientation following the strategy described below and sequenced. One fragment (1,859 bp) was amplified with primers NDA2–5F-EcoRV and NDA2–6R (Table S1) and digested by EcoRV and PstI (internal restriction site of the cDNA fragment). The 1,015-bp EcoRV-PstI fragment was cloned in pPN10 at the same restriction sites (pPN10-NDA2S). The other fragment (841 bp) was amplified with primers NDA2–7F-SpeI and NDA2–8R-PstI (Table S1). The pPN10-NDA2S was then digested by PstI and SpeI and the 841-bp fragment digested by the same enzymes was ligated, giving plasmid pRNAi-NDA2.

Transformation of C. reinhardtii and Selection of RNAi Clones.

Transformation of the cw15 mt- strain was made by electroporation (39) with 1 μg of pHyg3 (40) and 3 μg of pRNAi-NDA2. Transformants selected on TAP + hygromycin b were then analyzed by PCR for the presence of the 841-bp cDNA fragment of pRNAi-NDA2 with primers NDA2–5F-EcoRV and NDA2–8R-PstI. This PCR is discriminative because the genomic fragment amplified with these primers contains a 5,917-bp intron. The percentage of cotransformation was estimated to be 25%.

PCR, DNA, and RNA Analyses.

C. reinhardtii total nucleic acids were prepared as described in ref. 41. PCR fragments were amplified from total DNA, a cDNA library or directly on Chlamydomonas colonies as described in ref. 41. Sequencing of amplified products or cloned fragments was performed by Genome Express. RNA blot analyses were performed according standard protocols. Digoxigenin-labeled PCR products were used as gene probes and detected with anti-digoxigen-AP conjugates and CPD-Star as substrate (Roche). List of primers used to synthesize probes are listed in Table S1.

Mitochondria and Chloroplast Isolation.

Crude mitochondrial fraction was obtained according to (42) and loaded on a discontinuous 13%/21%/45% Percoll gradient in MET Buffer (Mannitol 280 mM, Tris·HCl pH 7 10 mM, EDTA 0.5 mM, and 0.1% BSA). Purified mitochondria were recovered at the 21/45 interface and washed two times in MET buffer by 10-min centrifugation at 11,000 × g. Final pellet was suspended in 20 mM Hepes-KOH (pH 7.2), 150 mM mannitol, 0.8 mM EDTA, and 4 mM MgCl2. Percoll-purified chloroplasts were isolated as described in ref. 43 except that a nebulizor (BioNeb, Cell disruption System, Glas-Col) was used for cell disruption.

Protein Analyses.

The protein content was determined by the Bradford method. Equal amounts (35 μg) of mitochondria and chloroplasts were loaded on 10% SDS-gels and electroblotted according to standard protocols on PVDF membranes (Amersham GE Healthcare). Immunodetection was performed by using the BM Chemiluminescence Western blotting kit (Roche) with anti-rabbit peroxidase-conjugated antibodies. The following antisera raised in rabbits were used: polyclonal antibodies raised against C. reinhardtii Aox1, Nda2, or cytochrome F proteins at dilution 1:54,000, 1:10,000, and 1:1,000, respectively.

Production of an Antibody Against the Nda2 Protein.

Total RNA was isolated from TAP cultures of C. reinhardtii with the RNeasy Plant Mini Kit (Qiagen). First-strand cDNA synthesis was performed by using OmniscriptTM Reverse Transcriptase system (Qiagen). cDNAs were amplified by PCR with Turbo Pfu polymerase enzyme (Stratagene) using primers NDA2–1Aand NDA2–1B. For cloning purpose, EcoRI and SmaI sites were respectively inserted upstream and downstream the start and the stop codons and a (His)6-tag coding sequence was added right upstream the start codon by using primers NDA2–1A–EcoRI and NDA2–1B-SmaI (Table S1) for amplification of NDA2 cDNA. The amplified fragment was digested by EcoRI and SmaI, and ligated into a digested pSD80 plasmid (44). After transformation of E. coli (strain DH10I^2) by the resultant plasmid (pSD80-NDA2) and selection on ampicillin (100 μg/ml), bacterial strains were screened by PCR by using NDA2–1A and NDA2–1B primers. Bacterial cells expressing His-tagged Nda2 were grown at 37°C in a LB medium supplemented with ampicillin to an OD600 of 0.5. After induction by 100 μM IPTG and overnight culture at room temperature, bacteria were pelleted by centrifugation and lysed by using a French press (16,000 p.s.i.) in a buffer containing 25 mM imidazole (pH 7.5), 20 mM amine-triethanol, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. The supernatant was recovered by centrifugation (12,000 × g for 20 min) and used for further purification of His-tagged Nda2 by metal-chelating chromatography. A rabbit serum was raised against the purified Nda2 protein (Agro-Bio).

