Chlamydomonas reinhardtii wild-type strains CC400 (mt-), 137c (mt+), CC4051 (mt+) and cgld1 mutant (strain CAL029_02_05) was obtained from the Chlamydomonas Genetics Center¹. The x32 mutant was isolated from an insertion mutant library constructed in our laboratory (Zhao et al., 2017) described in more detail below. The algal cells were cultured in TAP (Gorman and Levine, 1965) or in high-salt minimal (HSM) medium under continuous cool-white fluorescent light (60 μmol photons m^-2 s^-1) at 25°C. For all experiments, cells were grown to mid-exponential phase and harvested by centrifugation followed by adjusting the cell density to 2–4 × 10⁶ cells ml^-1. High-light stress treatment (1,300 μmol photons m^-2 s^-1) was performed according to (Zhao et al., 2013) except that the culture was transferred to flasks (25 ml) and the cell density was adjusted to 2 × 10⁶ cells ml^-1. ROS stress treatments were done as described (Chen et al., 2016). For mRNA and protein analysis, cells were harvested by centrifugation at 2500 g (4°C) for 5 min. After washing once with 0.01 M sodium phosphate buffer (pH 7.4), the cell pellets were stored at -70°C.

The insertion mutant library construction and photosynthetic mutants screen were described (Zhao et al., 2017). Briefly, the wild-type strain (CC400) was used and the mutant library was constructed by transforming this strain via the glass bead method with KpnI linearized plasmid pSI103 containing the aphVIII gene conferring paromomycin resistance (Kindle, 1990). Transformants that grew on TAP plates with 10 μg ml^-1 paromomycin (Sigma) were isolated for photosynthetic mutants screening. Mutant screening was based on chlorophyll a fluorescence measurements. Sample preparation was done as previously (Zhao et al., 2013) and the measurements were taken with a chlorophyll fluorometer (Maxi-Imaging PAM; Walz, Effeltrich, Germany) by following the manufacturer’s instructions. Among the mutants with both the lowest F_v/F_m and Y(II) values, x32 was chosen for subsequent characterization.

Oxygen evolution rate of Chlamydomonas was measured as previously described (Sun et al., 2013) with a Chlorolab-2 oxygen electrode (Hansatech, Norfolk, United Kingdom). 77K fluorescence emission spectra were measured with a fluorescence spectrophotometer (F-2500; Hitachi, Japan) as described (Yang et al., 2014) with minor changes (Zhao et al., 2017). An excitation wavelength of 435 nm (5 nm bandwidth) was used to induce chlorophyll a fluorescence. The emission spectra were recorded (600–750 nm) and normalized at 716 nm. Light-induced redox changes of P700 were monitored by measuring absorbance at 820 nm using PAM101 fluorometer equipped with a dual-wavelength P700 unit (ED800T). Sample preparation was done according to Berry et al. (2011). A far-red light illumination (FR, 720 nm, 24 μmol photons m^-2 s^-1) was provided for 45 s to enable oxidation of P700 to a steady state then turned off to monitor the initial rate of P700⁺ dark reduction according to Klughammer and Schreiber (1998).

Genetic analysis and complementation was done as described (Zhao et al., 2017) with minor modifications. For DNA blot analysis, genomic DNA was isolated from wild-type (CC400) and x32 mutant using the Plant Genomic DNA Kit by following the manufacturer’s instructions (Tiangen Biotech; Beijing, China). About 10 μg of genomic DNA was digested overnight with the restriction endonucleases KpnI and Hind III (New England Biolabs). The fragments resulting from the digestion were separated by 0.8% agarose gel electrophoresis followed by blotting onto nitrocellulose membranes and hybridizing with the DIG high prime DNA labeling and detection starter kit II from Roche (Catalog NO. 11585614910). The detection was achieved using a chemiluminescent substrate CSPD (Disodium 3-(4-methoxyspiro {l,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^3,7]decan}-4-yl) phenyl phosphate) (Catalog NO. 11 755 633 001).

For gene mapping, genomic DNA flanking the aphVIII gene was isolated using high efficiency TAIL-PCR with the specific primers listed in Supplementary Table 1 according to (Liu and Chen, 2007) with slight modifications. The primary amplification reactions (20 μL) was composed of 2 μL of PCR buffer (Takara Bio Inc, Otsu, Shiga; Japan), 200 μM of dNTPs (TransGen Biotech; Beijing, China), 1 μM of any of the LAD primers, 0.3 μM of SP0, 0.5 μL of LA Taq (Takara Bio Inc, Otsu, Shiga; Japan) and 20–30 ng of DNA. Each 25-μL secondary reaction contained 2.5 μL of PCR buffer, 200 μM each of dNTPs, 0.3 μM of AC1 and SP1, 0.5 μl of LA Taq, and 1 μL of 50-fold diluted primary product. The amplified products from the secondary reactions were analyzed by agarose gel electrophoresis and were purified prior to sequencing. Sequencing reactions were performed by Sunbiotech (Beijing Sunbiotech; China) and the data were used to search the Chlamydomonas genome.

