Greencut Protein CPLD49 of Chlamydomonas Reinhardtii Associates with Thylakoid Membranes and Is Required for Cytochrome B6f Complex Accumulation

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The Plant Journal (2018) 94, 1023–1037 doi: 10.1111/tpj.13915 GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6f complex accumulation

Tyler M. Wittkopp1,2,† , Shai Saroussi2,† , Wenqiang Yang2 , Xenie Johnson3 , Rick G. Kim1,2 , Mark L. Heinnickel2, James J. Russell1, Witchukorn Phuthong4 , Rachel M. Dent5,6 , Corey D. Broeckling7 , Graham Peers8 , Martin Lohr9 , Francis-Andre Wollman10 , Krishna K. Niyogi5,6 and Arthur R. Grossman2,* 1Department of Biology, Stanford University, Stanford, CA 94305, USA, 2Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA, 3Laboratoire de Bioenerg etique et Biotechnologie des Bacteries et Microalgues, CEA Cadarache, Saint Paul lez Durance, France, 4Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA, 5Department of Plant and Microbial Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720- 3102, USA, 6Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA, 7Proteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO 80523, USA, 8Department of Biology, Colorado State University, Fort Collins, CO 80523, USA, 9Institut fur€ Molekulare Physiologie – Pﬂanzenbiochemie, Johannes Gutenberg-Universitat,€ 55099 Mainz, Germany, and 10Institut de Biologie Physico-Chimique, CNRS-UPMC, Paris, France

Received 27 October 2017; revised 23 February 2018; accepted 6 March 2018; published online 30 March 2018. *For correspondence (e-mail [email protected]). †These authors contributed equally to this work.

SUMMARY The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The

mutant exhibits a nearly 90% reduction in the level of the cytochrome b6f complex (Cytb6f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts

Cytb6f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our ﬁndings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of

the Cytb6f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or

cofactor that inﬂuences the assembly and activity of Cytb6f.

Keywords: GreenCut, photosynthesis, cytochrome b6f, electron transport, chloroplast, thylakoid membranes, CPLD38, dehydrogenase.

INTRODUCTION Oxygenic photosynthesis involves the capture of solar absorbed light to chemical bond energy. Solar energy is energy by thylakoid membrane pigment–protein com- absorbed by light-harvesting complexes I (LHCI) and II plexes and coupled electron transfer reactions that convert (LHCII) that are associated with photosystems I and II (PSI

and PSII, respectively) reaction centers. Excited PSII reac- DAC may interact with the PetD subunit of the Cytb6f com- tion centers generate the oxidative environment that plex, the mechanism by which it impacts the levels of the + enables the splitting of water, which liberates O2,H and complex is not understood (Xiao et al., 2012). electrons. The electrons are transferred from PSII to PSI via To identify additional proteins involved in synthesis, the plastoquinone (PQ) pool, the cytochrome b6f complex assembly and stability of photosynthetic complexes, we

(Cytb6f) and the mobile electron carrier plastocyanin (PC), examined Chlamydomonas mutants disrupted in genes to ultimately reduce ferredoxin and NADP+, which function encoding various GreenCut proteins. The GreenCut is a as electron donors in anabolic metabolism. This electron bioinformatically derived protein inventory present in transport pathway is known as linear electron flow (LEF). green lineage (photosynthetic) organisms, but absent or In contrast, cyclic electron flow (CEF) involves the transfer poorly conserved in heterotrophic (non-photosynthetic) of electrons in a cycle around PSI. Although CEF does not organisms (Merchant et al., 2007; Karpowicz et al., 2011; generate NADPH, like LEF it is coupled to the establish- Heinnickel and Grossman, 2013; Wittkopp et al., 2016). ment of a transmembrane H+ gradient that drives ATP for- Many GreenCut proteins function within chloroplasts, mation by the chloroplast ATP synthase; both LEF and CEF where they are involved in diverse processes ranging from require Cytb6f. assembly of photosynthetic complexes to influencing elec- Assembly of the photosynthetic machinery involves tron transport and carbon fixation (Schult et al., 2007; coordinated synthesis of nucleus- and chloroplast-encoded Dobakov a et al., 2009; Armbruster et al., 2010; Karamoko polypeptides along with the pigment molecules with which et al., 2011; Kirst et al., 2012; Link et al., 2012; Mininno they associate (Eberhard et al., 2008; Lyska et al., 2013). et al., 2012; Xiao et al., 2012; Calderon et al., 2013; Hein- Recently, several factors necessary for assembly of PSII, nickel et al., 2013, 2016; Fristedt et al., 2014, 2015; Bhuiyan

PSI and Cytb6f were identified in cyanobacteria, algae and et al., 2015; Wittkopp et al., 2016). Mutants defective for plants (Rochaix, 2004, 2011; Schottler€ et al., 2011; Chi genes encoding GreenCut proteins have been isolated et al., 2012; Heinnickel et al., 2013, 2016; Nickelsen and using both forward (Dent et al., 2005, 2015) and reverse Rengstl, 2013; Hartings et al., 2017; Wittenberg et al., (Pootakham et al., 2010; Gonzalez-Ballester et al., 2011) 2017). For the unicellular green alga Chlamydomonas rein- genetic screens. In this work, we analyze a Chlamy- hardtii (Chlamydomonas throughout), Cytb6f assembly domonas strain that is unable to synthesize the GreenCut requires a class of proteins that facilitates integration of protein CPLD49, and demonstrate that this protein plays a heme into apocytochrome b6 and apocytochrome f (Inoue critical role in: (i) maintaining normal photosynthetic elec- et al., 1997; Xie et al., 1998; Kuras et al., 2007; Gabilly tron transport; (ii) the accumulation of the Cytb6f complex; et al., 2010; Cline et al., 2017). Many Chlamydomonas and (iii) survival during exposure of cells to HL under pho- mutants have been identified that are defective for the toautotrophic conditions. Furthermore, CPLD49 associates synthesis of Cytb6f, which involves both translational and with thylakoid membranes where it may interact with post-translational control (Kuras and Wollman, 1994; CPLD38, a GreenCut protein also critical for Cytb6f complex Choquet et al., 1998). Interestingly, regulation of transla- accumulation. tion of mRNAs for the chloroplast-encoded Cytb6f subunits operates through a process designated CES (‘controlled by RESULTS epistatic synthesis’; Wollman et al., 1999; Choquet and CPLD49 is necessary for photoautotrophic growth in HL Vallon, 2000; Choquet and Wollman, 2002; Choquet et al., 2003), in which a free subunit of the complex depresses The Chlamydomonas Cre16.g666050 locus encodes translation initiation of the mRNA encoding that subunit. A CPLD49, a GreenCut protein of 448 amino acids with a pre- similar mechanism controls the biogenesis of PSI dicted N-terminal chloroplast transit peptide (Tardiff et al., (Wostrikoff et al., 2004). 2012), two saccharopine dehydrogenase domains (March- We previously identified a thylakoid membrane protein ler-Bauer et al., 2015) and no predicted transmembrane designated CPLD38, which is necessary for proper accumu- sequences (Figure 1a). A partial characterization of the lation of Cytb6f (Heinnickel et al., 2013). The Chlamy- CAL014.01.15 mutant first suggested a critical role for domonas cpld38 mutant accumulates approximately 25% CPLD49 (Cre16.g666050) in photosynthesis (Dent et al., of wild-type (WT) levels of Cytb6f, potentially the conse- 2015). This mutant, which has an insertional disruption in quence of accelerated degradation of this complex. This Cre16.g666050, was described as acetate-requiring, sug- mutant is also sensitive to high light (HL), and has a gesting impairment in photoautotrophic growth (Dent growth defect under photoautotrophic conditions (Hein- et al., 2015). Based on sequence analysis of CAL014.01.15, nickel et al., 2013). An Arabidopsis thaliana (Arabidopsis we determined that the pMS188 cassette (used for random hereafter) mutant lacking the CPLD38 ortholog, designated mutagenesis) was inserted into chromosome 16, creating a

DAC (Defective Accumulation of Cytochrome b6f complex), 26 918 bp deletion at the insertion site that completely displayed a similar phenotype (Xiao et al., 2012). While deleted four genes and disrupted two others (Figures 1b

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Figure 1. Loss of CPLD49 in CAL014.01.15 causes compromised photoautotrophic growth in high light (HL). (a) Protein domains of CPLD49 showing the chloroplast transit peptide (cTP) and saccharopine dehydrogenase NADP binding and C-terminal saccharopine dehydrogenase domains. The number of the first and last amino acid of each domain is indicated. (b) Site of insertion of the pMS188 cassette into the Chlamydomonas genome in the cpld49 mutant (CAL014.01.15; the insertion displaced the shaded boxed region). Four gene models on chromosome 16 were completely deleted, and two others, including CPLD49 (Cre16.g666050), were truncated. The sites at which the vector caused truncation of Cre16.g665800 and Cre16.g666050 genes are shown. Additional details of the insertion are given in Figure S1(a). (c) Growth of the cpld49 mutant under photoheterotrophic conditions [Tris–Acetate–Phosphate (TAP) agar, left] and photoautotrophic conditions [Tris–Phos- phate (TP) agar, right] at different light intensities. Medium containing 105 cells was spotted onto agar plates and grown for 7 days. Growth light intensities are indicated. (d) Immunoblot of CPLD49 accumulation in wild-type (WT), cpld49 and cpld49-CPLD49. Tubulin was used as a loading control. and S1). Among the genes deleted or disrupted by the mutant did not exhibit a growth defect regardless of the insertion event in CAL014.01.15 is CPLD49 (Cre16.g666050), light intensity to which the cells were exposed [i.e. 30, which is the only gene of the disrupted cluster that 100 and 300 lmol photons m2 sec1; hereafter low light encodes a protein confirmed by proteomic analysis to be (LL), moderate light (ML) and HL, respectively; Figure 1c, chloroplast-localized (Table S1; Terashima et al., 2011). left]. To test whether cpld49 requires an external carbon DNA blot hybridizations revealed that a single pMS188 cas- source for normal growth, we simultaneously performed sette had incorporated into the genome of the mutant the spotting assay using photoautotrophic (TP) medium. (Figure S2). Importantly, the photosynthetic phenotype of Growth of cpld49 on TP under LL or ML was similar to the mutant was also observed in two independent cpld49 that of WT, while it was unable to grow photoautotrophi- mutant alleles (see below) and fully rescued by ectopic cally when exposed to HL (Figure 1c, right). Introduction expression of CPLD49; therefore, the CAL014.01.15 strain is of a WT genomic copy of CPLD49 into the cpld49 mutant hereafter referred to as the cpld49 mutant. restored photoautotrophic growth in HL on solid medium To evaluate growth and light sensitivity of cpld49,it (Figure 1c; complemented strain designated cpld49- was backcrossed three times to WT (see Experimental CPLD49), suggesting that disruption of CPLD49 in Procedures) and spotted onto Tris–Acetate–Phosphate CAL014.01.15 is responsible for the observed growth (TAP) and Tris–Phosphate (TP) agar medium. When defect on TP medium. Additionally, an antibody was cpld49 was grown heterotrophically or photoheterophi- raised to CPLD49 and immunoblots were performed cally on TAP medium (in darkness or light, respectively, using total protein extracts from WT, cpld49 and the with acetate as an external carbon source) for 7 days, the cpld49-CPLD49 rescued strain. The results confirmed that

© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 1023–1037 1026 Tyler M. Wittkopp et al. cpld49 does not accumulate CPLD49 protein (apparent for low photosynthetic yields associated with elevated light MW ~49 kDa), while cpld49-CPLD49 showed restoration levels, enabling HL growth on TAP but not TP. of CPLD49 accumulation to a level comparable to that of Loss of CPLD49 impacts photosynthetic electron transport WT cells (Figure 1d). Growth of the cpld49 mutant was also impaired in liquid The inability of the cpld49 mutant to grow photoautotroph- medium. In the presence of acetate, the cpld49 mutant ically in HL suggests that the strain is photosynthetically grew at a similar rate to that of WT and cpld49-CPLD49 in defective. To evaluate photosynthetic activity, all strains LL, achieving a slightly higher cell density in HL (Fig- were grown photoheterotrophically under LL and HL, and ure S3a, left panels). On a per Chl basis, all strains exhib- photoautotrophically in LL, and photosynthetic electron ited similar growth rates in TAP (Figure S3b, left panels). transport was assayed using Chl fluorescence induction In the absence of acetate, growth of cpld49 was indistin- kinetics (Figures 2a,b and S4a). When grown photo- guishable from WT and cpld49-CPLD49 in LL with respect heterotrophically in LL, the cpld49 mutant had a slightly to both cell density and Chl concentration (Figure S3a,b, higher level of steady-state fluorescence (F’) than WT and right panels). However, as observed on solid medium, cpld49-CPLD49 (Figure 2a), suggesting a constriction in cpld49 failed to grow photoautotrophically in HL (Fig- electron transport downstream of PSII (Baker, 2008). The F’ ure S3a,b, right panels, and c). These results suggest that level was even higher for HL, photoheterotrophically a defect in photosynthesis limits growth of cpld49, espe- grown cpld49 (relative to WT), suggesting a more pro- cially under conditions of high photon flux. An external nounced defect in electron flow (Figure 2b). Additional carbon source (i.e. acetate) may energetically compensate photosynthetic parameters derived from Chl fluorescence

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Figure 2. The cpld49 mutant is impaired in photosynthetic electron transport and deficient in Cytb6f accumulation. (a, b) Chl fluorescence measurements showing Fo,Fm values (left panels) and Chl fluorescence induction kinetics (right panels) for wild-type (WT), cpld49 and cpld49-CPLD49 cultures grown photoheterotrophically in low light (LL) and high light (HL). The LL (a) and HL (b) grown strains were dark-adapted prior to mea- 2 1 suring Fo,Fm and fluorescence induction kinetics at 156 lmol photons m sec actinic light. A saturating light pulse was administered to determine Fm and Fm’. Data were normalized to Fm values for each strain. Dark (filled) and light (unfilled) periods are indicated above each set of curves. Curves are slightly offset on x-axis. Fo, minimum fluorescence in the dark; Fo’, minimum fluorescence in the light; F’, steady-state fluorescence in the light; Fm, maximum fluorescence in the dark; Fm’, maximum fluorescence in the light. (c) Immunoblot analysis of representative photosynthetic pigment–protein complex subunits from cells grown photoheterotrophically in LL and HL. A dilution series is provided for WT grown in LL (10–100%). Tubulin was used as a loading control.

Table 1 Measurement of photosynthetic parameters after photoheterotrophic growth

Growth light Parameter WT cpld49 cpld49-CPLD49

LL Fv/Fm 0.689 0.013 0.683 0.040 0.666 0.027 ΦPSII (AL: 156) 0.479 0.007 0.405 0.046 0.473 0.004 ΦPSII (AL: 530) 0.322 0.006 0.177 0.043 0.328 0.024

HL Fv/Fm 0.539 0.038 0.571 0.007 0.576 0.024 ΦPSII (AL: 156) 0.472 0.026 0.354 0.028 0.464 0.015 ΦPSII (AL: 530) 0.352 0.003 0.129 0.013 0.348 0.006

Strains were grown in TAP medium in either LL (30 lmol photons m2 sec1) or HL (300 lmol photons m2 sec1). Assays were performed 2 1 using actinic light (AL) of either 156 or 530 lmol photons m sec .Fv/Fm and ΦPSII values were obtained from Chl ﬂuorescence measurements. Average SD values are reported for three biological replicates. HL, high light; LL, low light; WT, wild-type. measurements under photoheterotrophic conditions are photosystem stoichiometry. Hence, the loss of CPLD49 provided in Table 1. The maximum quantum yield of PSII speciﬁcally affects Cytb6f accumulation with little impact on

(Fv/Fm) is similar for all strains grown in LL and HL other major photosynthetic complexes. The reduced rate of

(Table 1), suggesting that reduced electron flow in cpld49 electron transport because of decreased Cytb6f levels in the is not the consequence of altered PSII activity, but rather a mutant is therefore likely to cause HL sensitivity under pho- block downstream of PSII. In accord with this finding, the toautotrophic conditions. Moreover, while increasing the quantum yield of PSII (ΦPSII) in photoheterotrophically incident excitation energy had a marked impact on electron grown cpld49 decreases with increasing growth light inten- transport of the mutant (Figure 2a,b), it did not appear to sity (Table 1). Importantly, these mutant phenotypes were further alter levels of Cytb6f subunits (Figure 2c). We also rescued in the cpld49-CPLD49 complemented strain (Fig- observed reduced levels of Cytb6f subunits in cpld49 grown ures 2, 3 and S4a; Table 1). photoautotrophically in LL (Figure S4b), indicating that CPLD49 is required for Cytb f accumulation regardless of cpld49 has reduced Cytb f accumulation 6 6 whether or not an external carbon source is present in the To determine whether the photosynthetic characteristics growth medium. To more firmly establish whether or not observed in cpld49 were a consequence of altered accumu- the phenotypes of CAL014.01.15 were caused by disruption lation of specific proteins or pigment–protein complexes of Cre16.g666050, encoding CPLD49 (e.g. no impact of dis- associated with photosynthetic function, we isolated total ruption of neighboring genes), we examined two additional protein from cells grown photoheterotrophically in LL and mutant lines containing insertions in Cre16.g666050 (cpld49- HL, and photoautotrophically in LL (Figures 2c and S4b). 2 and cpld49-3). These mutants also exhibited reduced

Proteins were resolved by sodium dodecyl sulfate–polyacry- levels of Cytb6f (Figure S5), conﬁrming conclusions from lamide gel electrophoresis (SDS–PAGE), and immunoblots the complementation results. performed using antibodies speciﬁc for the protein subunits Loss of CPLD49 impairs electron transport, but does not of the major photosynthetic complexes. Subunits of PSII markedly impact state transitions (PsbA or D1), the oxygen evolving complex (PsbO or OEE1),

LHCII (LHCBM2; LHCB4 or CP29), LHCI (LHCA1), the chloro- Cytb6f is a major hub for LEF and CEF, and forms a docking plast ATP synthase (AtpB or CF1-b) and Rubisco (RbcL, large site for the STT7 kinase, which is associated with redox- subunit) accumulated to near WT levels in cpld49 under dependent activation of state transitions (Gal et al., 1987; both LL and HL conditions (Figure 2c). Some reduction of Depege et al., 2003). The state transition component of PSI (PsaA) was observed in cpld49, but only in LL (Fig- non-photochemical quenching helps balance light absorp- ure 2c). In contrast, cpld49 exhibited strongly diminished tion between the photosystems and reduces redox pres- levels of Cytb6f subunits (~10% of WT levels) during growth sure on electron transport (Wollman, 2001). Therefore, we in both LL and HL, as represented by PetA (Cyt f), PetB (Cyt probed whether diminished levels of Cytb6f in cpld49 dur- b6) and PETC (Rieske iron-sulfur protein; Figure 2c). We also ing photoheterotrophic growth impacted the various activi- determined if the loss of CPLD49 had a broader impact on ties associated with this complex. As a proxy for LEF (i.e. the architecture of the photosynthetic apparatus by electron flow from water through PSII), we measured rates quantifying the PSI:PSII ratio using the single laser-flash of light-dependent O2 evolution, as well as rates of O2 electrochromic shift assay. For cells grown photo- uptake in the dark, at different light intensities for HL- heterotrophically in HL, the PSI:PSII ratio for all three strains grown WT, cpld49 and cpld49-CPLD49 cells and deter- was similar (1.73 0.17, 1.64 0.21 and 1.5 0.26 for WT, mined levels of gross O2 evolution. While all strains cpld49 and cpld49-CPLD49, respectively; t-test P-value >0.05; showed a light-dependent increase in gross O2 evolution, n = 6 for each strain), implying no significant alteration in the cpld49 mutant had significantly lower rates than those

