Molecular Plant ReseaRch aRticle

Retrograde signaling and Photoprotection in a gun4 Mutant of Chlamydomonas reinhardtiiCinzia Formighieria,2, Mauro Ceolb,2, Giulia Bonentea, Jean-David Rochaixb and Roberto Bassia,1

a Dipartimento di Biotecnologie, Università di Verona, strada le Grazie 15, i-37134 Verona, italyb Departments of Molecular Biology and Plant Biology, University of Geneva sciences iii 30, Quai ernest-ansermet ch-1211, Geneva 4, switzerland

ABSTRACT GUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. Here, we report the first characterization of a gun4 insertion mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll as compared to wild-type and accumulates ProtoIX. In contrast to the increase in LHC transcription, the accumulation of most LHC proteins is drastically diminished, implying posttranscriptional down-regulation in the absence of transcriptional coordination. We found that 803 genes change their expression level in gun4 as compared to wild-type, by RNA-Seq, and this wide-ranging effect on transcription is apparent under physiological conditions. Besides LHCs, we identified transcripts encoding enzymes of the tetrapyrrole pathway and factors involved in signal transduction, transcription, and chromatin remodeling. Moreover, we observe perturbations in electron transport with a strongly decreased PSI-to-PSII ratio. This is accompanied by an enhanced activ-ity of the plastid terminal oxidase (PTOX) that could have a physiological role in decreasing photosystem II excitation pressure.

Key words: green algae; Chlamydomonas; genome uncoupled (gun) mutants; gun4; retrograde signaling; PTOX.

1 to whom correspondence should be addressed. e-mail roberto.bassi@univr.it, tel. 390458027915, fax 390458027929.2 these two authors contributed equally to the work.

© the author 2012. Published by the Molecular Plant shanghai editorial Office in association with Oxford University Press on behalf of csPB and iPPe, siBs, cas.doi: 10.1093/mp/sss051Received 11 October 2011; accepted 2 april 2012

INTRODUCTIONin plants and algae, tetrapyrrole metabolism occurs mainly in the chloroplast, but is mostly catalyzed by nucleus-encoded enzymes. this metabolic pathway leads to the synthesis of chlo-rophyll, heme, siroheme, and phytochromobilins. the flux and partitioning of tetrapyrroles in the pathway need to be tightly regulated because the requirement of specific tetrapyrroles var-ies depending on different internal and external conditions, and tetrapyrrole intermediates and products act as photosensitizers, leading to the synthesis of reactive oxygen species (ROs) (tanaka and tanaka, 2007; Masuda, 2008; Masuda and Fujita, 2008).

Pigment mutants have proven to be useful tools for the analysis of the tetrapyrrole pathway and several mutants of the unicellular green alga Chlamydomonas reinhardtii defi-cient in various steps of chlorophyll biosynthesis have been identified (Wang et al., 1974; Malnoe et al., 1988; choquet et al., 1992; suzuki and Bauer, 1992; li et al., 1993; li and timko, 1996; tanaka et al., 1998; timko, 1998; cahoon and timko, 2000; chekounova et al., 2001; Meinecke et al., 2010). Photoautotrophic and/or photosensitive mutants can be propagated due to the ability of C. reinhardtii to grow het-erotrophically in the dark. Moreover, the available sequence information for the three genomes (nuclear, plastid, and mitochondrial) makes C.  reinhardtii an attractive model organism for studies in functional genomics.

the first committed step in chlorophyll biosynthesis is the insertion of a Mg2 in the tetrapyrrolic ring of Protoporphyrin iX (ProtoiX) by the magnesium chelatase (Mgch) enzyme (Wang et al., 1974; chekounova et al., 2001), generating Mg–Protoporphyrin iX (Mg–ProtoiX). this is also one of the most extensively regulated steps in the tetrapyrrole biosynthetic pathway. Mgch is composed of three subunits, conserved from prokaryotes to plants, and requires MgatP plus free Mg2 ions for its activity (Jensen et al., 1996, 1998, 1999; Karger et al., 2001). the chlh subunit has been assumed to be the ProtoiX binding and catalytic site (Jensen et al., 1998; Karger et al., 2001). Mgch is subjected to various levels of regulation. transcription and protein accumulation are up-regulated after a dark-to-light shift both in plants, where the oscillation is diurnal (Gibson et al., 1996; Papenbrock et al., 1999), and in C. reinhardtii (chekounova et al., 2001), reflecting the rate of chlorophyll biosynthesis. the enzymatic

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activity is then controlled by several chloroplast cues, such as the redox state (ikegami et al., 2007; Kobayashi et al., 2008), the concentration of stromal Mg2, and the atP/aDP ratio (Reid and hunter, 2004).

Recently, the GUN4 protein was reported to interact in vivo with chlh (larkin et al., 2003; sobotka et al., 2008) and to stimulate Mg-chelation in vitro (larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). a gun4 mutant was first identified in Arabidopsis thaliana by screening for mutants affected in plastid-to-nucleus retrograde signaling upon nor-flurazon treatment—a condition that disrupts the photosyn-thetic apparatus and induces oxidative stress. in wild-type, but not in gun (genome uncoupled), mutants, this condition leads to transcriptional repression of LHC genes (susek et al., 1993; Mochizuki et al., 2001; larkin et al., 2003). Most of the gun mutations perturb the tetrapyrrole biosynthetic path-way, suggesting that tetrapyrrole metabolism plays a role in the control of nuclear gene expression and that this ret-rograde signaling pathway is impaired in the gun mutants. chlorophyll biosynthesis in the chloroplast is tightly coordi-nated with the expression of photosynthesis-related genes such as the nucleus-encoded light-harvesting chlorophyll-binding (lhc) subunits of photosystems (Johanningmeier and howell, 1984; Johanningmeier, 1988; leister et al., 2011). chlh was suggested to have a specific function in plastid-to-nucleus signaling, sensing the flux of tetrapyrroles at the chlorophyll branch (Mochizuki et al., 2001). the tetrapyr-role intermediate Mg–ProtoiX was also proposed to act as a signal molecule (Kropat et al., 1997, 2000; strand et al., 2003; von Gromoff et al., 2006). however, the correlation between Mg–ProtoiX and nuclear gene expression origi-nally found in Arabidopsis thaliana plants (strand et al., 2003) was later unambiguously and independently refuted by two other groups (Mochizuki et al., 2008; Moulin et al., 2008). consistently, C. reinhardtii mutants defective in Mg–ProtoiX methyltransferase were shown to accumulate Mg–ProtoiX and yet retained up-regulated LHC transcript levels (Meinecke et al., 2010). these data contradict the model of an inverse correlation between Mg–ProtoiX level and LHC transcription proposed by strand et al. (2003). aside from Mg–ProtoiX, heme may serve as a plastid signal to regulate the expression of nuclear genes. in Arabidopsis, a gain-of-function mutation overexpressing the conserved plastid fer-rochelatase (Fech) 1 led to an enhanced flux of tetrapyrroles through the heme branch and an increased expression of photosynthesis-associated nuclear genes (Woodson et al., 2011). consistently, Mgch mutants of C.  reinhardtii exhib-ited two to fivefold elevated heme levels (chekounova et al., 2001; von Gromoff et al., 2008) and preserved light induction of hsP70a that was previously shown to be mediated by Mg–ProtoiX (von Gromoff et al., 2006). however, C.  reinhardtii mutants defective in Mg–ProtoiX methyltransferase exhib-ited twofold reduced heme, suggesting different regulation of heme synthesis as compared to Mgch mutants, despite up-regulated LHC transcription (Meinecke et al., 2010).

surprisingly, the gun4 mutation in Synechocystis decreased the activities of both Mgch and Fech, suggesting a role of GUN4 in maintaining a balanced, optimized porphyrin dis-tribution to both chelatases and in stabilizing these enzymes (sobotka et al., 2008). although tetrapyrrole metabolism is thought to regulate plastid-to-nucleus communication, the identity of the signal molecule is still under debate. Yet gun4 mutants are uncoupled, suggesting GUN4 might be involved in a still uncharacterized retrograde signaling pathway coor-dinating chlorophyll biosynthesis with LHC transcription in the nucleus.

in A. thaliana and Synechocystis, loss of GUN4 compromises chlorophyll accumulation under normal growth conditions (larkin et al., 2003; sobotka et al., 2008; Peter and Grimm, 2009). this result indicates that, even if GUN4 is not essential for chlorophyll synthesis, it is required for regulating its rate to an appropriate physiological level that sustains optimal photoautotrophic growth. the diurnal oscillation of GUN4 expression in A. thaliana (Peter and Grimm, 2009) resembles that of GlutR and chlh (Papenbrock et al., 1999), pointing to a tight correlation between GUN4 expression and the rate of chlorophyll biosynthesis mediated through posttransla-tional regulatory mechanisms (Peter and Grimm, 2009) such as activation and stabilization of the Mgch enzyme (larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). the crystal structure of GUN4 from cyanobacteria (Davison et al., 2005; Verdecia et al., 2005) showed that GUN4 has one por-phyrin binding pocket that can accommodate either ProtoiX or MgProtoiX. in particular, GUN4 was reported to stimulate Mg-chelation in vitro (larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). GUN4 enhances the sensitivity of Mgch to Mg2 in a range suitable for the physiologically low Mg2 concentration. Moreover, in the absence of GUN4, Mgch is inefficient at low concentrations of ProtoiX and the rate of enzyme–substrate complex formation and Mg incorporation is decreased. Finally, GUN4, upon porphyrin binding, was pro-posed to stabilize interactions between the catalytic subunit of Mgch and the chloroplast membranes, the site of chlo-rophyll synthesis, thereby presenting Mgch to complexes of enzymes involved in steps further downstream in the path-way (adhikari et al., 2009, 2011). since GUN4 orthologs are found only in oxygenic photosynthetic organisms, its regu-latory role appears to have evolved concomitantly with the production of oxygen and ROs generated by photosyn-thesis. consistently, the gun4 mutants of A.  thaliana and Synechocystis have a photosensitive phenotype (larkin et al., 2003; sobotka et al., 2008).