For tetrad analysis, genetic crosses between x32 and the wild-type (137c, mt+) and zygote dissection were performed accordingly to (Harris et al., 1989). The mutant was backcrossed twice and four progeny from distinct zygotes of the second generation (T2-x32) were obtained. To complement the x32 mutant, the gene was amplified from wild-type (CC400) with primers listed in Supplementary Table 1. The amplification product was digested with NdeI and EcoRI and subcloned into similarly treated vector pDble (Fischer and Rochaix, 2001). The constructed plasmids were introduced into x32 mutant by transformation. The transformants were then analyzed by chlorophyll fluorescence measurements. Colonies that displayed a wild-type phenotype were also analyzed for the integration of x32 by PCR with the specific primers listed in Supplementary Table 1.

Cloning and heterologous expression were performed as previously described (Chen et al., 2010). Total RNA isolation/purification and reverse transcription reactions were done as described (Sun et al., 2013). Briefly, the coding region of the CGLD1 gene without transmembrane sequence was amplified by PCR with PrimeSTAR HS DNA Polymerase (Takara, Ohtsu, Japan) using specific primers Pet28-CGLD1-F/R (Supplementary Table 1). The amplified fragment was cloned directly into pEasy-blunt vector (Beijing TransGen Biotech, China), which was then transformed into competent Escherichia coli DH5α cells. Positive clones containing the recombinant plasmid were selected and sequenced to ensure the authenticity of the ORFs (Beijing Sunbiotech, China). Extraction and purification of E. coli proteins were done as described (Zhou et al., 2008). A rabbit serum was produced by MBL (MBL, Nagoya, Japan) using the purified recombinant CGLD1 protein as immunogen. Specificity of the antibody was verified by immunoblotting using proteins extracted from the E. coli and Chlamydomonas cells, respectively.

Cells breakage, isolation of membrane proteins for blue-native (BN)/SDS-PAGE and SDS-PAGE were done as described (Chen et al., 2016). BN gel was prepared and electrophoresis was performed at 4°C with the running program previously reported (Yang et al., 2014) and immunoblot analysis were done according to (Zhao et al., 2013, 2017). The antibodies against D1, D2, CP43, CP47, Cytb₆f, and AtpB were from Agrisera and those against PsaB, Lhcb4, and LhcII were from J.-D. Rochaix (University of Geneva). The dilutions for the specific antibodies used in this study were: anti-CGLD1 (1:1000), anti-CP43 and CP47 (1:3000), anti-AtpB (1:4000), anti-D1 and D2 (1:5000), anti-PsaB, Cytb₆, LhcII and Lhcb4 (1:10000). The immuno-signal was detected using the Pro-light HRP ECL detection system (Tiangen Biotech; Beijing, China). The blots were scanned using a UMAX Power-Look 2100XL scanner (Willich, Germany). Protein content was determined according to Peterson (1977) using BSA as standard.

Quantitative real-time reverse transcription-PCR (qRT-PCR) was done as described (Zhao et al., 2013) using CBLP as the internal control. Gene-specific PCR primer pairs for psbA and psbC were designed using the online program Primer3². The primer pairs previously reported for APX1, CAT1, GSTS1, GSTS2, and GPXH (Fischer et al., 2012; Pokora et al., 2017) were used and listed in Supplementary Table 1. Relative abundance was expressed as the fold change in expression level relative to the reference, calculated as 2^-ΔΔC_T.

For enzyme activity assay, crude extracts preparation and determination of SOD and CAT activity was done as described (Chen et al., 2016).

