3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-Dibromo-6-isopropyl-3-methyl-1,4-Benzoquinone (DBMIB), 2E-Hexenal, Hydrogen peroxide (H₂O₂), Methyl Viologen (MV), Neutral red (NR), and Rose Bengal (RB) were purchased from Sigma-Aldrich.

Chlamydomonas reinhardtii wild type CC124 (137c mt-), 4A+ (137c background) and mutant CC4604- sor1 (mt+) (Fischer et al., 2012) were obtained from the Chlamydomonas Center. UVM4, a UV mutant derived from CC4350 (cw15 arg7-8 mt+) known to efficiently express nuclear transgenes (Neupert et al., 2009) was kindly provided by R. Bock (MPI for Molecular Plant Physiology, Potsdam-Golm). The MOC1 knock-out mutant stm6 was generated via random insertion of plasmid pArg7.8 (Debuchy et al., 1989), carrying the Arg7 gene, into the nuclear genome of the arginine auxotrophic strain, CC1618. The MOC1-complemented strain B13 (Schönfeld et al., 2004) was generated by co-transforming stm6 with a 37-kb Moc1-containing cosmid isolated from a cosmid library and the Cry1 gene as a dominant selectable marker conferring resistance to emetine (plasmid p613; Nelson et al., 1994). All strains were grown photoheterotrophically in TAP (tris acetate phosphate) medium (Harris, 1989) at 25°C with continuous white light of 100 μmol m^-2 s^-1. Experiments were performed by using the cells from mid-log phase. For hydrogen production, cells were harvested and washed three times with TAP-S medium. The cells were finally suspended in TAP-S to the tune of ∼25 μg/ml of chlorophyll. Hydrogen setup and gas measurement was carried out as described previously (Doebbe et al., 2010). After 20 h of anaerobic conditions, the effect of DCMU on H₂ production was assessed by adding 20 μM DCMU to the sealed bioreactors. Quantitative analysis of RES and ROS stress tolerance was evaluated by growing 2 × 10⁶ cells/ml in TAP at 100 μmol m^-2 s^-1 with following chemicals: 5 μM DBMIB, 500 μM 2E-Hexenal, 4 μM RB, 15 μM NR, 0.5 μM MV and 7 mM H₂O₂. Cell growth was determined by analyzing OD₆₈₀ and cell count (Z2 cell and particle counter, Beckman Coulter) at 0 and 24 h and 10 μl of culture was spotted on TAP agar plate for recovery.

The artificial microRNA sequence for generating IRL knock-down was designed with a web based tool WMD3¹. The amiRNA sequences were generated to target exons 2 (forward: ctagtCAGGTCCAGGAGATTGATATAtctcgctgatcggcaccatg ggggtggtggtgatcagcgcaTATAACAATCTCCTGGACCTGg; reverse: ctagcCAGGTCCAG GAGATTGTTATAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTATATCAATCTCCTGGACCTGa) and 4 (forward: ctagtGAGCACGCTATTAAGGTCGTAtctcgctgatcggcaccatgg gggtggtggtgatcagcgctaTACGGTCTTA-ATAGCGTGCTCg and reverse: ctagcGAGCACGC TATTAAGACCGTAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTACGACCTTAATAG GTGCTCa) of the coding region and cloned into vector pChlamiRNAi3int (Molnar et al., 2009). CC124 was transformed by electroporation (Jaeger et al., 2017) and stm6 was transformed via glass beads as mentioned previously (Kindle, 1990). Transformants were selected on paromomycin (10 μg/ml) TAP agar plates and transferred to sulfur depleted medium for screening.

The polyclonal antiserum directed against a 17 aa polypeptide IFR1 was raised in rabbit (Agrisera, Sweden). To heterologously express IFR1 in Escherichia coli, a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the NdeI and XhoI restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Real-time RT-PCR was performed with DNaseI (RQ1 RNase-free DNase, Promega)-digested total RNA samples which was subjected to reverse transcription and PCR amplification using the SensiFAST^TM SYBR No-ROX One-Step Kit (BIOLINE, Germany). SYBR Green I fluorescence was recorded on a DNA Engine Opticon (Bio-RAD, Germany). Per sample 100 ng total RNA were used and RPL13 (Gene ID: 5718254) as well as RACK1 (GeneID: 5723548) served as housekeeping genes. The following primers were used within the study: IFR1 (5′-ATGGCGACTAAGAAGCACAC-3′ and 5′-CGAAGCCTGCTCATTGTAGT-3′), RPL13 (5′-ATTCTTGCCGGGCAGCAGATTGTG-3′ and 5′-TTGCGCAGGAAG CGGTCATACTTC-3′) and RACK1 (5′-TCAACATCACCAGCAAGAAGG-3′ and 5′-CTGGGCATTTACAGGGAGTG-3′). Relative mRNA expression levels were calculated according to Pfaffl (Pfaffl, 2001).