the nuclear genome of C.  reinhardtii contains a GUN4 ortholog, present in single copy. in the present study, we report the characterization of the first knockout gun4 mutant of C. reinhardtii, obtained through random insertion mutagenesis of the nuclear genome and by screening for pale green mutants. GUN4 regulates tetrapyrrole flow and chlorophyll biosynthesis and a gun4 phenotype was observed both in the dark and in the light. in contrast to higher plants,

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C.  reinhardtii is able to synthesize chlorophyll and assemble functional photosynthetic complexes also in the dark. this is possible thanks to the presence of a light-independent protochlorophyllide reductase (li-POR) (Malnoe et al., 1988; choquet et al., 1992; suzuki and Bauer, 1992; li et al., 1993; cahoon and timko, 2000), in addition to the light-dependent POR (li and timko, 1996; timko, 1998) typical of plants. although chloroplast differentiation and biogenesis of the photosynthetic apparatus present differences as compared to higher plants, we still refer to a genome uncoupled phenotype of C. reinhardtii gun4 because of lack of coordination between chloroplast state and nuclear gene expression, as reflected by posttranscriptional down-regulation in the absence of transcriptional coordination for photosynthesis-related genes. Remarkably, while the genome uncoupled phenotype of Arabidopsis gun mutants was only observed upon norflurazon treatment that disrupts chloroplast function (larkin et al., 2003; strand et al., 2003), a wide-ranging different transcription profile in C.  reinhardtii gun4 as compared to wild-type is detectable under physiological conditions. in particular, we have determined the nuclear transcriptome in dark-grown cells to identify novel transcripts whose expression is possibly regulated by the tetrapyrrole-dependent plastid signal. in addition, it appears that a posttranscriptional process is also active, affecting accumulation, assembly, and function of the photosynthetic apparatus.

RESULTSIdentification of the gun4 Mutant of Chlamydomonas reinhardtii

the C. reinhardtii gun4 mutant was isolated in a screen for pale green/low fluorescence mutants after random insertion mutagenesis of a cw15 strain of C.  reinhardtii (also called wild-type in the present paper) with linearized psl18 plasmid containing the paromomycin resistance cassette (Bonente et al., 2011). We mapped the site of insertion by inverse PcR in the second exon at position 589 of the nuclear single-copy GUN4 gene (Figure 1a). the insertion caused deletions of 184  bp in the second exon and of 489  bp in the psl18 plasmid (dashed line in Figure  1a). the knockout was con-firmed by Rt–PcR with primers specific for the GUN4 tran-script. No GUN4 mRNa could be detected in the mutant under the same conditions (Rt in Figure 1B). analysis of the offspring from a backcross of gun4 with wild-type revealed co-segregation between paromomycin resistance and the mutant phenotype, indicating that the mutant is tagged by the transforming DNa (100 random progeny colonies were examined; Figure 2 reports an example of such an analysis). the mutant was then transformed with the wild-type GUN4 coding sequence tagged with ha under the control of the constitutive PSAD promoter. two clones expressing different levels of the protein (R1 and R2 in Figure  3a) rescued the mutant phenotype (Figure 3B and table 1).

alignment of the deduced amino acid sequence of Chlamydomonas GUN4 with those of Arabidopsis thali-ana, Synechocystis sp. Pcc 6803, and Thermosynechococcus elongatus revealed sequence conservation in these species (Figure 1c).

Tetrapyrrole Composition Is Affected in the gun4 Mutant

the gun4 mutant accumulates reduced amounts of chloro-phyll per cell, about 50% of the wild-type level in the dark (table 1) and in low light conditions (supplemental table 1). the level of the GUN4 protein correlates with chlorophyll accumulation, as shown by the analysis of complemented clones R1 and R2, expressing different amounts of GUN4–ha (Figure 3a and table 1). Both chlorophyll a and chloro-phyll b are reduced without affecting the carotenoid content (supplemental table 1). lutein is present as in the wild-type, while β-carotene, neoxanthin, and loroxanthin accumulate to slightly lower levels in the mutant. in contrast, the amount of pigments involved in the xanthophyll cycle (violaxanthin, antheraxanthin, and zeaxanthin) is increased in gun4 com-pared to the wild-type (24 versus 18% of total carotenoid content; supplemental table 1) consistently with an increased level of oxidative stress (havaux and Niyogi, 1999; Baroli et al., 2003 , 2004; Dall’osto et al., 2007, 2010).

Next, we examined whether the mutant accumulates high amounts of the chlorophyll precursor ProtoiX, the substrate of Mgch, as reported for the Synechocystis gun4 knockout (sobotka et al., 2008). ProtoiX was quantified using fluores-cence spectroscopy (table 1). the gun4 mutant accumulates ~20 times more ProtoiX than the wild-type on a per-chloro-phyll basis. even the low amount of GUN4 protein in clone R2 (Figure 3a) was sufficient to prevent accumulation of this chlorophyll precursor (table 1). since GUN4 acts at the branch point between chlorophyll and heme synthesis, it could influence tetrapyrrole flow in both pathways. We therefore measured the heme content, which was unchanged when expressed on a per-cell basis (table 1).

Loss of GUN4 Leads to a Photosensitive Phenotype at Increasing Light Irradiance and to Impaired Photoautotrophic Growth

We performed a growth test on acetate-containing medium (taP) and minimal medium (hsM) in different light conditions in order to analyze the light sensitivity and the ability of the gun4 mutant of C. reinhardtii to grow photoautotrophically. Growth of gun4 and wild-type was similar in the dark and under dim light conditions in taP medium. at 50  µmol photons  m2  s1, however, growth of the mutant was impaired. at higher irradiance, cells bleached and died (taP in Figure 3B). the gun4 mutant retained a significant amount of chlorophyll (supplemental table 1) and was able to grow photoautotrophically under conditions where the photo-oxidative stress was not the limiting factor (hsM in Figure 3B) although with a slower rate than wild-type. the two

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complemented clones (R1 and R2) were subjected to the same light treatments. Under all conditions, these strains grew like the wild-type, confirming that the light sensitivity and the poor photoautotrophic growth are due to the absence of the GUN4 protein (Figure 3B). it is interesting to note that clone R1, expressing higher levels of GUN4 (Figure 3a), was more resistant to high light on minimal medium than clone R2, although no ProtoiX accumulation was detected in both complemented strains (table  1). thus, the photosensitive phenotype at increasing light intensities correlated with GUN4 protein level but not with the amount of ProtoiX, suggesting a distinct role of GUN4 in photoprotection.

A.  thaliana gun4 was reported to be partially rescued under continuous light compared to photoperiodic condi-tions of the same light intensity, pointing at a role of GUN4 in regulating tetrapyrrole flow in a changing light environment (Peter and Grimm, 2009). in the case of C. reinhardtii gun4, only a minor improvement in growth was observed under continuous versus periodic light conditions (Figure 3B).

Accumulation of LHC Polypeptides Is Decreased in the gun4 Mutant

accumulation of polypeptides of both photosystems was investigated by immunoblot analysis in cells grown in the

Figure 1. Mapping of the insertion site and identification of the Mutation in gun4.(A) schematic representation of the GUN4 genomic region. Rectangles represent the two exons and lines non-coding regions. the site of insertion of the psl18 vector is indicated (+589) and the deletion caused by the insertion is marked by dashed lines. the arrows indicate the position of the primers used for the Rt–PcR in Figure 1B.(B) Rt–PcR with GUN4-specific primers and control primers (CBL) from gun4 and cw15 RNa. asterisk indicates a non-specific amplification product, verified by sequencing of the excised band. Markers size from top to bottom: 1000, 800, 600, 400 pb.(C) alignment of the predicted C. reinhardtii GUN4 protein sequence with the corresponding sequences from Arabidopsis thaliana, Synechocystis sp. Pcc 6803, and Thermosynechococcus elongatus. the secondary structure proposed for the Synechocystis GUN4 domain (Verdecia et al., 2005) is depicted: cylinders represent α-helices (α1 to α8) and lines represent unstructured regions. the loops putatively involved in porphyrin binding are indicated (α2/α3 and α6/α7). the location of the insertion in the C. reinhardtii gun4 mutant is shown by the open triangle and the dashed line marks the deletion.