Chlorophyll Fluorescence and Photosynthesis Measurements.

Post-illumination fluorescence transients were monitored by using a MFMS pulse-modulated fluorimeter (Hansatech Instruments). Analytical light was provided by light-emitting diodes at 580 nm (0.5 μmol/m2/s). Actinic light at 650 nm was provided by light-emitting diodes (25-nm half band-width). Cell suspensions were dark-adapted aerobically in minimal medium for 2 h before measurements.

Oxygen evolution was measured with a Hansatech Oxygen Electrode System using the same actinic light as above.

Low temperature (77 K) emission spectra were measured with a LS50B luminescence spectrometer (Perkin-Elmer) under 440-nm excitation light, using a Chl (a+b) concentration of 5 ± 1 μg/ml. Spectra were corrected for wavelength-dependent variations of photomultiplier sensitivity and were normalized to 685 nm. For anaerobic treatment, 40 units/ml glucose oxidase, 40 units/ml catalase and 6 mM glucose were added to the suspension.

Fast fluorescence rise kinetics associated with PQ photoreduction were recorded by using a home-made equipment. Fluorescence was measured at 685 nm under actinic excitation light at 633 nm (200 μmol/m2/s). Time resolution at the onset of light was 1 ms.

Hydrogen Evolution Measurements.

Cells were grown in TAP medium supplemented with 10 mM NaHCO3 under 250 μmol/m2/s white light, until a cell density of 9×106 cells per ml was reached. Cells were then washed and resuspended in sulfur-deplete TAP medium. After a further 24-h aerobic incubation in light, cell suspensions were transferred to a photobioreactor set-up, in which simultaneous monitoring of H2 photoproduction by wild-type and transformed cells (each in duplicate) was performed. The experimental set-up included four parallel tubular reactors, in which algal suspensions (640 ml per reactor) were continuously stirred by a moving glass ball. For this purpose, the reactors were fixed on an oscillating table. Each reactor was equipped with a home-made Clark-type electrode for continuous O2 concentration monitoring, and with a home-made gasometer for measurements of evolved gas volumes at atmospheric pressure. White light intensity was 200 μmol/m2/s and temperature was 24°C. Analysis of evolved gas samples was performed with a HP 5890 Series II chromatograph.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank M. Radoux and F. Magnette for expert technical assistance, Dr. S. Hilligsman for gas chromatography measurements, Dr. R. Matagne for critical reading of the manuscript, and Dr. S. Merchant (University of California, Los Angeles) for the kind gift of antibodies. This work was supported by Fonds National de la Recherche Scientifique (FNRS) Grants 2.4582.05 (to F.F. and C.R.), 1.5.255.08 and 2.4638.05 (to C.R.), 1.5.204.06.F (to F.F.), Fonds Spéciaux ULG (to C.R.), and Action de Recherche Concertee ARC07/12-04 (to F.F., C.R., and E.M.). The French Agence Nationale pour la Recherche (PHOTOBIOH2 project) and the European FP7-Energy-RTD program (SOLARH2 project no. 212508) are acknowledged for financial support (S.C., L.C., and G.P.). F.J. and P.A.H. are supported by Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture. P.C. and F.F. are research associate and senior research associate of the FNRS, respectively.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. K.K.N. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0806896105/DCSupplemental.

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