Cells were pelleted (3000 × g, 3 min) and suspended in lysis buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol and freshly added 1 mM Pefabloc). Total proteins were extracted via freeze-thaw cycle in liquid N₂ and quantified by Lowry assay (BioRAD). The proteins were separated by a 12% Tris-glycine SDS-PAGE and blotted on to a nitrocellulose membrane. After overnight blocking (5% Milk powder in TBST with 0.1% Tween), the membrane was incubated at room temperature for 1.5 h with IFR1-specific antiserum (1:2500), washed and then incubated for 1 h with a peroxidase-conjugated anti-rabbit antibody (Agrisera, Sweden) for chemiluminescence detection (ECL: GE Healthcare). Signals were visualized using the FUSION-FX7 detection system (Peqlab, Germany). Protein bands were quantified with MyImageAnalysis software (ThermoFisher Scientific).

To determine the maximum quantum yield (F_v/F_m), 2 ml samples of a culture were incubated in the dark and aerated for 20 min. Chlorophyll fluorescence changes were recorded during a 10 min induction curve with actinic light (800 μmol photons m^-2 s^-1) using a Mini PAM (Waltz) and F_v/F_m calculated according to the following equation (Maxwell and Johnson, 2000):

The IFR1 coding sequence, codon-optimized for the nuclear codon bias of C. reinhardtii was cloned into vector pOpt-mVenus_Paro (Lauersen et al., 2015) by using the NdeI::BglII and EcoRV::EcoRI restriction sites to obtain C-terminal and N-terminal fusions, respectively. Fluorescence imaging was accomplished with a confocal laser scanning microscope (LSM780, Carl Zeiss GmbH, Germany) with specific filters for chlorophyll and mVenus as described before (Lauersen et al., 2015).

The significance of results was evaluated with a student’s two-tailed t-test for independent samples. The significance threshold was set between p < 0.05 to p < 0.1. Error bars represent standard error (SE) and in case of box plots the whiskers represent variability within the first and third quartile.

A previous study (Nguyen et al., 2011), demonstrated that a transcript encoding a putative isoflavone reductase (IFR1) accumulated significantly in hydrogen-producing cells of C. reinhardtii. An NCBI-BLAST search using the amino acid sequence of IFR1 (Phytozome locus name Cre11.g477200; C. reinhardtii v5.5) revealed that this protein contains a conserved phenylcoumaran benzylic ether reductase (PCBER) like domain (specific hit/e-value 1.55e^-68). PCBERs are NADPH-dependent aromatic alcohol reductases, and are described as atypical members of the short-chain dehydrogenase/reductase (SDR) family (Min et al., 2003). Atypical SDRs possess an N-terminus characteristic of NAD(P)-binding proteins and a small C-terminal domain presumed to be involved in substrate binding (Filling et al., 2002; Persson et al., 2003; Kavanagh et al., 2008). In contrast to classical SDRs, they do not have the conserved active site tyrosine residue typically found in SDRs and contain an atypical glycine-rich NADP-binding motif reading GXGXXG or G[GA]XGXXG (Supplementary Figure S1). The amino acid sequence of IFR1 shows identities to other members of the protein family in the range of 20–30% (Supplementary Table S1), with the highest similarity found for isoflavone reductases (Babiychuk et al., 1995) and IRL proteins (Petrucco et al., 1996) (Supplementary Figure S1 and Table S1).

The C. reinhardtii IFR1 gene encodes a 32 kDa protein whose localization was predicted to be cytosolic by the in silico prediction tool PredAlgo (Tardif et al., 2012). To confirm that IFR1 indeed resides in the cytosol, IFR1 was C- and N- terminally fused with YFP (mVenus variant; Kremers et al., 2006) and expressed in the C. reinhardtii cell line UVM4 (Neupert et al., 2009). Two strains, stably expressing either full length IFR1-YFP (C) or IFR1-YFP (N) were identified via immunoblots (Supplementary Figure S2; C and N). YFP fluorescence could be detected in both strains expressing YFP, either N- or C- terminally fused to IFR1 (Figure 1; N and C), while the parental control strain (PCS) only emitted red chlorophyll auto-fluorescence from the cup-shaped structure representing the chloroplast. Superimposition of the chlorophyll and YFP fluorescence demonstrated that the YFP-tagged IFR1 variants displayed a distribution of the YFP signal identical to that observed in the control strain (Cyto), expressing YFP in the cytosol (Lauersen et al., 2015). YFP-tagging of IFR1 in conjunction with confocal laser-scanning microscopy demonstrated that the localization of IFR1 is indeed cytosolic.