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Figure 2. Random Progeny analysis of the cross between cell Wall-less gun4 mt- and cell Wall-containing Wild-type s34 mt+. Progeny colonies were tested on taP either in dim light (6 µmol photons m–2 s–1) in the presence of paromomycin (15 µg ml–1, left panel) or in high light (500 µmol photons m–2 s–1, right panel). Marked with an asterisk are colonies with the gun4 photosensitive phenotype, dying in high light (right panel). Only these colonies grew on taP-paromomycin (left panel). this result shows co-segregation between the mutant phenotype and the paromomycin resistance carried by the insertion cassette, indicating that the gun4 mutant is tagged by the transforming DNa. independent segregation of the cell wall phenotype was checked to verify that crossing had indeed occurred. Parental strains are marked with an arrow on the same plate as references.

dark or transferred to 60 µmol photons m2  s1 for 24 h, in order to verify the effect of limited amounts of chlorophyll on the accumulation of pigment-binding proteins (Figure  4a). accumulation of polypeptides of the photosystem  ii core (D1 and the inner antennae cP43 and cP47) was the same in gun4 and wild-type based on protein amount. in contrast, the amount of Psaa subunit of the photosystem i core and cYt f of cytochrome b6f complex was lower in the mutant, especially in the light (Figure  4a). Moreover, the mutant accumulated significantly lower amounts of antenna proteins of both photosystem ii (major trimeric lhcii, monomeric cP26 and cP29) (Figure  4a) and photosystem  i (lhca subunits; supplemental Figure  1), although to a different extent. stauber and colleagues (2009) showed that lhca3 is the most abundant lhca subunit in C. reinhardtii, followed by lhca1, lhca4, and lhca7. in the gun4 mutant, the amount of lhca3 was strongly reduced, while the levels of lhca1, lhca4, and lhca7 were only partially affected in the dark. their levels further decreased upon transfer from dark to light. among the remaining subunits, lhca2, lhca8, and lhca9 were only partially decreased, while lhca5 virtually disappeared (supplemental Figure  1). the level of other chloroplast proteins like phosphoribulose kinase (PRK), involved in the calvin-Benson cycle, DNa-K, hsP70B chaperone, and the FlU-like protein (FlP), a negative regulator of chlorophyll synthesis, were similar in gun4 and wild-type (Figure  4a) on a total protein basis. in Figure 4B, the polypeptide levels

were analyzed during a time course experiment. the cultures were transferred from the dark to the light and samples were collected at different time points upon illumination. in the wild-type, we could observe an initial decrease in the levels of cP29, cP26, and lhcBM5 followed by an increase after 24 h in the light. No oscillation of this type was observed for D1 and Psaa of the photosystem core complexes (Figure 4B, cw15). in the mutant, the initial decrease of the minor antennae cP29 and cP26 and of the major lhcBM5 was not followed by an increase during further illumination, resulting in a lower level of protein accumulation (Figure 4B, gun4). cP29 decreased more slowly than cP26 in gun4 (Figure 4B) and a band of lower molecular weight recognized by the α-cP29 appeared after 24 h of illumination, which may represent a cP29 breakdown product.

The gun4 Mutation Affects the Photosynthetic Apparatus

a green-native electrophoretic separation of mildly solubi-lized thylakoids was performed in order to assess the steady-state level of chlorophyll–protein complexes (Figure 5). since the mutant is likely to have a higher lipid/chlorophyll ratio, higher amounts of detergent might be required for thy-lakoid solubilization and this parameter can influence the green band separation pattern. We therefore compared results obtained upon solubilization with different concen-trations of αDM (n-dodecyl-α-D-maltoside). the densito-metric analysis of green bands reported in Figure 5c shows

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Figure 3. complementation of the gun4 Mutant with a Recombinant GUN4–ha construct.

(A) immunoblot analysis of total proteins extracts of cw15 (wild-type), gun4, and two complemented clones (R1 and R2) with α-GUN4 and α-PRK antibodies. the GUN4-ha protein in R1 and R2 is visible at a higher molecular weight compared to the native GUN4 in cw15. GUN4 protein level in the different lines is normalized to the PRK signal and expressed as percentage with respect to the wild-type.(B) Growth test of cw15, gun4, R1, and R2. Five µl containing 107, 106, 105, and 104 cells per ml of each strain were spotted on acetate-contain-ing medium (taP) or minimal medium (hsM) and grown for 2 weeks under the indicated light conditions (µe  µmol photons m–2 s–1).

Table 1. steady-state levels of tetrapyrroles in cw15, gun4, and two complemented clones (R1 and R2) Grown in the Dark.

pmol 106 cellsa

strains analyzed ProtoiX heme chlorophyll

cw15 0.38 0.17 8.4 2.8 3022.2 333.3

gun4 3.80 1.32 9.2 2.9 1466.7 366.7

R1 0.25 0.11 10.4 0.9 2488.9 42.2

R2 0.07 0.05 n 2022.2 244.4

a the data represent the mean with standard deviation of independent biological replicates. n, not measured.

Figure 4. immunoblot analysis on a Per-Protein Basis.

(A) immunoblot analysis of the indicated polypeptides in gun4 and cw15. D, dark acclimated cells; cl, after 24 h of illumination at 60 µmol photons m–2 s–1. (B) time course analysis of the changes in protein content of gun4 and cw15 after a shift of dark-adapted cells to 60 µmol photons m–2 s–1 of light. samples were collected at time 0 (Dark) and after 20 min, 40 min, 1 h, 3 h, 6 h, 12 h, and 24 h of continuous illumination at 60 µmol photons m–2 s–1.

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a higher amount of Ps core complexes compared to anten-nae in gun4, yielding an antennae/core ratio of three versus eight in wild-type. the gun4 mutant lacks the green band corresponding to the Psi–lhci supercomplex in thylakoids extracted from cells grown in low light (Figure 5a); instead,

the Psii core band at lower MW was enhanced, suggesting that the Psi core moiety co-migrates with the photosystem ii core complex. in order to specifically detect the Psi contribu-tion to the Psi core/Psii core band, the latter was excised from the gel and probed with a specific anti-Psaa antibody upon

Figure 5. Native electrophoretic separation of chlorophyll-containing Photosynthetic complexes.

(A) DeRiPhat–PaGe, gradient 4.5–12%. Unstacked thylakoids were extracted from cultures grown at 30 µmol photons m2 s1. thylakoids were then solubilized with different amounts of αDM (n-dodecyl-α-D-maltoside). 30 µg of chlorophylls are loaded in each lane. thylakoids from Arabidopsis thaliana are shown in the first lane for comparison.(B) Psaa subunit of photosystem i core was detected in the green gel-eluted bands with a specific antibody (1 µg of thylakoid chlorophylls is loaded as a positive control).(C) Densitometric analysis of green bands. the reported values are in arbitrary units (a.U.) and refer to the sum of the bands in the lane, the sum of monomeric and trimeric antennae, Psi/Psii core, Psi/Psii supercomplexes.(D) chlorophyll fluorescence emission at 77 K. spectra were recorded both in cells dark-adapted in state i before the measurement (dark) and in samples dark-adapted and then pre-illuminated at 760 µmol photons m2 s1 (light). chlorophylls were excited at 475 nm (width of the beam 17 nm). the peaks at 686 and 694 nm originate from photosystem ii, whereas the emission around 710 nm is attributed to photosystem i. the spectrum is normalized with respect to the 686-nm peak.

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re-electrophoresis in denaturing conditions. the presence of Psaa was detected in the Psi–lhci band in the wild-type and in the lower MW Psii core band in gun4 (Figure 5B), imply-ing that Psi was present in the mutant in a form lacking its antenna moiety. LHCA transcript levels were not reduced in the mutant (supplemental Figure  2) and yet lhca protein accumulation was decreased, implying a posttranscriptional regulation (supplemental Figure 1). Further information on the level of Psi holoproteins was obtained by fluorescence emission spectra at 77  K, which enhances the Psi fluores-cence quantum yield (Figure 5D). the emission spectrum of the wild-type showed three major peaks with maxima at 686, 694, and 710 nm (cw15 in Figure 5D): the bands at 686 and 694  nm originate from photosystem  ii, whereas the emis-sion around 710 nm is attributed to photosystem i (Bassi et al., 1992). interestingly, gun4 showed very low Psi emission relative to Psii irrespective of whether excitation occurred at 475  nm (chlorophyll  b) or 440  nm (chlorophyll  a) (gun4 in Figure 5D), implying that Psi holoproteins were strongly decreased, to an even higher extent than expected from the immunoblot analysis (Figure 4a).

Measurement of P700 photo-oxidation in vivo provides information about the photosynthetic electron transport (Joliot and Joliot, 2005). Wild-type and gun4 cells in state i were pre-illuminated before the measurement to fully acti-vate photosynthesis, after which P700 oxidation level was analyzed following irradiance with saturating actinic light (saturating    independent from the antenna size), in the presence or absence of DcMU (Figure 6). in wild-type, DcMU treatment, which blocks electron transfer on the photosys-tem  ii acceptor side, increased P700 oxidation (Figure  6a),

hence implying that linear electron transport between pho-tosystems is the major electron transfer pathway active in this condition. interestingly, P700 oxidation was insensitive to DcMU in gun4 (Figure 6B). this result suggests that photo-system  ii and photosystem  i are largely disconnected in the mutant.