(a) (b) ) –1 sec –1 Cyclic electron flow (electrons PSI

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Figure 3. Loss of Cytb6f restricts electron transport but does not compromise the ability of cpld49 to perform state transitions. (a) Measurements of gross photosynthetic O2 evolution. Strains were grown photoheterotrophically in high light (HL), pelleted and then resuspended in photoautotrophic [Tris–Phosphate (TP)] medium; 2 mM NaHCO3 was added to the cell suspension as an electron acceptor. The cells were maintained for 2 min at each light intensity, followed by a 2 min dark period, to generate the given rates. All O2 evolution rates were corrected for respiratory O2 uptake (see Experimen- tal Procedures). PAR, photosynthetically active radiation. Average SD shown for three biological replicates. Statistical analysis was performed using Student’s t-test. P-values indicate statistical significance between wild-type (WT) and cpld49 (black asterisks) or cpld49 and CPLD49-CPLD49 (blue asterisks). ns, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (b) Measurements of cyclic electron flow (CEF). Strains were grown photoheterotrophically under HL, pelleted and resuspended in fresh Tris–Acetate–Phosphate (TAP) medium supplemented with 10% ficoll PM400. Measurements were performed on cultures exposed to 156 lmol photons m2 sec1 actinic light, as indicated in Experimental Procedures. CEF rates are an average of four biological replicates SD. Statistical analysis was performed using Student’s t-test. P-values indicate statistical significance between the values of WT and cpld49 (black asterisks) or cpld49 and CPLD49-CPLD49 (blue asterisks). Notations are as in (a). (c) 77K fluorescence emission spectra. Strains were grown in TAP liquid medium in low light (LL) and collected during exponential growth phase. State 1 conditions: strong light (SL; 800 lmol photons m2 sec1) in the presence of DCMU. State 2 conditions: dark, anoxia. A single biological replicate is shown, although essentially identical results were obtained for three biological replicates. of WT and cpld49-CPLD49 cells at light intensities above emission measurements. Similar to WT and cpld49- 100 lmol photons m2 sec1, with an approximately two- CPLD49, the mutant was in state 1 when the cells were fold decrease at light intensities above 300 lmol pho- maintained in LL or treated with DCMU in HL (a control tons m2 sec1 (Figure 3a). These results suggest that condition that favors state 1; Figure 3c). Furthermore, when grown at light intensities below 100 lmol pho- when the cells were transferred to dark, anoxic conditions 2 1 tons m sec , decreased accumulation of Cytb6f in (control condition that induces state 2), all strains under- cpld49 does not limit LEF. However, at higher photon flux went a transition from state 1 to state 2 (Figure 3c). These densities the loss of CPLD49 impacts LEF. These results results suggest that the reduced levels of Cytb6f in cpld49 could explain the mutant’s growth defect during photoau- restrict LEF, with some reduction in CEF, but do not alter totrophic growth in HL (Figures 1c and S3). We also mea- the ability of the cells to undergo state transitions. sured rates of CEF for WT, cpld49 and cpld49-CPLD49 CPLD49 is dispensable for Cytb f transcript accumulation following growth in HL conditions. The steady-state rate of 6 CEF in cpld49 was slightly decreased relative to that in WT To determine whether CPLD49 influences expression of and cpld49-CPLD49 (t-test, P < 0.05), although this dimin- chloroplast and nucleus genes encoding subunits of ished activity was not as severe as the loss of LEF activity Cytb6f, we measured levels of these transcripts in WT,

(Figure 3b). Lastly, because Cytb6f serves as a docking site cpld49 and cpld49-CPLD49 (Figure 4). Total RNA was iso- for the STT7 kinase, we assessed the ability of cpld49 to lated from cells grown photoheterotrophically in HL and undergo state transitions based on 77K fluorescence the levels of specific transcripts quantified by reverse

© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 1023–1037 Impact of Chlamydomonas CPLD49 on photosynthesis 1029 transcriptase-quantitative polymerase chain reaction (RT- from WT cells and found that CPLD49 was present in the qPCR). Expression of three chloroplast genes (PetA, PetB thylakoid fraction, which was also highly enriched for inte- and PetD) and two nucleus genes (PETC and PETO) encod- gral thylakoid subunit PetA (Figure 6a). RbcL, a frequent ing subunits of Cytb6f was similar among the three strains contaminant of thylakoid membranes, was present at high

(Figure 4). Hence, the reduced accumulation of Cytb6f in levels in total protein lysates but not detected in thylakoid cpld49 is not the consequence of a transcriptional defect. preparations (Figure 6a). In a similar experiment in which total membrane proteins were separated from soluble pro- Cytb f remaining in cpld49 is not degraded more rapidly 6 tein by centrifugation, we observed only a small amount of than in WT CPLD49 in the soluble fraction; the distribution of CPLD49

To determine whether the reduced level of Cytb6f in cpld49 between the soluble and membrane fractions was similar was caused by accelerated protein degradation, we trans- to that of PetA (Figure 6b). ferred LL, photoheterotrophically grown cells to HL for up to Salt washes of isolated thylakoid membranes were used 24 h in the presence of chloramphenicol to inhibit chloro- to examine the association between CPLD49 and the mem- plast translation. Cells were collected at several time points branes. Puriﬁed thylakoids were treated with solutions of following administration of the inhibitor, total cellular pro- salt having a range of chaotropic strengths (Figure 6c). tein solubilized and resolved by SDS–PAGE, and the levels Treatment with the weaker chaotropic salts (NaBr, NaCl of PsbA (D1) and PetA (Cyt f) were immunologically exam- and Na2CO3) did not extract CPLD49 from the membranes, ined. This analysis revealed a similar rate of PsbA turnover while the stronger ones (NaSCN and NaOH) did (Fig- in WT, cpld49 and cpld49-CPLD49 (Figure 5a,b). Further- ure 6c). This pattern was also observed for NDA2, a thy- more, the rate of Cyt f turnover did not appear to be strongly lakoid membrane-associated NAD(P)H dehydrogenase affected by the loss of CPLD49 (Figure 5a,b), although its ini- involved in reduction of the PQ pool and in chlororespira- tial level was lower in cpld49 than in WT or cpld49-CPLD49. tion (Jans et al., 2008; Desplats et al., 2009; Saroussi et al.,

Therefore, the decreased accumulation of Cytb6f in cpld49 2016); like CPLD49, the NDA2 protein lacks predicted inte- does not appear to be a consequence of accelerated protein gral membrane domains. In contrast, the known integral degradation, but instead suggests a post-transcriptional membrane protein PsbA did not completely dissociate defect in synthesis/assembly of the complex. from the membranes even after treatment with the strong chaotropes (Figure 6c). CPLD49 is associated with thylakoid membranes CPLD49 co-migrates with CPLD38 on sucrose density CPLD49 has a predicted amino-terminal chloroplast transit gradients peptide (amino acids 1–33; Figure 1; Tardiff et al., 2012) and was experimentally demonstrated to be chloroplast- We sought to determine whether CPLD49 interacts with localized in Chlamydomonas (Terashima et al., 2011). To speciﬁc thylakoid membrane protein complexes involved validate the subcellular localization of CPLD49, we ana- in photosynthesis. Thylakoid membranes isolated from WT lyzed whole-cell lysates and puriﬁed thylakoid membranes cells grown photoheterotrophically in LL were solubilized with n-dodecyl-b-D-maltoside and solubilized complexes

108 separated on a continuous sucrose gradient. Gradient fractions were collected and proteins in these fractions 107 WT cpld49 resolved by SDS–PAGE. Immunoblot analysis revealed that 6 10 cpld49-CPLD49 CPLD49 did not co-migrate with PSI, PSII or Cytb6f (Fig- 105 ure 7a). Instead, it was present in a less dense fraction that contained free LHCs (released from PSI–LHCI or PSII–LHCII 4 10 supercomplexes as a consequence of solubilization). 103 CPLD49 was also present in the same fractions as CPLD38

102 (Figure 7a), another GreenCut protein implicated in Cytb6f

Log relative expression Log relative accumulation (Heinnickel et al., 2013), raising the possibil- 101 ity that the two proteins are components of the same com- 100 plex. PetA PetB PetD PETC PETO CPLD49 association with thylakoid membranes is

Figure 4. CPLD49 is dispensable for Cytb6f transcript accumulation. eliminated in the cpld38 mutant mRNA was collected from cells grown in high light (HL) for 24 h. Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis of To further explore the possibility that CPLD49 associates speciﬁc Cytb6f transcripts, including those from chloroplast (PetA, PetB and with CPLD38, we repeated thylakoid membrane fractiona- PetD) and nucleus (PETC, PETO) genes. Levels of individual transcripts were normalized to the housekeeping gene CBLP. Data represent average SD tion experiments using the cpld49 mutant and a mutant from three independent replicates. devoid of CPLD38 (cpld38; Heinnickel et al., 2013). When

(a) Figure 5. Loss of CPLD49 does not cause accelerated turnover of Cytb6f subunits. (a) Immunoblot assessment of the turnover of PSII

and Cytb6f subunits. Strains were grown photoheterotrophically in low light (LL), diluted to 5 lg Chl ml1 in fresh Tris–Acetate–Phosphate (TAP) medium, and transferred to high light (HL); 100 lgml1 chloramphenicol was added to inhibit chloroplast translation. The characteristic turnover of the PsbA (D1) protein is shown as a positive control. PetA was used as the representative subunit of

the Cytb6f complex. Tubulin was used as a loading control. (b) Time course of change of PsbA and PetA abundances (following addition of chloramphenicol) normalized to tubulin levels (loading control) at each time point. Data represent average SD from three (b) independent replicates.