The x32 mutant was isolated during a genetic screen for Chlamydomonas insertion mutants with decreased photosynthetic activity as described previously (for details see Zhao et al., 2017). Figure 1 shows that, compared to wild-type, growth of x32 in TAP and high salt minimal (HSM) medium (Figure 1A) was severely repressed, suggesting impaired photosynthesis in the mutant. This was confirmed by the measurements of in vivo chlorophyll a fluorescence (Imaging PAM, Heinz Walz, Germany). The maximal and effective quantum yield of PSII, F_v/F_m and Y(II), was reduced to the minimal (6.6 and 3.0% of the wild-type level, respectively) (Figure 1B). In line with these, the rate of photosynthetic oxygen evolution in x32 mutant was undetectable (Figure 1C). To obtain further functional insights of photosystems in x32, low temperature fluorescence (77K) emission spectra of x32 and wild-type cells were then compared (Figure 1D). A differential reduction of the fluorescence emission peak at 686 nm, which is characteristic for PSII mutants (Össenbuhl et al., 2004), was observed in x32 (Figure 1D). This indicates the level of functional PSII in the mutant was significantly lower than wild-type. Since no difference in the light-induced redox kinetics of PSI was revealed between wild-type and the mutant (Figure 1E), we conclude that only PSII is affected in the Chlamydomonas mutant x32.

DNA blot analysis revealed a single insertion of the paromomycin resistance cassette in the genome of x32 (Figure 2A). The insertion site was mapped on the genome of Chlamydomonas via sequencing the flanking regions of the insert through thermal asymmetric interlaced PCR (Liu and Chen, 2007). Analysis of this genomic region in x32 revealed a DNA rearrangement that resulted in the deletion of five genes, i.e., Cre02.g084250, Cre02.g084300, Cre02.g084350, Cre02.g084400, and Cre02.g084450 (Figure 2B). Prediction by PredAlgo software³ shows that only the gene Cre02.g084350 encoding CGLD1 in the GreenCut2 (Karpowicz et al., 2011) is localized in the chloroplast. We therefore introduced the gene into x32 for complementation. The x32 mutant phenotype could be fully rescued in the complemented strains (C15, C19) based on growth, F_v/F_m and Y(II) values (Figure 1A). Analysis of genetic crosses between x32 mutant and wild-type was in agreement with these data (Supplementary Figures 1A,B). The photosynthetic defect of x32 co-segregated with paromomycin resistance in six complete tetrads analyzed, suggesting that the phenotype is linked to this genetic disruption.

To further confirm the candidate gene, we compared the phenotype of x32 and another cgld1 mutant obtained from the Chlamydomonas Resource Center⁴, which was derived from wild-type strain CC4051 with an insertion in exon2 of the CGLD1 gene (Dent et al., 2015; Schneider et al., 2016), in the presence of additional manganese (Mn²⁺) and calcium (Ca²⁺) (Supplementary Figure 1C). The similar results of growth profiles and F_v/F_m values of the two cgld1 mutants (Schneider et al., 2016; Supplementary Figure 1C) verify that disruption of CGLD1 is the cause of the phenotype observed in x32.

In the genome of Chlamydomonas, CGLD1 is annotated as a predicted protein (Phytozome v12.1⁵). To determine the expression of CGLD1 protein in wild-type Chlamydomonas and verify the lack of CGLD1 in x32, we generated an antibody against recombinant CGLD1 protein and verified its specificity by immunoblotting using protein extracts from the wild-type Chlamydomonas (Figure 2C). Heterologous expression and purification of the recombinant CGLD1 protein was performed as previously reported (Zhou et al., 2008; Sun et al., 2013; Zhao et al., 2017). As shown in Figure 2C, the E. coli Transetta (DE3) cells produced a substantial amount of the expected recombinant protein possessing an estimated molecular mass of 15 kDa. This corresponds to the molecular mass calculated from the coding region of Chlamydomonas CGLD1 gene without the transmembrane sequences (Figure 2C, left panel). Total proteins extracted from the E. coli cells and Chlamydomonas cells were analyzed by immunoblotting with the antibody. A single band at 28 and 15 kDa was detected in wild-type Chlamydomonas and E. coli expressing the recombinant CGLD1 protein (Figure 2C, right panel), respectively. The former corresponds to the molecular mass that was calculated from the coding sequence of CGLD1 gene in Chlamydomonas. Since no band was detected in x32 mutant (Figure 2C, right panel), this antibody was therefore used for quantification of CGLD1 in wild-type, x32 and the complemented (C15, C19) strains (Figure 2D).