In a previous study (Nguyen et al., 2011) we compared the transcriptomes of the C. reinhardtii wild type (wt) cc406 and the high hydrogen production mutant stm6glc4 (Doebbe et al., 2007) [derived from stm6 (Kruse et al., 2005)] during photosynthetic hydrogen production triggered by sulfur deprivation (Melis et al., 2000). The transcriptome data revealed that within the peak hydrogen production phase, IFR1 transcripts accumulated to a high extent (∼10–40-fold compared to sulfur-replete condition) in both strains. In order to confirm that the accumulation of IFR1 transcript is indeed translated into elevated protein amounts, we analyzed protein samples taken at distinct points from a hydrogen-producing culture of a C. reinhardtii wild type (Figure 2A). A strong induction of IFR1 protein expression was observed in the wild type under S-deprived anaerobic H₂ production conditions. IFR1 accumulation started before the onset of anaerobiosis and H₂ production conditions (from 24 h onward), indicating that sulfur deprivation rather than anaerobiosis is required for IFR1 induction. An inspection of published RNAseq data sets using AlgaePath (Zheng et al., 2014) revealed that sulfur deprivation alone triggers IFR1 mRNA accumulation [∼8-fold induction 6 h after withdrawal of sulfur; González-Ballester et al., 2010; gene expression omnibus (GEO) series GSE17970]. An even stronger induction was observed within a transcriptome study analyzing the modulation of the C. reinhardtii transcriptome in response to nitrogen depletion (∼46-fold after 48 h; Miller et al., 2010; GSE24367), but IFR1 protein expression could not be detected under nitrogen-deplete conditions (data not shown), demonstrating that IFR1 accumulation is not generally observed as a response to macronutrient limitation. While effects of carbon dioxide limitation on IFR1 transcript accumulation were comparably small (∼2-fold; Fang et al., 2012; GSE33927), exposure of wild type C. reinhardtii cells to hydrogen peroxide led to a rapid accumulation of IFR1 transcript (∼19-fold within 1 h; Blaby et al., 2015; GSE34826). In addition, a previous transcriptome study indicated that IFR1 belongs to the set of genes overexpressed in the mutant singlet oxygen resistant 1 (sor1), which shows a constitutively higher expression of genes implicated in the detoxification of reactive oxygen and electrophile species [9.8 in sor1 vs. 0 in parental (4A+); Fischer et al., 2012; GSE33548]. Overexpression of IFR1 mRNA in sor1 vs. its parental strain (4A+) could be confirmed by RTqPCR experiments [median 17.8; lower quartile (Q1) 13.9; upper quartile (Q3) 25.4; IFR1 mRNA level in 4A+ set to 1; Figure 2B]. The higher mRNA level was also translated into higher IFR1 protein amounts found in sor1 (Figure 2C; 48 and 72 h; sor1 vs. 4A+). Analysis of the IFR1 promoter region led to the identification of an 8 bp palindromic motif (CAACGTTG) (Figure 2D) which was identified as an electrophile response element (ERE) in nuclear promoters of C. reinhardtii genes overexpressed in the mutant sor1 and whose expression is activated by reactive electrophile species (RES) (Fischer et al., 2012).

Indeed, treatment of C. reinhardtii WT cultures with the RES-compounds DBMIB (2,5-Dibromo-6-isopropyl-3-methyl-1,4-benzoquinone) and 2-(E)-hexenal triggered a strong accumulation of IFR1 mRNA [median fold-induction vs. control: 250.5 for DBMIB and 403.5 for 2-(E)-hexenal; Figure 2E]. In contrast to DBMIB, 2-(E)-hexenal is a RES (oxylipin) that occurs naturally in high light-stressed cells of C. reinhardtii (Roach et al., 2017) and is formed from polyunsaturated fatty acids via peroxidation and subsequent enzymatic cleavage (Mosblech et al., 2009). Addition of DBMIB to sulfur-replete cultures of a C. reinhardtii WT in the dark induced a strong accumulation of IFR1 protein (Figure 2F). However, IFR1 protein expression could not be observed (data not shown) in TAP grown cultures supplemented with DCMU [3-(3,4-Dichlorophenyl)-1,1-dimethylurea; PSII forward electron inhibitor; Metz et al., 1986], indicating that inhibition of photosynthetic electron transport by DCMU or DBMIB can be excluded and noted effects can be mainly attributed to DBMIB’s action as a reactive electrophile.