Photochemical efficiency of photosystem  ii was then investigated. Wild-type and gun4 cells grown in low light (30  µmol photons  m2  s1) and dark-adapted before the measurement showed similar Fv/Fm (0.7    0.1). Under an actinic illumination of 100 µmol photons m2 s1, the wild-type showed a Psii quantum yield (ϕPsii) of 0.46, while a lower value of 0.32 was detected in the mutant, indicating a less efficient electron transfer (Figure 7a). in order to discriminate different components of the electron transport, cells were submitted to an hyper-osmotic stress (0.5 M sucrose) that blocks plastocyanin diffusion and thus electron transfer between cytochrome b6f and Psi (cruz et al., 2001) and allows better study of the contribution of the plastid terminal oxidase (PtOX) to ϕPsii. PtOX was shown to catalyze reduction of oxygen by oxidizing plastoquinol and to be sensitive to the quinone analogue n-propyl-gallate (pgal) in C. reinhardtii (cournac et al., 2000). after hyper-osmotic stress, gun4 retained ϕPsii of 0.055 and this activity was sensitive to pgal (Figure  7B). the residual activity in the presence of pgal could be due to limited pgal diffusion into the chloroplast and/or to partial inhibition of PtOX. the reduction in ϕPsii after addition of pgal in Figure 7a (gun4, gray bar) is consistent with ϕPsii after hyper-osmotic stress (gun4, Figure  7B) and implies that PtOX contributes to 20% of Psii electron transport in the mutant. in contrast, PtOX contribution to ϕPsii in the wild-type is only 2% (cw15

Figure 6. analysis of P700 Photo-Oxidation In Vivo.

cells in state i were pre-illuminated for 20 min at 400 µmol photons m2 s1 before the measurement. Data were normalized with respect to the value at the end of the actinic light phase (60 s, 940 µmol photons m2 s1).(A) P700 oxidation in cw15 without (untreated) or with 10 µM DcMU.(B) P700 oxidation in gun4 without (untreated) or with 10 µM DcMU.

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in Figure 7a and 7B). the behavior of the complemented clone R1 was more similar to the wild-type, suggesting that the PtOX-related phenotype observed in gun4 is a consequence of the gun4 mutation (Figure 7B).

two PTOX genes, encoding putative plastid-targeted plas-toquinol oxidases, are present in the nuclear genome of Chlamydomonas (PTOX1, cre07.g350750, predicted molecu-lar mass of the processed protein 47.5  kDa; PTOX2, cre03.g172500, predicted molecular mass of the processed protein 45  kDa; signal peptide prediction by chloroP (emanuelsson et al., 1999)). in contrast to PtOX1, PtOX2 has one predicted transmembrane domain (www.cbs.dtu.dk/services/tMhMM-2.0/) and the corresponding polypeptide was detected in the thylakoid membrane fraction (Figure  8). accumulation of both PtOX proteins is preserved in gun4 (Figure 8) together with Psii core (Figure 4) to comparable levels to the wild-type, on a per-cell basis.

The gun4 Mutation Affects Transcription of Nuclear Genes

transcription of nuclear LHC genes was investigated during a dark-to-light shift (Figure 9a) with a time course experiment as in Figure 4B. the level of the transcripts for cP29, cP26, and lhcBM5 (Figure 9a) and the level of the corresponding

polypeptides (Figure  4B) displayed a similar pattern in the wild-type, with an initial decrease upon illumination followed by a recovery after a period of light acclimation. in contrast, although the accumulation of the corresponding polypeptides was decreased in the gun4 mutant (Figure 4B, gun4), the LHC transcript level was similar or even higher than in the wild-type (Figure  9a, gun4) and a higher steady-state level of transcripts in light-acclimated cells of gun4 was determined by q-PcR (ll in Figure 9B). since the transcript level of the LHC genes was not reduced in gun4, it is likely that accumulation of chlorophyll-binding lhc subunits of both photosystems was decreased through a posttranscriptional mechanism because of limited chlorophyll availability. During the dark-to-light shift, regulation of nuclear gene expression appeared to be maintained in gun4 as suggested by the similar RNa profiles of mutant and wild-type. however, the level of the nuclear LHC transcripts was no longer coordinated with the accumulation of chlorophyll in the chloroplast. Nevertheless, plastid-to-nucleus signaling was not completely suppressed in the mutant. transcription of LHC genes is down-regulated in the wild-type in excess light (teramoto et al., 2002). the gun4 mutant was still able to down-regulate LHCBM1 and CP29 transcription upon transfer to high light (hl, 1 h 400 µmol photons m2 s1, Figure 9B).

Figure 7. Photochemical Yield of Photosystem ii (ϕPsii).

it was calculated as (Fm’ – Fs)/Fm’ with an actinic light of 100 µmol photons m2 s1 in the absence (white bars) or after addition (gray bars) of pgal.(A) cells grown at 30 µmol photons m2 s1 and dark-adapted before the measurement.(B) cells grown as in (a) but submitted to a hyper-osmotic stress (0.5 M sucrose).

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to further characterize transcripts, beside LHC genes, whose expression is possibly regulated by tetrapyrrole-dependent plastid signal(s), transcriptome sequencing was performed in dark-grown cells. We chose to use dark-grown cells to avoid effects on gene expression due to other signaling pathways such as those triggered by ROs and the reduction/oxidation state of the photosynthetic electron transport chain in the light. thus, under these conditions, transcript accumulation in the dark likely depends on the impaired tetrapyrrole flow; 803 genes changed their expression level more than threefold (508 induced and 295 repressed) in gun4 as compared to wild-type (supplementary Data). in particular, we found 69 nuclear

genes encoding predicted chloroplast proteins (based on Arabidopsis orthology, http://pathways.mcdb.ucla.edu, cellular compartment annotation; lopez et al., 2011) that are differen-tially expressed in gun4 by more than threefold as compared to wild-type (listed in table 2). interestingly, these genes are all up-regulated in the mutant except for two genes that are down-regulated (cre16.g681251.t1.1 with no functional anno-tation and cre12.g517700.t1.1, a predicted chlorophyll(ide) b reductase). the gun4 mutation led to an up-regulation of all LHC family members and of several genes encoding enzymes of the tetrapyrrole pathway as compared to wild-type (table 2 lists only those that changed their expression more than three-fold in the dark). in addition, we found genes involved in the metabolism of carbohydrates and nitrogen compounds, as well as transcripts encoding metabolite transporters between chloroplast and cytoplasm, redox, and calcium sensing agents (table 2). Different nuclear gene transcript levels could be asso-ciated with different activities of transcription factors and/or chromatin remodeling factors that, in turn, could be themselves differentially regulated at the transcriptional level. indeed, we found genes involved in signaling (two MaPKK-related pro-tein kinases, a calcium/calmodulin- dependent protein kinase, a taP42-like protein), control of gene expression (a MYB-like transcription factor, a F-BOX/WD40 domain protein), and chro-matin reorganization (histone h1, h2B, h3, and a nucleosome assembly protein) that are differentially expressed in gun4 as compared to wild-type (listed in table 3). in particular, protein

Figure 9. transcript levels in cw15 and gun4.

(A) RNa blot analysis during light acclimation of gun4 and cw15. samples were collected at time 0 (Dark) and after 30 min, 1 h, 2 h, 8 h, and 24 h of continuous illumination at 60 µmol photons m2 s1.(B) Real-time Rt–PcR analysis of the transcript content of lhcBM1 and cP29 in cw15 and gun4. in the histogram are reported the differences between the cycles in which the syBR Green fluorescence crosses a threshold (–ΔΔct). the error bars refer to different biological replicates for each genotype and condition. Values around zero mean that the expression of the gene is the same in the samples analyzed. Positive values indicate an up-regulation and negative values a down-regulation. Data are normalized with respect to the constitutive expression of 18s rRNa. gun4/cw15 ll, comparison of transcription in gun4 with respect to cw15 at 30 µmol photons m2 s1. cw15 hl/ll and gun4 hl/ll, comparison of transcription in cw15 and gun4, respectively, after exposure to 400 µmol photons m2 s1 for 1 h relative to the sample in low light.

Figure 8. analysis of PtOX accumulation by immunoblot from total Protein extracts or thylakoids.

the amount (μg) of chlorophyll loaded in each lane is indicated. PRK is used as internal control in total protein extracts, while it is not present in thylakoids since it is a soluble protein. Numbers 1 and 2 on the left indicate the putative PtOX1 and PtOX2 signals.