the same separations of solubilized thylakoid membranes 2015), catalyzing the condensation of a-aminoadipate-d- from the cpld38 mutant were examined, we no longer semialdehyde and glutamate to form saccharopine, which observed thylakoid association of CPLD49, even though it is then hydrolyzed to lysine and a-ketoglutarate (Broquist was still present in the total cell extract (Figure 7b). This and Trupin, 1966). To explore whether CPLD49 has in vivo raised the possibility that maintenance of CPLD49 on thy- saccharopine dehydrogenase activity, we quantified lakoid membranes required association with CPLD38, a metabolites that would be expected to accumulate in the protein of the thylakoid membranes with two predicted absence of a functional saccharopine dehydrogenase (e.g. transmembrane domains (TMDs; Heinnickel et al., 2013), lysine, saccharopine, glutamate, a-aminoadipate), but did and can explain the finding that CPLD49 does not contain a not observe any significant differences between WT and predicted TMD and yet is not easily removed from the cpld49, except for some change in cysteine levels membranes (Figure 6c). To determine whether CPLD49 (Table S2). Therefore, CPLD49 does not likely function as a impacts thylakoid localization of CPLD38, we performed saccharopine dehydrogenase in Chlamydomonas, at least the reciprocal experiment with thylakoid membranes from under our experimental conditions. the cpld49 mutant and found that the absence of CPLD49 We detect no impact of CPLD49 loss on carotenoid did not alter the thylakoid association of CPLD38 (Fig- accumulation ure 7c). To further test a potential interaction between CPLD38 and CPLD49, we performed the yeast mating- The Arabidopsis genome encodes three putative proteins based split ubiquitin assay with these proteins and found with homology to saccharopine dehydrogenase and each that they strongly interact (Figure 7d). Overall, these has a distinct subcellular localization: AT4G33150 in the results indicate that CPLD38 and CPLD49 may be part of a cytosol; AT5G39410 in mitochondria; and AT1G50450 in novel thylakoid membrane protein complex that impacts chloroplasts. While AT1G50450 encodes the protein most accumulation of Cytb6f and possibly affects other regula- similar to Chlamydomonas CPLD49 (Tang et al., 1997; Cun- tory aspects of photosynthesis. ningham and Gantt, 2011), AT4G33150 is the only one of these related proteins for which saccharopine dehydroge- Targeted metabolomics suggest that CPLD49 is not a nase activity was experimentally demonstrated (Tang saccharopine dehydrogenase et al., 1997). Furthermore, Chlamydomonas CPLD49 and Bioinformatic analyses raised the possibility that CPLD49 is Arabidopsis AT1G50450 have homology to a dehydroge- a saccharopine dehydrogenase (Marchler-Bauer et al., nase in Adonis aestivalis that is involved in converting b-

(a) (b) (a)

Figure 6. CPLD49 strongly associates with thylakoid membranes. (a, b) Immunoblot analysis of total proteins (Total) and purified thylakoid (c) membranes (Thylakoids) (a), and total protein, membrane protein and soluble proteins (b), all purified from low light (LL), photoheterotrophically grown wild-type (WT) cells. Equal Chl loaded in each lane. (c) Immunological analysis of membrane versus soluble protein fractions from LL, photoheterotrophically grown WT cells after salt washes to assess the strength of the CPLD49 association with thylakoid membranes. Thylakoid membranes were purified from LL, photoheterotrophically grown WT cells and treated on ice for 30 min with H2O (control), 1 M NaBr, 2 M NaCl, 0.1 M Na2CO3,2M NaSCN or 0.1 M NaOH. Dissoci- ated proteins (S, supernatant) were separated from thylakoid membrane proteins (P, pellet) by low-speed centrifugation (1000 g). Immunoblot (d) analyses were performed for: CPLD49, 0 predicted transmembrane domains (TMDs); NDA2, thylakoid-associated protein involved in cyclic electron flow (CEF) and chlororespiration, 0 predicted TMDs; PsbA, D1 reaction center subunit of PSII, 5 TMDs.

carotene to the xanthophyll astaxanthin (Cunningham and Figure 7. CPLD49 co-migrates with CPLD38, but not with PSI, PSII or Cytb6f Gantt, 2011). To explore whether CPLD49 is involved in the on sucrose gradients. synthesis of a major or minor carotenoid, we made use of (a) Sucrose gradient fractionation of thylakoid membranes purified from b three highly sensitive high-performance liquid chromatog- wild-type (WT) cells grown photoheterotrophically in low light (LL). After - DM treatment, solubilized thylakoids were overlaid on a 0.1–1.3 M continu- raphy (HPLC) systems, each capable of detecting the pres- ous sucrose gradient and separated by ultracentrifugation. Fractions of ence of low-abundance pigments. All of the pigments that equal volume were collected and analyzed by sodium dodecyl sulfate–poly- – we detect in WT cells accumulated to similar levels in acrylamide gel electrophoresis (SDS PAGE) and immunoblotting. (b) Sucrose gradient fractionation of thylakoid membranes from the cpld38 cpld49 (Figure S6a–c). These results suggest that the mutant grown photoheterotrophically in LL. Total protein from WT and the defect in photosynthesis and Cytb6f accumulation is not cpld38 mutant was also probed (right). related to the absence of a specific carotenoid, although if (c) Sucrose gradient fractionation of thylakoid membranes from cpld49 grown photoheterotrophically in LL. Dashed boxes in PetA and CPLD38 it is an abundant carotenoid of which only one molecule immunoblots represent fractions 5–17 that were probed separately. Total localizes to the Cytb6f complex in WT cells but not in the protein from WT and the cpld49 mutant was also examined (right). mutant, we would likely not be able to detect the change. (d) Interaction between CPLD49 and CPLD38 using the yeast mating-based split ubiquitin system (Grefen et al., 2009). CubMETYC/NubG, negative con- We were also unable to detect a change in the pigment trol; CubCPLD38/NubI and CubCPLD38/NubWT, positive controls for strong profile of the cpld38 mutant relative to WT cells interactions; CubCPLD38/NubG, control for weak interactions; CubCPLD38/ (Figure S6a–c). NubCPLD49, for specific interactions. Permissive medium, diploid plates. Selective medium, MET0 plates. DISCUSSION

The assembly and maintenance of photosynthetic pig- protein in the GreenCut, in the biogenesis of the photosyn- ment–protein complexes are highly regulated, and must be thetic apparatus. A strain devoid of CPLD49 is unable to coordinated with cellular and environmental cues. Here, we grow without a ﬁxed carbon source when exposed to HL. describe the involvement of CPLD49, a highly conserved Moreover, CPLD49 associates with thylakoid membranes,

© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 1023–1037 1032 Tyler M. Wittkopp et al. and has a critical role in maintaining photosynthetic activity to interact with subunit IV (chloroplast petD gene) of the and accumulation of the Cytb6f complex. complex (Xiao et al., 2012). We did not ﬁnd evidence sug-

A loss of nearly 90% of the Cytb6f complex is evident in gesting that either CPLD38 or CPLD49 interacts with Cytb6f cpld49 under both LL and HL (Figures 2c, S4 and S5). The in Chlamydomonas, though we cannot rule out the possi- complex remaining in the mutant transfers electrons (Fig- bility that these proteins interact in a transient manner ures 2a,b and 3a,b), supports state transitions (Figure 3c), (e.g. during assembly of the complex) or that this interac- and sustains photoautotrophic growth under LL and ML tion is disrupted by the procedures employed for puriﬁca- (Figures 1c and S3), suggesting that the complex that tion and solubilization of thylakoid membranes. assembles in cpld49 is functional. The observation that Among green lineage organisms, the closest homologs state transitions are not perturbed in cpld49 is reasonable of CPLD49 are annotated as saccharopine dehydrogenases. as previous work has shown an approximate stoichiometry In plants and animals, saccharopine dehydrogenases cat- of 1 STT7 kinase per 20 Cytb6f complexes (Lemeille et al., alyze the synthesis of saccharopine from a-ketoglutarate 2009). Growth of cpld49 was not impaired when the cells and L-lysine (Serrano et al., 2012). This reaction may be were illuminated with LL or ML under photoheterotrophic critical for preventing accumulation of toxic levels of free and photoautotrophic growth conditions (Figures 1c and lysine in plants (Arruda et al., 2000). In contrast, fungi use S3). Similarly, others have demonstrated that codon initia- saccharopine dehydrogenase to synthesize lysine (Serrano tion mutants of the chloroplast petD gene accumulate et al., 2012). While Chlamydomonas may have multiple

10–20% of Cytb6f, but are still able to grow photoau- pathways for lysine synthesis (Vallon and Spalding, 2009), totrophically in LL (Chen et al., 1993). Moreover, LEF was based on our metabolite analysis, we were unable to not impaired when HL-grown cpld49 was illuminated with establish a role for CPLD49 in either lysine degradation or LL (Figure 3a). However, when the growth light was synthesis (Table S2). increased above ~250 lmol photons m2 sec1, the capac- CPLD49 is also related to an enzyme (carotenoid ity to evolve O2 in HL was almost twofold lower in cpld49 4-hydroxy-b-ring 4-dehydrogenase, HBFD) involved in than in WT and cpld49-CPLD49 (Figure 3a), suggesting that astaxanthin biosynthesis, a carotenoid found at high levels the decreased levels of Cytb6f in the mutant are insufficient in the petals of some species of the genus Adonis (Cun- to drive high rates of electron transfer. Interestingly, CEF ningham and Gantt, 2011). HBFD in A. aestivalis is rates under similar conditions were only 25–30% lower involved in the biosynthesis of astaxanthin from b-carotene than those of the WT and cpld49-CPLD49 (Figure 3b). (Cunningham and Gantt, 2011). Astaxanthin and other Therefore, it appears that under elevated light conditions ketocarotenoids accumulate in Chlamydomonas zygos- and in the presence of acetate, the Cytb6f complex in the pores where they may act as antioxidants (Lohr, 2009), but cpld49 mutant can support CEF more efficiently than LEF, have not been identified in vegetative cells. Interestingly, which may afford future opportunities to investigate elec- Cytb6f does bind pigments; each Cytb6f monomer binds tron partitioning between the two pathways. both a Chl a and a carotenoid molecule, probably b-caro- Loss of the GreenCut protein CPLD38 in Chlamy- tene (Zhang et al., 1999; Kurisu et al., 2003; Stroebel et al., domonas resulted in phenotypes similar to those of 2003). Based on crystal structures, the carotenoid within cpld49, including defects in accumulation of Cytb6f and the Cytb6f complex is not close enough to the Chl to partic- compromised photoautotrophic growth. However, unlike ipate in triplet quenching and, thus, the roles of these pig- cpld49, the turnover rate of Cytb6f subunits in cpld38 ments remain enigmatic (Dashdorj et al., 2005). appeared more rapid than in WT (Heinnickel et al., 2013). A Furthermore, in certain cyanobacterial genomes the puta- specific function for CPLD38 has not yet been determined. tive ortholog of CPLD49 is contiguous to and probably in However, we demonstrate here that CPLD38 associates the same operon as a gene involved in carotenoid biosyn- with thylakoid membranes and co-migrates with CPLD49 thesis. This gene (crtD) encodes a carotenoid dehydroge-