To functionally characterize IFR1 of C. reinhardtii, we applied a reverse genetics approach, employing a nuclear expression vector for the expression of artificial microRNAs (amiRNA) (Molnar et al., 2009). Screening of transformants based on immunoblots with the IFR1-specific antiserum led to the identification of two knock-down strains. When grown in S-deplete medium, IFR1_1 and IFR1_6 accumulated ∼65 and ∼95% less IFR1 protein, respectively as compared to the parental strain (Figure 3A).

Diminished accumulation of IFR1 in both knock-down strains was also observed, when cells were treated with the RES compound DBMIB (Figure 3B). In line with the strong IFR1 transcript accumulation observed after treatment with 2-(E)-hexenal (Figure 2E), addition of this compound to liquid cultures triggered a strong accumulation of IFR1 protein in the parental strain, which was diminished in knock-down strains (Figure 3C). Prompted by the finding that IFR1 protein accumulates following the exposure of C. reinhardtii cells to RES and the reported requirement of IRL proteins for oxidative stress tolerance in higher plants (Babiychuk et al., 1995; Kim et al., 2010), we analyzed the tolerance of IFR1 knock-down strains toward various compounds which either act as reactive oxygen/electrophile species (ROS/RES) or induce their cellular accumulation (Figures 3D–F). To this end, compounds inducing ROS-stress [rose bengal (RB); neutral red (NR), and methyl viologen (MV)] or acting as RES [DBMIB and 2-(E)-hexenal] were added to the cultures and the growth retarding-effect was quantified after 24 h via measurement of optical densities (oD_{680 nm/750 nm}) and cell densities based on cell counting. The cultures were also spotted on TAP agar plates for recovery (Supplementary Figure S3). Significant differences reflected by all growth parameters applied could not be observed regarding the susceptibility of knock-down strains vs. parental strain toward methyl viologen, which triggers superoxide formation in vivo (Babbs et al., 1989) (Figure 3D; MV). Although neutral red and rose bengal, which act as photosensitizers and trigger the formation of singlet oxygen in live cells (Fischer et al., 2004), exerted a greater growth-inhibiting effect on both knock-down strains (Figure 3D), differences between the parental and IFR1 knock-down strains were not statistically significant according to a two-tailed Student’s t-test (p < 0.05). In contrast, statistically robust (p < 0.05; two-tailed Student’s t-test) differences could be seen when cells were treated for 24 h with DBMIB or 2-(E)-hexenal (Figures 3E,F), which caused a more pronounced growth inhibition in the knock-down strains. At least when cell counts were used as a growth parameter, a significantly higher susceptibility of knock-down strains toward RES could already be seen 9 h after the addition of DBMIB or 2-(E)-hexenal. A diminished availability of IFR1 in C. reinhardtii therefore reduces the tolerance toward RES.

IFR1 protein accumulates strongly in hydrogen producing cultures following sulfur-depletion (Figure 2A), indicating a potential role of this protein during the acclimation to sulfur depletion or anaerobiosis. The effect of IFR1 knock-down was assessed by measuring H₂ production of the knock-down strains (Figure 4A). Parental strain, IFR1_1 and IFR1_6 were grown in sulfur-replete TAP medium to a mid-log phase and transferred to sulfur-deplete TAP medium by adjusting them to the same starting chlorophyll concentration (∼25 μg/ml). Hydrogen production was first notable 48 h after the onset of sulfur depletion and at the beginning hydrogen yields in the PCS exceeded those of the knock-down strains by ∼35–40%. During the course of H₂ production, production rates declined in the PCS from 48 h onward, while rates in the knock-down strains increased toward t_{72 h} and started declining notably beyond the time point t_{120 h} (Figure 4B). The H₂ production phase in PCS ceased at 96 h with a production phase (time between the first detection and the end of H₂ production) of 3 days as compared to 5 days by IFR1 knock-down strains. Although the highest rate of hydrogen production (2.98 ± 0.25 ml L^-1 h^-1) was reached in the PCS strain, the prolonged hydrogen production in the knock-down strains eventually led to final hydrogen yields that were about 68 ± 10% (SE) (IFR1-1) and 93 ± 12% (IFR1-6) higher than the yield from the wild type.