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transcript log2fold changea

Nitrogen compound metabolism

cre06.g308500.t1.1 carbamoyl phosphate synthase

–3.245025928

cre28.g776100.t1.1 carbamoyl phosphate synthase

–3.237441823

cre14.g627850.t1.1 Dihydrodipicolinate reductase

–3.095094459

cre09.g410750.t1.1 Nitrite reductase –4.387402846

Carbon metabolism

cre09.g405750.t1.1 carbonic anhydrase –3.012447268

cre12.g513200.t1.1 enolase, Phosphopyruvate hydratase

–3.85435735

cre13.g598750.t1.2 Phosphoglucomutase –3.646195271

Metabolite transport

cre41.g786600.t1.1 Plastidic aDP/atP translocase

–3.325191845

cre17.g713350.t1.1 Oxoglutarate:malate antiporter

–3.119655126

cre01.g045550.t1.1 triose phosphate transporter

–3.297233366

Pigments metabolism

cre04.g215050.t1.1 Beta-carotene hydroxylase

–4.512326991

cre01.g050950.t1.1 geranylgeranyl reductase –3.39672765

Protein folding and proteolisis

cre13.g572900.t1.2 Oligopeptidase a, Protease M3 thimet

–3.835627197

cre10.g466850.t1.1 Peptidyl-prolyl cis-trans isomerase

–3.501406902

Redox reaction

cre02.g087700.t1.1 ascorbate peroxidase –3.865233505

cre10.g422300.t1.1 thioredoxin dependent peroxidase

–5.077206895

Calcium sensing

cre01.g038400.t1.1 calreticulin 2, calcium-binding protein

–3.438606654

cre12.g497300.t1.1 Rhodanese-like ca-sensing receptor

–3.584259821

Other processes

cre02.g145050.t1.1 4-diphosphocytidyl-2-c-methyl-D-erythritol kinase

–3.372730995

cre09.g393200.t1.1 heat-shock protein 70c –4.105406714

cre09.g414000.t1.1 homogenitisate phytyltransferase

–3.816217467

cre01.g002400.t1.2 lipase (class 3) –3.055194732

cre14.g625450.t1.1 MPBQ/MsBQ methyltransferase

–5.252752318

cre14.g628500.t1.2 NaD dependent epimerase/dehydratase

–3.966404735

cre12.g521650.t1.1 Predicted hydrolase/acyltransferase

–3.943291702

Table 2. Nuclear Genes encoding Predicted chloroplast Proteins that change expression more than threefold in gun4 compared to Wild-type in Dark-Grown cells.

transcript log2fold changea

Photosystem, light-harvesting

cre06.g283950.t1.1 lhcBM4 –3.655115217

cre03.g156900.t1.1 lhcBM5 –4.079787046

cre16.g673650.t1.1 lhcB5 (cP26) –3.586547036

cre08.g365900.t1.1 lhcsR1 –4.841006618

cre06.g283050.t1.1 lhca1 –3.716795028

cre12.g508750.t1.1 lhca2 –3.232576085

cre18.g749750.t1.1 lhca3 –3.21382722

cre10.g452050.t1.1 lhca4 –4.11437604

cre10.g425900.t1.1 lhca5 –3.460965544

cre13.g598900.t1.1 lhca6 –4.011165361

cre16.g687900.t1.1 lhca7 –3.611632799

cre06.g272650.t1.1 lhca8 –3.245210149

cre07.g344950.t1.1 lhca9 –3.055814003

cre08.g372450.t1.1 Oxygen evolving enhancer protein 3 (PsbQ)

–3.298091695

cre10.g420350.t1.1 Psae –3.351080857

cre07.g330250.t1.1 Psah –3.432215772

cre17.g724300.t1.1 PsaK –3.077126563

cre07.g334550.t1.1 PsaO –3.323766867

cre07.g346050.t1.1 cRD1 –4.321872641

Tetrapyrrole metabolism

cre03.g158000.t1.1 Glutamate-1-semialdehyde aminotransferase

–3.646676675

cre16.g663900.t1.1 Porphobilinogen deaminase

–3.593326926

cre02.g133050.t1.2 Uroporphyrinogen iii methyltransferase

–4.333558028

cre02.g085450.t1.1 coproporphyrinogen iii oxidase

–3.507236066

cre01.g036950.t1.1 cobalamin-5-phosphate synthase

–3.979488431

cre07.g325500.t1.1 Magnesium chelatase subunit h

–3.607869373

cre12.g510800.t1.1 Magnesium chelatase subunit i

–3.339870277

cre12.g498550.t1.2 MgProtoiX O-methyltransferase

–3.178055711

cre01.g015350.t1.1 light-dependent POR1 –3.314650464

cre12.g517700.t1.1 chlorophyll(ide) b reductase

3.903749694

Nitrogen compound metabolism

cre08.g377100.t1.1 adenylate kinase –4.73351706

cre02.g097900.t1.1 aspartate aminotransferase

–3.588360142

cre12.g489700.t1.1 aspartate/ornithine carbamoyltransferase

–3.66165132

cre12.g528450.t1.1 l-aspartate oxidase –4.218077381

cre03.g180750.t1.1 Methionine synthase –4.475300351

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kinase transcripts were up-regulated, while those for chroma-tin-associated proteins were down-regulated.

DISCUSSIONin this study, we have characterized a gun4 knockout mutant of Chlamydomonas reinhardtii, isolated through random insertion mutagenesis and screening for a pale green/low fluorescence phenotype (Bonente et al., 2011). We confirmed the knockout by sequencing, Rt–PcR and immunoblot analy-sis (Figures 1 and 3a) and rescued the phenotype by trans-formation with a construct expressing GUN4 (Figure  3 and table 1), showing that the phenotype of the present mutant is only due to the identified insertion in the GUN4 gene.

Loss of GUN4 Perturbs the Tetrapyrrole Biosynthesis Pathway

in the present work, we observed a gun4 phenotype, with 50% decrease in chlorophyll content, both in the dark and in the light. Moreover, GUN4 expression directly correlates with chlorophyll accumulation, as indicated by clones R1 and R2, which accumulate different levels of the protein (Figure  3a and table 1). GUN4 accumulation is increased during a dark-to-light shift (supplemental Figure  3a), concomitantly with the activation of the light-dependent chlorophyll biosynthesis pathway. this result is consistent with a role of GUN4 in opti-mizing the rate of chlorophyll synthesis. interestingly, loss of GUN4 in C. reinhardtii seems to have a less severe effect on chlo-rophyll accumulation than in Arabidopsis and Synechocystis gun4 mutants reported to be nearly albino (larkin et al., 2003; sobotka et al., 2008; Peter and Grimm, 2009). therefore, in C.  reinhardtii, chlorophyll biosynthesis appears to be less

dependent on the stimulatory/regulatory role of GUN4, possi-bly due to the ability to synthesize chlorophyll also in the dark. in addition, by its binding of ProtoiX, GUN4 acts at the branch-ing point between chlorophyll and heme pathways and its role during evolution might be somehow different, depending on tetrapyrrole metabolism and partitioning, in spite of an essen-tially conserved physiological role.

transcript log2fold changea

Other processes

cre13.g582200.t1.1 thiamin pyrophosphokinase

–3.421971222

cre13.g607050.t1.1 thiosulfate sulfurtransferase

–3.109572545

cre02.g086100.t1.1 No functional annotation –3.414396092

cre03.g198950.t1.1 No functional annotation –3.486442009

cre05.g233950.t1.1 No functional annotation –3.034641391

cre08.g377950.t1.1 No functional annotation –3.526212298

cre10.g421750.t1.2 No functional annotation –3.498713599

cre10.g446350.t1.2 No functional annotation –4.260028152

cre12.g519300.t1.1 No functional annotation –4.316698183

cre16.g681251.t1.1 No functional annotation 3.45985982

a log2 of the ratio between wild-type and mutant expression level. a positive value means down-regulation in the mutant compare to wt, a negative value means up-regulation in the mutant compare to wt.

Table 3. Nuclear Genes involved in signaling, chromatin Modeling, and control of Gene transcription that change expression more than threefold in gun4 compared to Wild-type in Dark-Grown cells.

transcript log2fold changea

Signaling

cre01.g065750.t1.2 MaPKK-related protein kinase

–4.255087274

cre02.g112500.t1.2 calcium/calmodulin-dependent protein kinase

–4.021980044

cre02.g132150.t1.1 taP42-like family –3.022442077

cre09.g404750.t1.2 scavenger receptor cysteine rich (sRcR) protein

–4.74185701

cre12.g556250.t1.1 septin-like protein –5.626162031

cre13.g599850.t1.2 MaPKK- related protein kinase

–3.682797531

cre17.g702600.t1.2 Putative tyrosine kinase –3.343956592

cre20.g759500.t1.2 Mitogen-activated Protein Kinase

–3.612866459

Chromatin modeling and transcription

cre06.g275350.t1.2 transcription factor, Myb superfamily

7.950095918

cre06.g310550.t1.1 GiY-YiG catalytic domain, nuclease

5.129946377

cre07.g347900.t1.2 F-BOX and WD40 domain protein

3.0208827

cre09.g386400.t1.1 Ubiquitin-activating enzyme e1

–3.291160004

cre10.g419750.t1.1 DNa-directed RNa polymerase ii

–3.282754244

cre29.g778700.t1.1 DNa binding protein –4.101289471

cre64.g793000.t1.1 DNa PhOtOlYase/cryptochrome

–3.49089

Chromatin assembly

cre06.g274050.t1.2 histone h3 5.75639867

cre11.g471800.t1.2 Nucleosome assembly protein

3.226266801

cre13.g567450.t1.1 histone h1 4.073284805

cre17.g711750.t1.1 histone h2B inf

cre04.g216650.t1.2 No functional annotation –4.406782207

cre05.g247200.t1.1 No functional annotation –3.204357553

cre12.g551900.t1.1 No functional annotation –3.672451229

a log2 of the ratio between wild-type and mutant expression level. a positive value means down-regulation in the mutant compare to wt, a negative value means up-regulation in the mutant compare to wt.