(but not with Cytb6f) when purified thylakoid membranes nase related to the carotenoid isomerase CRTISO from are solubilized and the membrane complexes resolved by green algae and land plants (Grossman et al., 2004; Nisar sucrose gradient centrifugation (Figure 7a). Furthermore, et al., 2015), that in tomato is involved in carotenoid iso- in the cpld38 mutant, CPLD49 is not associated with thy- merization through a reversible redox reaction (Isaacson lakoids, suggesting that CPLD38 and CPLD49 may physi- et al., 2002, 2004). These links between CPLD49 and carote- cally interact (Figure 7b). This possibility was supported by noid biosynthesis raise the possibility that CPLD49 is the finding that CPLD49 and CPLD38 positively interact in involved in modifying the carotenoid associated with the yeast mating-based split ubiquitin assay (Figure 7d). Cytb6f, which could account for reduced accumulation of Future biochemical/genetic and structural studies will help this complex in the mutant. However, we did not observe clarify specific aspects of these and other potential interac- differences in the abundances of either the major or minor tions. In Arabidopsis, the ortholog of CPLD38 (DAC) was pigments of cpld49 relative to WT cells (Figure S6a–c), also shown to be necessary for accumulation of Cytb6f and making it unclear whether or not CPLD49 functions in the

© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 1023–1037 Impact of Chlamydomonas CPLD49 on photosynthesis 1033 synthesis of a carotenoid. If it is involved in the synthesis GreenCut protein CPLD38 may act as a scaffold on thy- of a minor carotenoid that co-migrates with other pig- lakoid membranes that brings CPLD49 into close proximity ments, it may not be detected in the chromatographic and to the Cytb6f complex. Although still speculative, CPLD38 spectral analyses used. Furthermore, it is possible that and CPLD49 may comprise a novel thylakoid complex CPLD49 is involved in the synthesis or modiﬁcation of involved in the synthesis/maturation of thylakoid lipophilic another class of chloroplast lipophilic compounds, such as molecules and their incorporation into photosynthetic pig- the lipid/quinone constituents of thylakoids that associate ment–protein complexes. Additional information on the with Cytb6f. Alternatively, CPLD49 may have a role in the catalytic activity of these proteins is necessary to develop a synthesis of cofactors needed for the biogenesis of Cytb6f. better understanding of their precise roles in maintenance

The assembly, structural stability and biological activity of photosynthetic function and Cytb6f accumulation. of membrane protein complexes depend on proper integration of lipids. Cytb6f from Chlamydomonas has been EXPERIMENTAL PROCEDURES successfully purified with its native lipids, revealing the Chlamydomonas strains and culture conditions presence of an associated acidic sulfoquinovosyldiacyl- glycerol (SQDG) molecule (Stroebel et al., 2003). Interest- A C. reinhardtii mutant with an insertion in the CPLD49 gene ingly, the sulfonate group of SQDG interacts with Lys272 of (Cre16.g666050) was generated by random insertional mutagene- + Cyt f, and Arg13 and Asn17 of the PETC Rieske iron-sulfur sis of the WT 4A strain (CC-4051; Dent et al., 2015). This mutant, 272 CAL014.01.15 (cpld49 throughout), was first identified in a forward subunit (Stroebel et al., 2003). Lys was previously genetic screen for strains affected in photosynthesis (Dent et al., shown to be a critical residue in controlling Cyt f synthesis, 2015). The insertion site in cpld49 was confirmed by PCR using 50 and its assembly and stability (Choquet et al., 2003; de and 30 border primers (Figure S1; Table S3). The cpld49 mutant Vitry et al., 2004). It was suggested that an extensive lipid- was backcrossed three times to 21gr mating type +/ (CC-1690 based network is required for the flexibility of the PETC and CC-5119, respectively), and all experiments were conducted with 21gr+ as the WT genetic background. The ability of the subunit, which is critical for the transfer of electrons from mutant to grow using nitrate as a sole N source was confirmed the PQ pool to Cyt f (Hasan et al., 2013; Hasan and Cramer, after each backcross, which mitigated artifacts related to muta- 2014). Cytb6f in Chlamydomonas also contains two lumi- tions in the NIT2 locus (Wei et al., 2014; Saroussi et al., 2016). For nal-side lipids, potentially monogalactosyldiacylglycerol complementation, the WT Cre16.g666050 (CPLD49) gene, includ- molecules (Stroebel et al., 2003). Furthermore, computa- ing 1000 bp upstream (to obtain the native promoter) and 500 bp downstream (to obtain the native terminator), was amplified from tional modeling approaches have implicated other lipids, WT genomic DNA. The PCR product was cloned into pSL18 (con- such as diacylglycerol in the structure of Cytb6f (Hasan and ferring resistance to paromomycin) and the linearized plasmid Cramer, 2014). The 2.5 A crystal structure of Cytb6f from used to transform the cpld49 mutant. Transformants were the cyanobacterium Nostoc PCC 7120 revealed the pres- selected on TAP agar containing 6 lgml 1 paromomycin (Fig- ence of at least 23 lipid-binding sites, which are thought to ure S1c). The cpld49 mutant (CC-5401), cpld49-CPLD49 complemented strain (CC-5402) and the cpld38 mutant (CC-5403) are have a number of critical roles in electron transfer, com- available from the Chlamydomonas Resource Center (www.chla plex and supercomplex formation, complex dimerization my.org). Two additional mutants disrupted in Cre16.g666050 were and facilitation of interactions with Chl a and carotenoid isolated in a large-scale mutant library (https://www.chlamycol molecules (Hasan and Cramer, 2014). These results high- lection.org/products/clip-strains/). These strains are referred to as light a critical and as yet poorly understood role for lipids cpld49-2 and cpld49-3, and are described in more detail in Figure S5. in the assembly and activity of Cytb6f, and suggest that certain enzymatic reactions involving oxidation–reduction, Growth on solid and in liquid media such as that predicted for CPLD49, may be critical for creat- To assess growth on solid medium, strains were first grown in ing the proper lipid context to sustain the activity and sta- TAP (Harris, 1989) liquid medium to mid-late exponential phase bility of macromolecular protein complexes integral to the (4–8 9 106 cells ml1) and subcultured for 24 h to maintain loga- membranes. On the other hand, short-chain dehydroge- rithmic growth. Cell densities were determined using a hemocy- nases like CPLD49 may impact photosynthetic complexes tomer or a Beckman Coulter Counter (results were consistent indirectly through the control of various functions that between the two methods) and collected by centrifugation at 3220 g for 10 min at 20°C. Cell pellets were resuspended in TP liq- occur in chloroplasts, such as translation initiation (Link uid medium (same as TAP, but lacking acetate, buffered with HCl), et al., 2012). 5 ll drops containing 1 9 105 cells were spotted onto either solid TP or TAP medium, and growth was assessed after 7 days. For Conclusions growth in liquid medium, the strains were inoculated from plates, – Overall, we have demonstrated a critical role for CPLD49, a allowed to reach mid late exponential phase and then subcultured for 24 h. Cells were centrifuged as above and resuspended in conserved GreenCut protein, in photoautotrophic growth fresh TAP and TP medium to either a density of 5 1 1 and accumulation of Cytb6f. Recent studies on CPLD38 and ~5 9 10 cells ml or a Chl concentration of ~1 lgml . For its Arabidopsis homolog (Xiao et al., 2012; Heinnickel growth curves, cells were counted using a Beckman Coulter Coun- et al., 2013), together with our results, suggest that the ter or a Countess automated cell counter (Invitrogen). Chl was

© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 1023–1037 1034 Tyler M. Wittkopp et al. determined as previously described following extraction in 100% using a Fluorolog-3 Fluorometer (Horiba Jobin-Yvon). Excitation methanol (Porra et al., 1989; Yang et al., 2014). of the sample was at 440 nm using a 4 nm excitation slit width; the emission was measured from 650 to 800 nm using a 2 nm Chlorophyll fluorescence emission slit width. Cells were grown in TAP at 30 (LL) or 300 lmol pho- RNA extraction and quantification of Cytb6f transcripts tons m2 sec1 (HL) or in TP at LL for 24 h, collected by centrifugation (as above), resuspended in fresh medium to a Cells were grown in TAP for 24 h under HL, concentrated by cen- concentration of 10 lg Chl ml1 and dark-adapted with vigorous trifugation (3000 g, 2 min, 4°C), and RNA was extracted from the shaking for at least 20 min. Chl fluorescence transient induction cells (equivalent to 40 lg Chl) using Direct-zol RNA MiniPrep Plus kit kinetics were measured using a JTS10 spectrophotometer (Zymo Research) according to the manufacturer’s instructions. To

(Biologic); 1 mM NaHCO3 was added as an electron acceptor. Fo, analyze the relative abundance of PetA, PetB, PetD, PETC and PETO minimum fluorescence; Fm, maximum fluorescence in the transcripts, 1 lg of RNA was reverse-transcribed using the iScript dark-adapted state; Fm’, maximum fluorescence at a given actinic cDNA synthesis kit (BioRad). Gene-specific PCR primers with a Tm light intensity; F’, fluorescence at a given actinic light intensity; Fv, of ~60°C, GC content of ~50%, and PCR amplicon length that ranged variable fluorescence. The maximum quantum yield of PSII was from 125 to 200 bp were manually designed (Table S3). The effi- measured in dark-adapted cultures grown in TAP, and is calcu- ciency of each primer pair was verified to be better than 98%. RT- lated as Fv/Fm = (Fm Fo)/Fm. The quantum yield of PSII was mea- qPCR was performed using a Lightcycler 480 (Roche). Reactions sured at different actinic light intensities and is calculated as were performed in a final volume of 20 ll containing 10 llSensiFast 0 0 0 ΦPSII = (Fm F )/Fm . SYBR mix (Bioline), 4 nM of each primer and 0.05 lg of cDNA. PCR conditions were as follows: 3 min at 95°C followed by 40 cycles of Photosynthetic oxygen evolution 10 sec at 95°C and 30 sec at 60°C. Fluorescence threshold (Ct) was analyzed using Lightcycler 480 SW version 1.5 (Roche). Relative O evolution and consumption were measured using a Clark-type 2 expression levels for all transcripts were compared with the CBLP electrode (Hansatech) at 20°C. Briefly, cells were grown photo- housekeeping gene under the same conditions. The values reported heterotrophically in HL for 24 h and collected as described above. for the expression data represent an average of three biological Prior to the measurements, cells were resuspended in photoau- replicates SD according to Pfaffl (2001). totrophic (TP) medium to a concentration of 10 lg Chl ml1. Cells (2 ml, totaling 20 lg Chl) were exposed to increasing light intensities (2 min for each intensity); each intensity was followed by a Protein isolation and immunoblot analysis dark period. O2 evolution and uptake were calculated using the Total protein was extracted from cultures during mid-exponential slope values of the light and dark periods, respectively. Gross growth phase by resuspending cells in 3 mM HEPES-KOH, pH 7.5, rates of photosynthetic O2 evolution (shown as lmol O2 mg 1 1 60 mM dithiothreitol, 60 mM Na2CO3, 12% sucrose, 2% SDS, pro- Chl h ) were obtained by subtracting the O2 uptake rate in the tease inhibitors, and boiling the sample for 50 sec. Samples were dark (negative values) from the rate of O2 evolution at the corre- normalized to Chl content and proteins separated by SDS–PAGE sponding light intensity. Three biological replicates were used for on 10% polyacrylamide gels. Resolved proteins were transferred each measurement. to PVDF membranes using a semidry blotting apparatus (Bio- Spectroscopic analysis and CEF Rad). For immunoblotting, membranes were blocked in TBS containing 0.1% Tween (TBST) and 5% milk for 1 h at 20°C. Primary