One of the reasons for a prolonged hydrogen production in the course of sulfur starvation, could be a high residual PSII activity, which is required for efficient hydrogen production (Volgusheva et al., 2013; Steinbeck et al., 2015). Indeed, both IFR1 knock-down strains displayed a higher residual activity of PSII, measured as the maximum quantum yield of dark-adapted cells (F_v/F_m), following the exposure to sulfur limitation under aerobic conditions (Figure 5A; -S/+O₂; Fv/Fm; t_{72-168 h}). The knock-down strain IFR1_6 was then selected for more detailed analyses regarding differences in PSII stability between knock-down and parental strain under hydrogen production conditions (sulfur deprivation under anaerobic conditions). Also during the course of hydrogen production, the knock-down of IFR1 causes an increased stability of PSII, as seen by higher F_v/F_m values from time point t_{29 h} onward (Figure 5B; -S/-O₂; left y-axis; PCS vs. IFR1_6). The lowered susceptibility of PSII toward photoinhibition in the IFR1_6 was also reflected by a lower relative decrease in the cellular chlorophyll content (∼30% vs. 50% in PCS; Figure 5B; right y-axis; green curves). In good agreement with the higher residual PSII activity found in IFR1 knock-down strains (Figures 5A,B), the cellular content of the PSII core subunit D1 declined more slowly within the course of hydrogen production in knock-down strain IFR1_6 compared to its parental strain (Figure 5C).

Specific inhibition of PSII with DCMU was used to confirm if the electrons for prolonged H₂ production indeed originated from residual PSII activity. DCMU was added directly into the H₂ bioreactors 30 h after the onset of sulfur deprivation. DCMU blocks the PSII-dependent pathway of hydrogen production based on residual water-splitting activity and linear electron transport toward the hydrogenase enzyme and inhibits H₂ production substantially, as reported before (Kruse et al., 2005; Volgusheva et al., 2007; Scoma et al., 2014). H₂ production dropped in both strains upon addition of DCMU (Figure 5D), but the relative effect of DCMU on hydrogen production was much stronger in the IFR1 knock-down strain (73% reduction in PCS vs. 163% reduction in IFR1_6). Furthermore total hydrogen production in strain IFR1_6 (16.6 ± 3.2%) was lower than the production observed for the parental strain (27.1 ± 9.4%), when DCMU was added to inhibit PSII. It can thus be concluded that the increased hydrogen production capacity caused by a knock-down of IFR1 mainly results from an enhanced activity of the PSII-dependent pathway, especially during the later stages of the hydrogen production pathway, when PSII activity in IFR1 knock-down strains exceeds the respective activity seen in the parental strain (Figures 5A–C).

To test whether the knock-down of IFR1 can be applied as a tool to improve the hydrogen production capacity in various C. reinhardtii strains, we selected the strain stm6, known to produce high amounts of hydrogen (Kruse et al., 2005; Doebbe et al., 2010; Nguyen et al., 2011). One of the created IFR1 knock-down strains, stm6_IFR1kd, displayed an IFR1 accumulation diminished to ∼20% of the IFR1 level found in the parental strain stm6 (Figure 6A; αIFR1; 1X stm6 vs. 1X stm6_IFRkd). Confirming the results obtained with knock-down strains derived from a wild type cell line (Figure 4), an IFR1 knock-down in the background of strain stm6 also had a tremendous impact on the time course of hydrogen production and the overall production capacity (Figure 6B). The onset of hydrogen production in strain stm6_IFRkd (Figure 6B; gray curve) was delayed by ∼20 h compared to the parental strain (black curve), but hydrogen production in the knock-down strain reached a plateau phase only at time point 168 h, while the parental strain reached this phase already before t_{120 h}. A prolonged hydrogen production phase together with an increased H₂ productivity rate (up to 3.07 ml⋅ L^-1⋅h^-1 at t_{72 h}), indicated by a steeper slope of the stm6_IFRkd curve, resulted in a final hydrogen yield of the knock-down strain which was 70% higher than the respective yield obtained with the parental strain.

In analogy to what has been observed for the IFR1 knock-down strains derived from a wild type, PSII activity (F_v/F_m; Figure 6C) declined more slowly in stm6_IFRkd vs. stm6 when cells were cultivated under aerobic sulfur-limiting conditions (0.48 + 0.01 in stm6_IFRkd vs. 0.35 + 0.02 in stm6 at t_{96 h}). In summary, these results demonstrate again that a diminished IFR1 level boosts hydrogen production in C. reinhardtii and that this effect is based on a sustained residual PSII activity which extends the hydrogen production phase significantly. The correlation between cellular amounts of IFR1 and hydrogen production capacity is further underscored by the diminished hydrogen yields obtained with the sor1 mutant (Supplementary Figure S4), which overexpresses IFR1 (Figures 2B,C), in comparison to its parental strain (4A+).