Table 2. continued

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although its effect on chlorophyll content was mild, the gun4 mutation in C. reinhardtii impaired tetrapyrrole flow in the biosynthetic pathway and led to accumulation of ProtoiX (table  1). accumulation of ProtoiX was also reported in C. reinhardtii strains mutated in the Mgch enzyme (100-fold increase in ProtoiX in the dark) (chekounova et al., 2001; von Gromoff et al., 2008) and in the Synechocystis gun4 mutant (sobotka et al., 2008). in contrast, gun4 and gun5 (for Mgch) mutants of Arabidopsis showed no or only slightly higher ProtoiX level than wild-type (Mochizuki et al., 2008; Moulin et al., 2008). this lack of ProtoiX accumulation in Arabidopsis was attributed to reduced levels of the enzyme activities involved in the early steps in tetrapyrrole synthesis medi-ated through a negative feedback control (Papenbrock et al., 2000). the recent discovery of a FLU-like gene (FLP) in C. rein-hardtii showed that FlP proteins regulate chlorophyll syn-thesis in response to light and tetrapyrrole level in algal cells (Falciatore et al., 2005), in a similar way to FlU in A. thaliana (Meskauskiene et al., 2001; Meskauskiene and apel, 2002). in particular, overexpression of FlPs was observed in mutants deficient in chlorophyll synthesis that over-accumulate chlo-rophyll precursors (Falciatore et al., 2005). however, in the present work, we show that C. reinhardtii gun4 accumulates high levels of Proto iX (table  1) but expression of FlP was not enhanced (Figure 4a). this result suggests a less efficient feedback control of the pathway in C. reinhardtii. this may be caused by the ability to synthesize chlorophyll in the dark, while higher plants have developed efficient feedback con-trols that become important upon shut-down of chlorophyll synthesis in the absence of light (chekounova et al., 2001).

impaired chlorophyll biosynthesis due to a mutation in Mgch led to an increased flow of tetrapyrroles in the heme branch and two to fivefold elevated heme levels in C.  rein-hardtii (chekounova et al., 2001; von Gromoff et al., 2008). however, in the present gun4 mutant, no increased heme level was observed on a per-cell basis (table  1). consistently, the gun4 mutation in Synechocystis decreased the activities of both Mgch and Fech (sobotka et al., 2008), suggesting different regulation of heme synthesis as compared to Mgch mutants.

Increased Photosensitivity in the Absence of GUN4

increasing light irradiance had negative effects on the growth of C. reinhardtii gun4 (Figure 3B). GUN4 orthologs are present only in species that carry out oxygenic photosynthesis (larkin et al., 2003; Verdecia et al., 2005), suggesting a role in protec-tion from photo-oxidation. accumulation of ProtoiX (table 1), a photosensitizer in its free form, may be responsible for this photosensitivity (sobotka et al., 2008). chlorophyll precursors are present in trace amounts in the dark in the wild-type, but they were not detected in light-grown C.  reinhardtii cells, since the rate of chlorophyll synthesis is increased under the latter condition. in the wild-type, we measured 0.008 pmol of GUN4 protein per nmol of chlorophyll ab (supplemental Figure 3B). this level of GUN4 expression is possibly enough to bind all or at least a major fraction of ProtoiX and MgProtoiX

in vivo, preventing their reaction with oxygen. however, an interesting observation derives from the complementation of the gun4 mutant. the amount of GUN4 protein in both R1 and R2 strains (Figure 3a) was sufficient to reduce the accu-mulation of ProtoiX to a level similar to that of the wild-type (table 1). Yet, the R1 strain accumulated more GUN4 protein (Figure 3a), had higher chlorophyll content (table 1), and was more light-resistant than the R2 strain (Figure 3B). the pho-tosensitive phenotype at increasing light intensities thus cor-relates with GUN4 protein level but not with ProtoiX amount, supporting a distinct role of GUN4 in photoprotection, inde-pendently from the accumulation of porphyrin metabolites. consistently, A. thaliana gun4 is light-sensitive as well (larkin et al., 2003), although no significant accumulation of ProtoiX occurs (Mochizuki et al., 2008; Moulin et al., 2008). at least in C. reinhardtii, the major effect of the gun4 mutation on the organization of the photosynthetic apparatus was the strong decrease in photosystem  i activity (Figure 6) and accumula-tion (Figures 4 and 5) without significantly affecting photo-system ii. although PtOX activity is effective in oxidizing the plastoquinol pool, its electron transport rate only accounts for 20% of photosystem ii electron transport in the mutant, thus leaving a large imbalance between the excitation pres-sure of photosystem ii and photosystem i activity, particularly at high light intensity. these observations suggest that the gun4 photosensitive phenotype is due not only to the accu-mulation of ProtoiX, but also to photoinhibition of photo-system ii. it is consistent with the photosensitive phenotype of Chlamydomonas photosystem  i-deficient mutants (Farah et al., 1995; hippler et al., 2000), although it is attenuated by the function of PtOX as electron sink (cournac et al., 2000).

Uncoupling of LHC Transcription from Chlorophyll Accumulation in the gun4 Mutant

Normally, chlorophyll biosynthesis in the chloroplast is coor-dinated with the expression of the nuclear genes encoding chlorophyll-binding lhc proteins (Pogson et al., 2008). GUN4 stimulates the rate of chlorophyll biosynthesis and could be involved, directly or indirectly, in coordinating tetrapyrrole metabolism with expression of photosynthesis-related genes in the nucleus. here, we show that, in the C. reinhardtii gun4 mutant, despite a lower chlorophyll content than in wild-type (table 1) and a reduced accumulation of lhc polypep-tides (Figure  4), LHC transcripts were not down-regulated. Remarkably, their steady-state level was higher than in wild-type under normal growth conditions (Figure 9a, ll in Figure 9B). these data support an impaired plastid-to-nucleus signaling in gun4, coupling nuclear genome expression to chloroplast metabolism that is evident under physiological conditions. it has been proposed that the activity of GUN4 in regulating transcription of nuclear genes could depend on its capacity to change the concentration of one or more tetrapy-rrole intermediates, namely Mg–Proto iX and/or heme (strand et al., 2003; Woodson et al., 2011). however, the level of nei-ther the substrate nor the product of Mgch can be correlated

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with LHC transcription (Mochizuki et al., 2008; Moulin et al., 2008; Meinecke et al., 2010). here, we report no correlation between up-regulation of LHC and heme level (table 1). thus, it seems unlikely that tetrapyrrole-dependent retrograde signaling relies on the tetrapyrrole species mentioned above.

although nuclear gene regulation is partially impaired in gun4, at least one distinct retrograde signaling pathway is still functional, since LHC RNa levels were down-regulated upon transfer to high light not only in the wild-type, but also in gun4 (hl in Figure 9B). Nuclear gene expression in gun4 could still be sensitive to other chloroplast signals such as ROs and/or the reduction/oxidation state of the photosynthetic electron transport chain as previously proposed (Pogson et al., 2008; Kleine et al., 2009).

the antenna components of both photosystems were reduced in gun4 (Figure  4 and supplemental Figure  1), despite increased transcript levels (Figure 9 and supplemental Figure 2). Possibly, the limitation in chlorophyll supply impairs pigment–protein stability—an effect previously observed in the case of limiting levels of chlorophyll  b (havaux et al., 2007) or xanthophylls (Dall’Osto et al., 2007). in gun4, both chlorophyll  a and chlorophyll  b are limited, thus impairing stability not only of lhc proteins, but also of the photosys-tem  i core complex (Figure  4). Depletion of lhca subunits was accompanied by further destabilization of photosystem i supercomplexes in the mutant, as observed by green gel anal-ysis (Figure 5a) and 77-K fluorescence spectra (Figure 5D). the photosystem  i supercomplex is synthesized in steps during which core and lhci subunits are assembled. chlorophyll-binding subunits at the interface stabilizing the association between the core and the antenna systems could be particu-larly affected by the restrained chlorophyll biosynthesis in the mutant (Ozawa et al., 2010).

We cannot exclude that GUN4 is indirectly involved in retrograde signaling in C.  reinhardtii. however, it is very unlikely that this effect is mediated through the decreased accumulation of chlorophyll because the albino3 mutant of Chlamydomonas, which accumulates less than 30% chloro-phyll compared to the wild-type, displays a very different phenotype (Bellafiore et al., 2002). in contrast to gun4, it is not light-sensitive and grows better under high light but only poorly under low light.