Absorbance changes associated with P700 oxidation–reduction antibodies were diluted in TBST 3% milk, and incubations of the were performed using a JTS10 spectrophotometer (Alric et al., membranes with the antibody solutions were performed over- 2010; Wittkopp et al., 2017). Cells were pelleted by centrifugation night at 4°C. CPLD49 and CPLD38 antibodies were generated from (1200 g, 5 min, 20°C) and resuspended to a concentration of 25– oligopeptides CKTQPGVWYPEEKEALQDRRQ-amide and CDAD 30 lgml1 Chl in fresh medium supplemented with 10% ficoll KQRKAAARAAAQQQQ-amide, respectively (Yenzym). All other PM400 (GE Healthcare). Dark-adapted cells were exposed to antibodies were obtained from Agrisera, with the exception of Cyt 156 lmol photons m2 sec1 during PSI oxidation followed by a f (from Francis-Andre Wollman) and NDA2 (from Xenie Johnson). saturating pulse, and a re-reduction period in the dark. Redox Proteins were detected by enhanced chemiluminescence. Protein changes for P700 were monitored at 705 nm. Absorption changes abundances were quantified using ImageJ (N.I.H.). measured at 740 nm were subtracted to correct for unspecific con- Isolation of thylakoid membranes tributions to the 705 nm measurements; 10 lM DCMU and 1 mM hydroxylamine were added to the suspension to inhibit LEF prior Cultures were grown as described above, and thylakoid mem- to the measurement. The electrochromic shift following a laser branes purified according to Takahashi et al. (2006). Briefly, l 2 1 flash or 4 ms continuous light (156 mol photons m sec ) was 250 ml of cells in mid-exponential growth phase was pelleted by monitored using a 520 nm filter on the same samples as centrifugation (4225 g, 10 min, 4°C), resuspended in Buffer 1 described above. CEF was calculated according to Takahashi et al. (25 mM HEPES-KOH, pH 7.5, 5 mM MgCl , 0.3 M sucrose, 0.2 mM (2013). The ratio between PSI and PSII was estimated by measur- 2 PMSF, 1 mM benzamidine, 5 mM e-amino-a-caproic acid) and bro- ing the ‘a’ phase of an electrochromic shift using a single turnover ken using a French Pressure Cell at 500 PSI. Membranes were sep- laser flash at 520 nm, both in the presence and absence of 10 lM arated from the soluble phase of the cell by centrifugation at DCMU and 1 mM hydroxylamine (Joliot and Delosme, 1974). 2316 g (10 min, 4°C), and then resuspended and homogenized in 77K fluorescence Buffer 2 (5 mM HEPES-KOH, pH 7.5, 10 mM EDTA, 0.3 M sucrose, protease inhibitors as above) and centrifuged at 68 588 g (20 min, Aliquots of cells were collected and adjusted to 0.1 0.01 absor- 4°C). The resulting pellet was resuspended in 5 ml Buffer 3 (5 mM bance at 440 nm by dilution in TAP medium before freezing in liq- HEPES-KOH, pH 7.5, 10 mM EDTA, 1.8 M sucrose, protease inhibi- uid N2. Fluorescence emission was measured on frozen material tors as above), and overlaid with 2 ml Buffer 4 (same as solution

3, except 1.3 M sucrose) and then 5 ml Buffer 5 (5 mM HEPES- Sciences of the National Institutes of Health under award number KOH, pH 7.5, 0.5 M sucrose). Thylakoid membranes were sepa- T32GM007276. S.S. was supported by a Vaadia-BARD Postdoc- rated from unbroken cells, plasma membrane and chloroplast toral Fellowship (United States-Israel Binational Agricultural envelope by sucrose step-gradient ultracentrifugation (247 605 g, Research and Development Fund, grant number FI-485-2013). Iso- 1h,4°C); the thylakoids were collected from the Buffer 3 (1.8 M lation and initial characterization of the cpld49 mutant was sup- sucrose)–Buffer 4 (1.3 M sucrose) interface, washed with 5 vol. of ported by the US Department of Energy, Office of Science, Basic Buffer 6 (5 mM HEPES-KOH, pH 7.5, 10 mM EDTA, protease inhibi- Energy Sciences, Chemical Sciences, Geosciences, and Bio- tors as above) and collected by centrifugation (68 588 g, 20 min, sciences Division under Field Work Proposal number 449B 4°C). The resulting thylakoid pellet was resuspended in 200 ll awarded to K.K.N. K.K.N. is an investigator of the Howard Hughes Buffer 6. Medical Institute, and the Gordon and Betty Moore Foundation (through Grant GBMF3070). Separation of membrane and soluble proteins CONFLICT OF INTEREST Cells were grown and pelleted as above, resuspended in Buffer 1 (25 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 0.3 M sucrose, 0.2 mM The authors declare no conflict of interest. PMSF, 1 mM benzamidine, 5 mM e-amino-a-caproic acid), and bro- ken with a French Pressure Cell at 500 PSI. Membranes were sepa- AUTHOR CONTRIBUTIONS rated from the soluble phase of the cell by centrifugation at 100 000 g for 1 h at 4°C. Soluble proteins were precipitated with T.M.W., S.S., X.J. and A.R.G. designed the experiments; saturated ammonium sulfate on ice for 30 min and collected by T.M.W., S.S. and A.R.G. wrote the article with contributions centrifugation at 2316 g for 10 min at 4°C. Membrane and soluble of all the authors; T.M.W., S.S., W.Y., X.J., R.G.K., M.L.H., – polypeptides were resolved by SDS PAGE and specific polypep- J.J.R., W.P., C.D.B., G.P. and M.L. performed experiments; tides detected by immunoblotting, as described above. T.M.W., S.S., W.Y., X.J., W.P., C.D.B., G.P., M.L. and A.R.G. Treatment of thylakoid membranes with chaotropic salt analyzed data; R.M.D., K.K.N. and F.A.W. provided materi- solutions als and/or technical assistance.

Thylakoid membranes were puriﬁed as described above and SUPPORTING INFORMATION resuspended in Buffer 6 to a concentration of 0.655 mg Chl ml1. Thylakoids (10 lg Chl total) were incubated on ice in the dark for Additional Supporting Information may be found in the online ver- 30 min in the following solutions: water (control); 1 M NaBr; 2 M sion of this article.

NaCl; 0.1 M Na2CO3;2M NaSCN; or 0.1 M NaOH. Soluble and Figure S1. Genotyping of the cpld49 mutant and cpld49-CPLD49 membrane phases were separated by centrifugation (16 100 g, complemented strain. 10 min, 4°C). The soluble phase (supernatant) was removed prior Figure S2. The cpld49 mutant is disrupted by a single pMS188 to detergent solubilization of the pelleted membrane proteins. cassette. Both soluble and membrane proteins were resolved by SDS– Figure S3. Photoheterotrophic and photoautotrophic growth. PAGE, and specific proteins detected by immunoblot analysis. Figure S4. Chlorophyll fluorescence measurements and immunoblots for photoautotrophically grown cells. Sucrose gradient fractionation of thylakoid membrane Figure S5. Disruption of Cre16.g666050 in two independent strains protein complexes results in reduced accumulation of Cytb6f. Figure S6. High-resolution HPLC analyses show no differences in 1 Purified thylakoid membranes (0.8 mg Chl ml ) were solubilized abundances of either major or minor pigments between WT and with 0.75% (w/v) n-dodecyl-b-D-maltoside (b-DM) on ice in the dark cpld49 or cpld38 mutants. for 40 min. Solubilized thylakoids were loaded onto a continuous Table S1. Predicted functions and localization of genes disrupted – sucrose density gradient (0.1 1.3 M sucrose, 5 mM Tricine-NaOH, in the CAL014.01.15 strain (cpld49 mutant). b – pH 8, 0.05% -DM) and the various pigment protein complexes Table S2. Targeted metabolomics analysis of cpld49. ° resolved by ultracentrifugation for 16 h at 288 000 g,4C. Table S3. Primers used in this study. Yeast mating-based split ubiquitin system REFERENCES