The decreased PSII activity of x32 could be due to reduced accumulation of thylakoid membrane proteins. To clarify this, we analyzed membrane protein complexes of x32 and wild-type cells by blue-native (BN)/SDS-PAGE using a slightly modified protocol (Yang et al., 2014) as described (Chen et al., 2016). As shown in Figure 3A, the overall BN-gel profile of thylakoid membranes isolated from wild-type was similar to earlier reported (Dewez et al., 2009; Chen et al., 2016), demonstrating high technical reproducibility of the BN-PAGE from different experiments. Comparision of the BN-PAGE gel pattern between wild-type and x32 revealed a clear reduction of the band corresponding to a PSII supercomplex, PII1 containing core subunits of PSII (D1, D2, CP43, CP47) (Rexroth et al., 2003; Dewez et al., 2009), in the mutant (Figure 3A). This was further confirmed by immunoblotting using the antibodies specific for key proteins of photosynthetic apparatus (Figure 3B). The levels of core subunits of PSII (D1, D2, CP43, and CP47) in x32 were 30% or less of that in wild-type whereas the levels of the key subunits of PSI (PsaB), Cytb₆f (Cytb₆), and ATP synthase (AtpB) as well as the LHCII proteins (LhcII and Lhcb4) remained unchanged in x32 (Figure 3B). To test whether the reduced levels of the core PSII proteins in x32 was due to limited transcription, we then compared the psbA and psbC mRNA levels between wild-type and x32 by quantitative real-time PCR (qRT-PCR) analysis. The transcript level of both genes was decreased 67.2% (psbA) and 68.3% (psbC) in relation to the wild-type (Figure 3C). Thus, these experimental data demonstrate a significant and specific impact of CGLD1 on PSII at both mRNA and protein level in Chlamydomonas.

To determine potential physiological roles of CGLD1 protein in Chlamydomonas under adverse condition, we performed various stress experiments. Because high-light is the most common stress condition occurring to photosynthetic organisms and Chlamydomonas is a key model system for studying photo-oxidative stress in unicellular eukaryotes, we compared CGLD1 levels of wild-type cells after treatments with high-light (1,300 μmol photons m^-2 s^-1) and different ROS, i.e., H₂O₂, neutral red (NR) and rose bengal (RB). The stress treatments were done as described (Zhao et al., 2013; Chen et al., 2016) and the CGLD1 protein was monitored using immunoblot analyses. As shown in Figure 4A, the amount of CGLD1 was in most cases increased after these stress treatments, indicating that this protein is also involved in photoinhibition/photoprotection against photo-oxidative stress in the organism. To confirm these, we then investigated the sensitivity of x32 to high-light stress treatment (Figure 4B). As expected, cell growth of x32 was significantly repressed during high-light stress treatment (1,300 μmol photons m^-2 s^-1) compared to wild-type (Figure 4B). The remarkable reduction of cell growth was observed upon exposure of x32 to high light for 40 min. This was more apparent based on F_v/F_m values (Figure 4B, right panel), suggesting increased photoinhibition in the mutant.

To determine whether the tolerance of x32 mutant to oxidative stress was changed due to loss of CGLD1, we also compared the sensitivity of x32 and wild- type cells to different ROS treatments. Similar results were obtained with wild-type strain in the present (Figure 4B) and earlier investigations (Chen et al., 2016). However, the x32 mutant was more sensitive to H₂O₂ and tert-Butyl hydroperoxide (t-BOOH) than wild-type (Figure 4C, upper panel). Correlated with this, both gene expression of ascorbate peroxidase 1 (APX1) and catalase 1 (CAT1) (Supplementary Figure 2), which are known to be H₂O₂-responsive marker genes (Wakao et al., 2014), as well as the increase of activity of superoxide dismutases (SOD) and catalases (CAT), which are the dominant ROS scavenging enzymes known to be present in the chloroplasts, was less pronounced in x32 mutant than in wild-type (Supplementary Figure 3). Interestingly, our experimental data shows that x32 was more resistant to neutral red (NR) and rose bengal (RB) than wild-type (Figure 4B, lower panel). These observations raise the possibility that anti-singlet oxygen response is enhanced in x32 mutant under such stress conditions.

To test the possibility mentioned above, we measured the mRNA levels of several key genes encoding glutathione-S-transferases and thioredoxin peroxidase, i.e., GSTS and GPXH, that are known to be involved in singlet oxygen-induced acclimation process in Chlamydomonas (Leisinger et al., 2001; Fischer et al., 2005, 2009; Ledford et al., 2007) using qRT-PCR. Total mRNA isolated from different strains treated with NR or RB was subjected for the analysis. Figure 5 shows that expression of these genes was in most cases increased in all strains. The fold-increase of expression in wild-type was similar to the earlier report (Fischer et al., 2005). In x32, a differential increase pattern of expression was observed compared to that in wild-type (Figures 5A–C). Higher expression of GSTS1 and GSTS2 in x32 was most remarkable under NR (2.3-fold) and RB (8.9-fold) treatment, respectively. Based on these experimental results, we conclude that the singlet-oxygen resistance observed in the mutant could be largely attributed to the up-regulated expression of GST genes.