Decrease of Linear Electron Transport between Photosystems Is Accompanied by Increased Electron Flow through PTOX in the gun4 Mutant

the gun4 mutation in C. reinhardtii affects the correct assem-bly, function, and/or organization of the photosynthetic apparatus, which explains the observed slower photoauto-trophic growth (Figure 3B). Destabilization of photosystem i supercomplexes (Figure  5a and 5D) and disruption of elec-tron transport between photosystem  ii and photosystem  i (Figure 6) were observed in gun4. ϕPsii, indicative of electron transport rate, is reduced in the mutant compared to wild-type (Figure 7a). Moreover, PtOX activity accounts for 20% of

total photosystem ii-generated electron flow in gun4 versus only 2% in wild-type (Figure 7a and 7B). PtOX is expressed in the wild-type as well (Figure 8); however, it only appears to play a significant role in decreasing photosystem  ii exci-tation pressure under conditions of limited linear electron flow in the mutant (Figure 7). Besides its function in oxygen uptake during chlororespiration (Peltier and cournac, 2002), a role of PtOX as an electron sink during photosynthesis has so far only been reported in Chlamydomonas mutants lack-ing either photosystem i or cytochrome b6f (cournac et al., 2000), in marine cyanobacteria, and in a marine strain of the green alga Ostreococcus isolated from a low-iron oligo-trophic marine environment that reduces photosystem i level (Bailey et al., 2008; cardol et al., 2008). a similar contribu-tion of PtOX to photoprotection was observed in mountain species as part of a stress response to high light and low temperature (streb et al., 2005). in contrast, an Arabidopsis photosystem i-depleted mutant was unable to avoid overre-duction of plastoquinone in the light and photoinhibition, indicating that the capacity of electron transfer to oxygen catalyzed by PtOX in higher plants plays a minor role as an alternative pathway for electron transport (Melis, 1999; haldrup et al., 2003; Kuntz, 2004). IMMUTANS and GHOST PtOX-defective mutants of Arabidopsis and tomato, respec-tively, revealed a crucial role of PtOX in carotenoid biosynthe-sis, namely for phytoene desaturation (Giuliano et al., 1993; carol et al., 1999; Wu et al., 1999). the present work shows that PtOX, although playing a minor role in photosynthesis in the Chlamydomonas wild-type, has the potential to sustain a significant electron flow rate under particular conditions. this flexibility of the photosynthetic apparatus contributes to overcoming the effects of limited chlorophyll availability and to attenuating the photo-oxidative stress to higher light intensity in gun4.

A Large Set of Nuclear Genes Is Differentially Transcribed in the gun4 Mutant, besides the LHC Family

a large alteration in the transcription profile occurs in C. rein-hardtii gun4 under physiological conditions: 803 genes change their expression more than threefold compared to wild-type in dark-grown cells (supplementary Data). in Arabidopsis, instead, de-repression of photosynthesis-related genes was only observed in gun mutants upon norflurazon treatment that disrupts chloroplast function. in this case, particularly responsive are LHC genes, RBCS1 and RBCS2, encoding small subunits of Rubisco, and HEMA, encoding the glutamyl–tRNa reductase, which catalyzes an early step in ala syn-thesis (strand et al., 2003; Mochizuki et al., 2008; Moulin et al., 2008). Remarkably, we show that RBCS and HEMA genes are not differentially expressed in C. reinhardtii gun4 as com-pared to wild-type, while the mRNas of all members of the LHC gene family and of several genes encoding enzymes of the tetrapyrrole pathway over-accumulate in the mutant (table 2). in Arabidopsis, GUN4 was proposed to positively reg-ulate tetrapyrrole biosynthesis at the posttranslational level,

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while no transcript regulation was observed under normal growth conditions (Peter and Grimm, 2009). here, we show that gene expression related to the tetrapyrrole pathway is modulated also at the level of transcription, but no longer coordinated to the rate of chlorophyll biosynthesis in the absence of GUN4 in C. reinhardtii.

When the analysis was extended to all the genes predicted to encode chloroplast proteins, we found transcripts encod-ing an ascorbate peroxidase (cre02.g087700.t1.1), a thiore-doxin-dependent peroxidase (cre10.g422300.t1.1), and two proteins involved in calcium sensing (calreticulin 2, calcium-binding protein cRt2, cre01.g038400.t1.1 and Rhodanese-like ca-sensing receptor, cre12.g497300.t1.1) to be up-regulated in gun4 as compared to wild-type. Many genes involved in nitrogen metabolism, but fewer genes encoding enzymes involved in carbohydrate metabolism, are also differentially expressed in gun4 (table  2). in particular, three metabolite transporters are up-regulated in gun4 as compared to wild-type, namely the oxoglutarate:malate antiporter (cre17.g713350.t1.1), the triose phosphate transporter (cre01.g045550.t1.1), and an aDP/atP translocase (cre41.g786600.t1.1). Photosynthates have been shown to affect nuclear gene expression (Jang and sheen, 1997; Rolland et al., 2006) and metabolite transport has been proposed to communicate the metabolic state of the chloroplast to the nucleus and to act in retrograde signaling (Rolland et al., 2006; Kleine et al., 2009). however, it remains to be seen whether the proteins of these genes are also up-regulated in the gun4 mutant.

in Arabidopsis, plastid-derived signals seem to converge into a common pathway and the negative aBi4 transcrip-tion factor, first studied for its involvement in the response to abscisic acid, as well as the positive atGlK1 transcription factor have been proposed to regulate expression of photo-synthesis-related genes (Koussevitzky et al., 2007; Kakizaki et al., 2009). however, the sequence motifs that mediate the response to tetrapyrrole metabolism in higher plants and C.  reinhardtii are not related and the binding factors to the plastid-response element in C. reinhardtii are still elu-sive (von Gromoff et al., 2008). in the present transcriptome analysis, we found some nuclear genes involved in signaling, chromatin remodeling and control of gene transcription that change their expression as compared to wild-type (table 3). these include the genes of several kinases, overexpressed in gun4, that might participate in signal transduction and of a taP42-like protein (cre02.g132150.t1.1). taP42-like proteins regulate ubiquitination and stability of protein phosphatases 2a involved in the regulation of numerous cell signaling pathways (McDonald et al., 2010). in contrast, other genes were down-regulated in gun4. they include a transcription factor belonging to the Myb superfamily (cre06.g275350.t1.2) and an F-BOX/WD40 domain protein (cre07.g347900.t1.2). as the activity of WD40-repeat proteins is regulated by photoreceptors and mediates repression of photomor-phogenesis in dark-grown Arabidopsis seedlings by control-ling ubiquitination and proteolysis of transcription factors

(Gruber et al., 2010; Nixdorf and hoecker, 2010; Fankhauser and Ulm, 2011), it will be interesting to test whether this particular WD40 domain protein is indeed down-regulated in the gun4 mutant and whether it could act in a similar way and control transcription of photosynthesis-related genes. consistently, transcripts of a ubiquitination enzyme e1 (cre09.g386400.t1.1), of three histones, and of a nucleo-some assembly protein are down-regulated in the mutant as compared to wild-type (table 3), raising the possibility that their products influence chromatin remodeling during de-repression of transcription.

METHODSChlamydomonas Strains and Culture Conditions

the wild-type strain utilized was cw15 mt– (mating type minus) (harris, 1989). the gun4 mutant was obtained by insertion mutagenesis of cw15 mt– with linearized psl18 plasmid con-taining the paromomycin resistance cassette (Bonente et al., 2011). Wild-type s34 mt, kindly provided by F.a. Wollman (UMR cNRs/UMPc institut de Biologie Physico-chimique Paris) was employed for backcross analysis in Figure 2 (harris, 1989). the insertion was mapped by sequencing the 5' flank-ing region by inverse PcR (Ochman et al., 1988; Bonente et al., 2011). the 3' flanking region was amplified with oligos promPsaDFw and G4p3'Rv (supplemental table 2).

all C. reinhardtii strains were grown at 25°c in taP or mini-mal hsM media (harris, 1989). the light conditions are speci-fied in the ‘Results’ section. For the growth tests in Figure 3B, 5 µl of 1·107, 1·106, 1·105, and 1·104 cells ml1 were spotted on solid taP or hsM medium and subjected to the light condi-tions indicated.

Cloning of C. reinhardtii GUN4 cDNA

the GUN4 encoding sequence was amplified by Rt–PcR with the specific oligos G4ORF–Fw and G4ORF–Rv (supplemental table 2), containing, respectively, a Ndei and a Nrui restric-tion site at the 5' end, and cloned downstream of the PSAD promoter in the psl18 vector (Fischer and Rochaix, 2001) as a fusion with ha-tag at the c-terminus. a control PcR was performed on CBL cDNa using GP-Fw and GP-Rv oli-gos (supplemental table  2). the GUN4 sequence encoding the mature protein (without the first 45 amino acids corre-sponding to the predicted chloroplast transit peptide) was amplified by Rt–PcR with the G4wt-Fw and G4wt-Rw prim-ers (supplemental table  2) and cloned into the BamHi and Xhoi restriction sites of the petMhys vector for expression in E. coli (derived from the pet28a, Novagen, it retains the c-terminal 6Xhis tag, preceded by a thrombin cleavage site).