CPLD49 and CPLD38 genes (full-length, including chloroplast transit Alric, J., Lavergne, J. and Rappaport, F. (2010) Redox and ATP control of peptide sequences) were amplified from cDNAs and cloned in photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aer- pENTR/D-TOPO (Invitrogen). The CPLD49 gene from pENTR/D- obic conditions. Biochim. Biophys. Acta, 1797,44–51. TOPO_CPLD49 was introduced into pNX22-DEST by LR cloning, Armbruster, U., Zuhlke,€ J., Rengstl, B. et al. (2010) The Arabidopsis thy- while the CPLD38 gene from pENTR/D-TOPO_CPLD38 was intro- lakoid protein PAM68 is required for efficient D1 biogenesis and photo- duced into pMetYC-DEST by LR cloning. An interaction between system II assembly. Plant Cell, 22, 3439–3460. CPLD49 and CPLD38 was assessed by the yeast mating-based split Arruda, P., Kemper, E.L., Papes, F. and Leite, A. (2000) Regulation of lysine catabolism in higher plants. Trends Plant Sci. 5, 324–330. ubiquitin system, as previously described (Yang et al., 2015). Baker, N.R. (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu. Rev. Plant Biol. 59,89–113. ACKNOWLEDGEMENTS Bhuiyan, N.H., Friso, G., Poliakov, A., Ponnala, L. and van Wijk, K.J. (2015) MET1 is a thylakoid-associated TPR protein involved in photosystem II This work was supported by NSF Grant MCB 0951094, and the supercomplex formation and repair in Arabidopsis. Plant Cell, 27, 262–285. Office of Biological and Environmental Research, GTL Program, Broquist, H.P. and Trupin, J.S. (1966) Amino acid metabolism. Annu. Rev. Office of Science, US Department of Energy Grants DE-FG02- Biochem. 35, 231–274. 07ER64427, DE-FG02-12ER16338 awarded to A.R.G. T.M.W. was Calderon, R.H., Garcıa-Cerdan, J.G., Malnoe,€ A., Cook, R., Russell, J.J., supported in part by the National Institute of General Medical Gaw, C., Dent, R.M., de Vitry, C. and Niyogi, K.K. (2013) A conserved

rubredoxin is necessary for photosystem II accumulation in diverse oxy- Grossman, A.R., Lohr, M. and Im, C.S. (2004) Chlamydomonas reinhardtii in genic photoautotrophs. J. Biol. Chem. 288, 26688–26696. the landscape of pigments. Annu. Rev. Genet. 38, 119–173. Chen, X., Kindle, K. and Stern, D. (1993) Initiation codon mutations in the Harris, E.H. (1989) The Chlamydomonas Sourcebook. A Comprehensive Chlamydomonas chloroplast petD gene result in temperature sensitive Guide to Biology and Laboratory Use. San Diego, CA: Academic Press. photosynthetic growth. EMBO J. 12, 3627–3635. Hartings, S., Paradies, S., Karnuth, B., Eisfeld, S., Mehsing, J., Wolff, C., Chi, W., Ma, J. and Zhang, L. (2012) Regulatory factors for the assembly of Levey, T., Westhoff, P. and Meierhoff, K. (2017) The DnaJ-like zinc-ﬁnger thylakoid membrane protein complexes. Philos. Trans. R Soc. B Biol. Sci. protein HCF222 is required for thylakoid membrane biogenesis in plants. 367, 3420–3429. Plant Physiol. 174, 1807–1824. Choquet, Y. and Vallon, O. (2000) Synthesis, assembly and degradation of Hasan, S.S. and Cramer, W.A. (2014) Internal lipid architecture of the het-

thylakoid membrane proteins. Biochimie, 82, 615–634. ero-oligomeric cytochrome b6f complex. Structure, 22, 1008–1015. Choquet, Y. and Wollman, F.A. (2002) Translational regulations as speciﬁc Hasan, S.S., Yamashita, E. and Cramer, W.A. (2013) Transmembrane signal-

traits of chloroplast gene expression. FEBS Lett. 529,39–42. ing and assembly of the cytochrome b6f-lipidic charge transfer complex. Choquet, Y., Stern, D.B., Wostrikoff, K., Kuras, R., Girard-Bascou, J. and Biochem. Biophys. Acta, 1827, 1295–1308. Wollman, F.A. (1998) Translation of cytochrome f is autoregulated Heinnickel, M.L. and Grossman, A.R. (2013) The GreenCut: re-evaluation of through the 5’ untranslated region of petA mRNA in Chlamydomonas physiological role of previously studied proteins and potentially novel chloroplasts. Proc. Natl Acad. Sci. USA, 95, 4380–4385. protein functions. Photosynth. Res. 116, 427–436. Choquet, Y., Zito, F., Wostrikoff, K. and Wollman, F.A. (2003) Cytochrome f Heinnickel, M.L., Alric, J., Wittkopp, T., Yang, W., Catalanotti, C., Dent, R., translation in Chlamydomonas chloroplast is autoregulated by its car- Niyogi, K.K., Wollman, F.A. and Grossman, A.R. (2013) Novel thylakoid boxy-terminal domain. Plant Cell, 15, 1443–1454. membrane GreenCut protein CPLD38 impacts accumulation of the cyto-

Cline, S.G., Laughbaum, I.A. and Hamel, P.P. (2017) CCS2, an octatricopep- chrome b6f complex and associated regulatory processes. J. Biol. Chem. tide-repeat protein, is required for plastid cytochrome c assembly in the 288, 7024–7036. green alga Chlamydomonas reinhardtii. Front. Plant Sci. 8, 1306. https://d Heinnickel, M., Kim, R.G., Wittkopp, T.M., Yang, W., Walters, K.A., Herbert, oi.org/10.3389/fpls.2017.01306. S.K. and Grossman, A.R. (2016) Tetratricopeptide repeat protein protects Cunningham, F.X. and Gantt, E. (2011) Elucidation of the pathway to astax- photosystem I from oxidative disruption during assembly. Proc. Natl anthin in the ﬂowers of Adonis aestivalis. Plant Cell, 23, 3055–3069. Acad. Sci. USA, 113, 2774–2779. Dashdorj, N., Zhang, H., Kim, H., Yan, J., Cramer, W.A. and Savikhin, S. Inoue, K., Dreyfuss, B.W., Kindle, K.L., Stern, D.B., Merchant, S. and Sodeinde,

(2005) The single chlorophyll a molecule in the cytochrome b6f complex: O.A. (1997) Ccs1, a nuclear gene required for the post-translational assem- unusual optical properties protect the complex against singlet oxygen. bly of chloroplast c-type cytochromes. J. Biol. Chem. 272,31747–31754. Biophys. J. 88, 4178–4187. Isaacson, T., Ronen, G., Zamir, D. and Hirschberg, J. (2002) Cloning of tanger- Dent, R.M., Haglund, C.M., Chin, B.L., Kobayashi, M.C. and Niyogi, K.K. ine from tomato reveals a carotenoid isomerase essential for the produc- (2005) Functional genomics of eukaryotic photosynthesis using insertional tion of beta-carotene and xanthophylls in plants. Plant Cell, 14, 333–342. mutagenesis of Chlamydomonas reinhardtii. Plant Physiol. 137,545–556. Isaacson, T., Ohad, I., Beyer, P. and Hirschberg, J. (2004) Analysis in vitro of Dent, R.M., Sharifi, M.N., Malnoe,€ A., Haglund, C., Calderon, R.H., Wakao, the enzyme CRTISO establishes a poly-cis-carotenoid biosynthetic path- S. and Niyogi, K.K. (2015) Large-scale insertional mutagenesis of way in plants. Plant Physiol. 136, 4246–4255. Chlamydomonas supports phylogenomic functional prediction of photo- Jans, F., Mignolet, E., Houyoux, P.-A., Cardol, P., Ghysels, B., Cuine, S., synthetic genes and analysis of classical acetate-requiring mutants. Plant Cournac, L., Peltier, G., Remacle, C. and Franck, F. (2008) A type II NAD J. 82, 337–351. (P)H dehydrogenase mediates light-independent plastoquinone reduc- Depege, N., Bellafiore, S. and Rochaix, J.D. (2003) Role of chloroplast pro- tion in the chloroplast of Chlamydomonas. Proc. Natl Acad. Sci. USA, tein kinase Stt7 in LHCII phosphorylation and state transition in Chlamy- 105, 20546–20551. domonas. Science, 299, 1572–1575. Joliot, P. and Delosme, R. (1974) Flash-induced 519 nm absorption change Desplats, C., Mus, F., Cuine, S., Billon, E., Cournac, L. and Peltier, G. (2009) in green algae. Biochim. Biophys. Acta, 357, 267–284. Characterization of Nda2, a plastoquinone-reducing type II NAD(P)H dehy- Karamoko, M., Cline, S., Redding, K., Ruiz, N. and Hamel, P.P. (2011) Lumen drogenase in Chlamydomonas reinhardtii. J. Biol. Chem. 284, 4148–4157. Thiol Oxidoreductase1, a disulfide bond-forming catalyst, is required for Dobakov a, M., Sobotka, R., Tichy, M. and Komenda, J. (2009) Psb28 protein the assembly of photosystem II in Arabidopsis. Plant Cell, 23, 4462–4475. is involved in the biogenesis of the photosystem II inner antenna CP47 Karpowicz, S.J., Prochnik, S.E., Grossman, A.R. and Merchant, S.S. (2011) (PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol. The GreenCut2 resource, a phylogenomically derived inventory of pro- 149, 1076–1086. teins specific to the plant lineage. J. Biol. Chem. 286, 21427–21439. Eberhard, S., Finazzi, G. and Wollman, F.A. (2008) The dynamics of photo- Kirst, H., Garcıa-Cerdan, J.G., Zurbriggen, A. and Melis, A. (2012) Assembly synthesis. Annu. Rev. Genet. 42, 463–515. of the light-harvesting chlorophyll antenna in the green alga Chlamy- Fristedt, R., Williams-Carrier, R., Merchant, S.S. and Barkan, A. (2014) A thy- domonas reinhardtii requires expression of the TLA2-CpFTSY gene. Plant lakoid membrane protein harboring a DnaJ-type zinc finger domain is Physiol. 158, 930–945.

required for photosystem I accumulation in plants. J. Biol. Chem. 289, Kuras, R. and Wollman, F.A. (1994) The assembly of cytochrome b6f com- 30657–30667. plexes: an approach using genetic transformation of the green alga Fristedt, R., Herdean, A., Blaby-Haas, C.E., Mamedov, F., Merchant, S.S., Chlamydomonas reinhardtii. EMBO J. 13, 1019–1027. Last, R.L. and Lundin, B. (2015) PHOTOSYSTEM II PROTEIN33, a protein Kuras, R., Saint-Marcoux, D., Wollman, F.A. and de Vitry, C. (2007) A speci- conserved in the plastid lineage, is associated with the chloroplast thy- ﬁc c-type cytochrome maturation system is required for oxygenic photo- lakoid membrane and provides stability to photosystem II supercom- synthesis. Proc. Natl Acad. Sci. USA, 104, 9906–9910. plexes in Arabidopsis. Plant Physiol. 167, 481–492. Kurisu, G., Zhang, H., Smith, J.L. and Cramer, W.A. (2003) Structure of the