RNA Analysis

total RNa was prepared from algal cells using the tri-Reagent™ siGMa according to the manufacturer’s manual. For the Northern blot in Figure 9a, RNa electrophoresis was

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performed as described in sambrook et al. (1989) and trans-ferred overnight to a N nylon membrane (Pharmacia). after transfer, RNa was UV-cross-linked and hybridized with spe-cific probes radiolabeled with α32P-dctP (hartmann analytic). hybridization and washing were performed at 65°c. signals were detected with a phosphorimager. the probes for the minor antennae were amplified by PcR on genomic DNa. the oligos used are listed in supplemental table 3 and the regions chosen have no homology with sequences of other antenna proteins. the CBL gene was used as a loading control and the probe was amplified with the same oligos as for the Rt–PcR (supplemental table 2). For the q-PcR in Figure 9B, total RNa was reverse-transcribed with random nonamers (siGMa). conditions and primers (supplemental table  4) for the fol-lowing PcR were the same as in teramoto et al. (2002, 2004). the amount of the amplified DNa was monitored by syBR Green fluorescence at the end of each cycle (Platinum® sYBR® Green qPcR super Mix-UDG with ROX, invitrogen). analysis of relative gene expression data was performed using the –ΔΔct method (livak and schmittgen, 2001). the relative abundance of 18s rRNa was used as the internal standard.

For RNa-seq (Mortazavi et al., 2008; Nagalakshmi et al., 2008), RNa quality and quantity were determined using a Nanodrop 2000 instrument (thermo scientific) and a Bioanalyzer chip RNa 7500 series ii (agilent). a non-direc-tional illumina RNa-seq library was prepared with truseq RNa sample Prep Kits (illumina). We modified the proto-col by adding a size selection step after the library enrich-ment. library fragments were separated on a 2% certified low-range ultra agarose gel (Biorad) and a slice of gel of 350–600  bp was extracted with a Qiaquick Gel extraction Kit (Qiagen). We modified the extraction step by dissolving excised gel slices at room temperature to avoid under-rep-resentation of at-rich sequences (Quail et al., 2008). library quality control and quantification were performed with a Bioanalyzer chip DNa 1000 series ii (agilent). libraries were sequenced on a hiseq 1000 sequencer (illumina) and paired end 100-bp sequences were generated. sequences were then aligned to the C. reinhardtii genome with tophat alignment software (trapnell et al., 2009), which is designed to analyze RNa-seq data and is able to perform spliced alignments. expression levels were evaluated with cufflinks (trapnell et al., 2010; Roberts et al., 2011). statistically significant dif-ferentially expressed genes were identified by using the R package Deseq (anders and huber, 2010). Functional anno-tation was based on the algal Functional annotation tool (http://pathways.mcdb.ucla.edu; lopez et al., 2011). We con-sidered transcripts showing a minimum log2 fold change of three (absolute value) between gun4 and wild-type and a P-value  0.05%.

Protein Analysis

total protein extracts were obtained from exponential grow-ing cells collected and re-suspended in lysis buffer (50  mM

tris-hcl, ph 6.8, containing 2% sDs and 10 mM eDta and a protease inhibitor cocktail (sigma-aldrich)). cells were incu-bated for 30  min at room temperature and centrifuged at 13 000 g for 30 min at 4°c. supernatants contain total protein extracts. concentrations of extracts were assayed by colori-metric measurement with bicinchoninic acid (sigma-aldrich). For analysis on a per-chlorophyll basis, cell pellets were directly re-suspended in loading buffer (1% running buffer, 2% sDs, 5% β-Me, 10% glycerol) and chlorophylls were extracted in 80% acetone for quantification (Porra et al., 1989). For immu-noblot analysis, proteins were separated by sDs–PaGe, trans-ferred to a nitrocellulose membrane, detected with specific anti-sera, and visualized by the ecl chemiluminescence sys-tem (Durrant, 1990) or by the colorimetric reaction of alkaline phosphatase. the recombinant C. reinhardtii GUN4 protein, expressed in E. coli in the present work, was used to obtain the anti-serum in rabbit. the antibody used to detect PtOX in Figure 8 was a kind gift by Marcel Kuntz. For the native electrophoretic separation of chlorophyll-containing photo-synthetic complexes in Figure 5a, unstacked thylakoids were obtained from cultures grown in taP at 30 µe. cells were col-lected during the exponential phase, frozen in liquid nitro-gen, and re-suspended in 0.1  M tricine KOh, ph  7.8, 0.5% milk powder before sonication (two cycles of 5 s for a strain without cell wall). the sample was centrifuged for 10 min at 10 000 g and the pellet re-suspended in 25 mM hepes KOh, ph 7.5, 10 mM eDta. Debris was removed by centrifugation for 1 min at 500 g and, finally, thylakoids were collected at 10  000  g and re-suspended in 25  mM hepes KOh, ph  7.5, 10 mM eDta, 50% glycerol. thylakoids were partially solubi-lized by different amounts of the detergent αDM (n-dodecyl-α-D-maltoside) at a final concentration of 0.5 mg chl ml1 and finally separated by a native DeRiPhat–PaGe according to Peter et al. (1991).

Pigment Analysis

chlorophyll and carotenoids were extracted in 80% acetone. separation and quantification were performed by RP-hPlc c18 column (lagarde et al., 2000) and by fitting of the absorb-ance spectrum of the acetone extract with spectra of indi-vidual pigments (croce et al., 2002) and recorded using an aminco DW-2000 spectrophotometer (slM instruments). For ProtoiX determination, recovered pellets (about 200  mg) were lysed by re-suspension in acetone: 0.1  M Nh4Oh (9:1, vol/vol), 7  ml  g1. these lysates were clarified by centrifu-gation at 16 000 g for 10 min at 4°c. chlorophylls and fully esterified tetrapyrroles were removed from the resulting supernatants by hexane extraction. Monocarboxylic and dicarboxylic tetrapyrroles were separated by diethyl ether extraction (Rebeiz, 2002). Proto iX was quantified using fluo-rescence spectroscopy as previously described (Rebeiz, 2002). Proto iX purchased from Frontier scientific was used to con-struct standard curves. the content of non-covalently bound

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heme was determined as previously described (Weinstein and Beale, 1983; von Gromoff et al., 2008).

Spectroscopy

Analysis of P700 Light-Induced Oxidation In Vivo

cells grown in taP under an irradiance of 30  µmol pho-tons  m2  s1 were collected during the exponential phase and re-suspended to a final OD680nm of 1.2 in 20 mM hePes, ph  7.5 with 20% Ficoll (w/v) to prevent sedimentation. all samples examined were dark-adapted, with continuous shak-ing (state i), for at least 1 h before use. samples were then subjected to white light of 400  µmol photons  m2  s1 for 20 min. the absorption change at 705 nm, due to oxidation of photosystem i reaction center, was monitored during 60 s of illumination (940 µmol photons m2 s1, Jts-10 Joliot-type spectrophotometer). When specified, just before the meas-urement, DcMU (10 µM) was added to inhibit linear electron transport between the photosystems. Data were normalized with respect to the value at the end of the actinic light phase.

Chlorophyll Fluorescence at 77 K

cells grown in taP (30 µmol photons m2 s1 light) were col-lected during the exponential phase and re-suspended in min-imal medium. all samples examined were dark-adapted, with continuous shaking, for at least 1 h before use to reach state i. Only the samples pre-illuminated were then transferred to white light at 400 µmol photons m2 s1 for 30 min. cells were immediately frozen in liquid nitrogen. When defrosted, they were collected and re-suspended in hepes ph  7.5, glycerol 85%. Fluorescence spectra were recorded at 77 K.

Analysis of Chlorophyll Fluorescence at Room Temperature

Measurements of fluorescence parameters were performed with a PaM 101 (Walz, effeltrich, Germany) fluorometer. Wild-type and gun4 cells were grown in taP at 30 µmol pho-tons  m2  s1. cells were collected, re-suspended in 20  mM hePes, ph  7.5 with 20% Ficoll (w/v), and dark-adapted for at least 1 h with continuous shaking (Finazzi et al., 2006). For the hyperosmotic stress (cruz et al., 2001), cells were treated with sucrose (final concentration 0.5 M) for 30 min before the measurement. the samples were then exposed for 2 min to weak illumination with far-red light and with the PaM meas-uring beam for determination of F0 (minimum fluorescence in the dark-adapted state). a saturating pulse (0.8 s) of white light (3000 µmol photons m2 s1) was applied for the determi-nation of the Fm (maximum fluorescence in the dark-adapted state) or Fm′ (maximum fluorescence in the light-adapted state). the quantum yield of Psii in the light (ϕPsii) is calcu-lated as (Fm′ – Fs)/Fm′, where Fs is the steady-state chlorophyll fluorescence level in the light (actinic illumination of 100 µmol photons m2 s1). the maximum quantum efficiency of Psii is

(Fm  –  F0)/Fm (or Fv/Fm). When specified, n-propyl-gallate was added to the sample 5  min before the measurement (final concentration 1 mM; cournac et al., 2000; Bailey et al., 2008).

SUPPLEMENTARY DATAsupplementary Data are available at Molecular Plant Online.

FUNDING

eec project sunbiopath and the caRiVeRONa foundation for grant BiOMasse Di OGGi e Di DOMaNi to R.B. Grant 3100aO-117712 from the swiss National Foundation to J.D.R.

ACKNOWLEDGMENTS

the authors thank centro di Genomica Funzionale of the University of Verona for RNa-seq; stefano cazzaniga for the production of the polyclonal antibody against C. reinhardtii GUN4; Manuela Mantelli, who contributed to the early effort in mutagenesis and screening of C.  reinhardtii; and Prof. Marcel Kuntz for the generous gift of the PtOX antibody. No conflict of interest declared.

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