Gabilly, S.T., Dreyfuss, B.W., Karamoko, M., Corvest, V., Kropat, J., Page, cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity. M.D., Merchant, S.S. and Hamel, P.P. (2010) CCS5, a thioredoxin-like pro- Science, 302, 1009–1014. tein involved in the assembly of plastid c-type cytochromes. J. Biol. Lemeille, S., Willig, A., Depege-Fargeix, N., Delessert, C., Bassi, R. and Chem. 285, 29738–29749. Rochaix, J.D. (2009) Analysis of the chloroplast protein kinase Stt7 dur- Gal, A., Shahak, Y., Schuster, G. and Ohad, I. (1987) Speciﬁc loss of LHCII ing state transitions. PLoS Biol. 7, e45. https://doi.org/10.1371/journal.pb

phosphorylation in the Lemna mutant 1037 lacking the cytochrome b6f io.1000045. complex. FEBS Lett. 221, 205–210. Link, S., Engelmann, K., Meierhoff, K. and Westhoff, P. (2012) The atypical Gonzalez-Ballester, D., Pootakham, W., Mus, F. et al. (2011) Reverse genet- short-chain dehydrogenases HCF173 and HCF244 are jointly involved in ics in Chlamydomonas: a platform for isolating insertional mutants. Plant translational initiation of the psbA mRNA of Arabidopsis. Plant Physiol. Methods, 7,7–24. 160, 2202–2218. Grefen, C., Obrdlik, P. and Harter, K. (2009) The determination of protein- Lohr, M. (2009) Carotenoids. In The Chlamydomonas Sourcebook: Organel- protein interactions by the mating-based split-ubiquitin system (mbSUS). lar and Metabolic Processes, 2 edn, Vol. 2 (Stern, D.B. and Harris, E.H., Methods Mol. Biol. 479, 217–233. eds). Dordrecht: Elsevier, pp. 799–817.

Lyska, D., Meierhoff, K. and Westhoff, P. (2013) How to build functional thy- Tang, G., Miron, D., Zhu-Shimoni, J.X. and Galili, G. (1997) Regulation of lakoid membranes: from plastid transcription to protein complex assem- lysine catabolism through lysine-ketoglutarate reductase and saccha- bly. Planta, 237, 413–428. ropine dehydrogenase in Arabidopsis. Plant Cell, 9, 1305–1316. Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R. et al. (2015) CCD: Tardiff, M., Atteia, A., Specht, M. et al. (2012) PredAlgo: a new subcellular NCBI’s conserved domain database. Nucleic Acids Res. 43, D222–D226. localization prediction tool dedicated to green algae. Mol. Biol. Evol. 29, Merchant, S.S., Prochnik, S.E., Vallon, O. et al. (2007) The Chlamydomonas 3625–3639. genome reveals the evolution of key animal and plant functions. Science, Terashima, M., Specht, M. and Hippler, M. (2011) The chloroplast proteome: 318, 245–250. a survey from the Chlamydomonas reinhardtii perspective with a focus Mininno, M., Brugiere, S., Pautre, V., Gilgen, A., Ma, S., Ferro, M., Tardif, on distinctive features. Curr. Genet. 57, 151–168. M., Alban, C. and Ravanel, S. (2012) Characterization of chloroplastic Vallon, O. and Spalding, M.H. (2009) Amino acid metabolism. In The Chlamy- fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in domonas Sourcebook: Organellar and Metabolic Processes,2edn,Vol.2 plants. J. Biol. Chem. 287, 21034–21044. (Stern, D.B. and Harris, E.H., eds). Dordrecht: Elsevier, pp. 115–158. Nickelsen, J. and Rengstl, B. (2013) Photosystem II assembly: from de Vitry, C., Ouyang, Y., Finazzi, G., Wollman, F.A. and Kallas, T. (2004) The cyanobacteria to plants. Annu. Rev. Plant Biol. 64, 609–635. chloroplast Rieske iron-sulfur protein: at the crossroads of electron trans- Nisar, N., Li, L., Lu, S., Khin, N.C. and Pogson, B.J. (2015) Carotenoid meta- port and signal transduction. J. Biol. Chem. 279, 44621–44627. bolism in plants. Mol. Plant, 8,68–82. Wei, L., Derrien, B., Gautier, A. et al. (2014) Nitric oxide-triggered remodeling Pfaffl, M.W. (2001) A new mathematical model for relative quantification in of chloroplast bioenergetics and thylakoid proteins upon nitrogen starva- real-time RT-PCR. Nucleic Acids Res. 29, e45. tion in Chlamydomonas reinhardtii. Plant Cell, 26,353–372. Pootakham, W., Gonzalez-Ballester, D. and Grossman, A.R. (2010) Identifi- Wittenberg, G., Jarvi,€ S., Hojka, M., Toth, S.Z., Meyer, E.H., Aro, E.M., cation and regulation of plasma membrane sulfate transporters in Schottler,€ M.A. and Bock, R. (2017) Identification and characterization of Chlamydomonas. Plant Physiol. 153, 1653–1668. a stable intermediate in photosystem I assembly in tobacco. Plant J. 90, Porra, R.J., Thompson, W.A. and Kriedemann, P.E. (1989) Determination of 478–490. accurate extinction coefficients and simultaneous equations for assaying Wittkopp, T.M., Saroussi, S., Yang, W. and Grossman, A.R. (2016) The chlorophylls a and b extracted with four different solvents: verification of Greencut: functions and relationships of proteins conserved in green lin- the concentration of chlorophyll standards by atomic absorption spec- eage organisms. In Chloroplasts: Current Research and Future Trends troscopy. Biochim. Biophys. Acta, 975, 384–394. (Kirchhoff, H., ed). Norfolk: Caister Academic Press, pp. 239–276. Rochaix, J.D. (2004) Genetics of the biogenesis and dynamics of the photo- Wittkopp, T.M., Schmollinger, S., Saroussi, S. et al. (2017) Bilin-dependent synthetic machinery in eukaryotes. Plant Cell, 16, 1650–1660. photoacclimation in Chlamydomonas reinhardtii. Plant Cell, 29, 2711– Rochaix, J.D. (2011) Assembly of the photosynthetic apparatus. Plant Phys- 2726. iol. 155, 1493–1500. Wollman, F.A. (2001) State transitions reveal the dynamics and flexibility of Saroussi, S.I., Wittkopp, T.M. and Grossman, A.R. (2016) The type II NADPH the photosynthetic apparatus. EMBO J. 20, 3623–3630. dehydrogenase facilitates cyclic electron flow, energy dependent Wollman, F.A., Minai, L. and Nechushtai, R. (1999) The biogenesis and quenching and chlororespiratory metabolism during acclimation of assembly of photosynthetic proteins in thylakoid membranes. Biochim. Chlamydomonas reinhardtii to nitrogen deprivation. Plant Physiol. 170, Biophys. Acta, 1411,21–85. 1975–1988. Wostrikoff, K., Girard-Bascou, J., Wollman, F.A. and Choquet, Y. (2004) Bio- Schottler,€ M.A., Albus, C.A. and Bock, R. (2011) Photosystem I: its biogene- genesis of PSI involves a cascade of translational autoregulation in the sis and function in higher plants. J. Plant Physiol. 168, 1452–1461. chloroplast of Chlamydomonas. EMBO J. 23, 2696–2705. Schult, K., Meierhoff, K., Paradies, S., Toller,€ T., Wolff, P. and Westhoff, P. Xiao, J., Li, J., Ouyang, M., Yun, T., He, B., Ji, D., Ma, J., Chi, W. and Zhang,

(2007) The nuclear-encoded factor HCF173 is involved in the initiation of L. (2012) DAC is involved in the accumulation of the cytochrome b6f com- translation of the psbA mRNA in Arabidopsis thaliana. Plant Cell, 19, plex in Arabidopsis. Plant Physiol. 160, 1911–1922. 1329–1346. Xie, Z., Culler, D., Dreyfuss, B.W., Kuras, R., Wollman, F.A., Girard-Bascou, Serrano, G.C., Rezende e Silva Figueira, T., Kiyota, E., Zanata, N. and J. and Merchant, S.S. (1998) Genetic analysis of chloroplast c-type cyto- Arruda, P. (2012) Lysine degradation through the saccharopine pathway chrome assembly in Chlamydomonas reinhardtii: one chloroplast locus in bacteria: LKR and SDH in bacteria and its relationship to the plant and and at least four nuclear loci are required for heme attachment. Genetics, animal enzymes. FEBS Lett. 586, 905–911. 148, 681–692. Stroebel, D., Choquet, Y., Popot, J.L. and Picot, D. (2003) An atypical haem Yang, W., Catalanotti, C., D’Adamo, S. et al. (2014) Alternative acetate pro-

in the cytochrome b6f complex. Nature, 426, 413–418. duction pathways in Chlamydomonas reinhardtii during dark anoxia and Takahashi, H., Iwai, M., Takahashi, Y. and Minagawa, J. (2006) Identiﬁcation the dominant role of chloroplasts in fermentative acetate production. of the mobile light-harvesting complex II polypeptides for state transi- Plant Cell, 26, 4499–4518. tions in Chlamydomonas reinhardtii. Proc. Natl Acad. Sci. USA, 103, 477– Yang, W., Wittkopp, T.M., Li, X. et al. (2015) Critical role of Chlamydomonas 482. reinhardtii Ferredoxin-5 in maintaining membrane structure and dark Takahashi, H., Clowez, S., Wollman, F.A., Vallon, O. and Rappaport, F. metabolism. Proc. Natl Acad. Sci. USA, 112, 14978–14983. (2013) Cyclic electron ﬂow is redox-controlled but independent of Zhang, H., Huang, D. and Cramer, W.A. (1999) Stoichiometrically-bound b-

state transition. Nat. Commun. 4, 1954. https://doi.org/10.1038/ncomm carotene in the cytochrome b6f complex of oxygenic photosynthesis pro- s2954. tects against oxygen damage. J. Biol. Chem. 274, 1581–1587.