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First published online March 10, 2006; 10.1105/tpc.105.040121 The Plant Cell 18:992-1007 (2006) © 2006 American Society of Plant Biologists A Soluble Carotenoid Protein Involved in Phycobilisome-Related Energy Dissipation in Cyanobacteria
a Unité de Recherche Associée 2096, Centre National de la Recherche Scientifique, Service de Bioénergétique, 91191 Gif sur Yvette, France 1 To whom correspondence should be addressed. E-mail diana.kirilovsky{at}cea.fr; fax 33-1-69088717.
Photosynthetic organisms have developed multiple protective mechanisms to survive under high-light conditions. In plants, one of these mechanisms is the thermal dissipation of excitation energy in the membrane-bound chlorophyll antenna of photosystem II. The question of whether or not cyanobacteria, the progenitor of the chloroplast, have an equivalent photoprotective mechanism has long been unanswered. Recently, however, evidence was presented for the possible existence of a mechanism dissipating excess absorbed energy in the phycobilisome, the extramembrane antenna of cyanobacteria. Here, we demonstrate that this photoprotective mechanism, characterized by blue light–induced fluorescence quenching, is indeed phycobilisome-related and that a soluble carotenoid binding protein, ORANGE CAROTENOID PROTEIN (OCP), encoded by the slr1963 gene in Synechocystis PCC 6803, plays an essential role in this process. Blue light is unable to quench fluorescence in the absence of phycobilisomes or OCP. The fluorescence quenching is not  pH-dependent, and it can be induced in the absence of the reaction center II or the chlorophyll antenna, CP43 and CP47. Our data suggest that OCP, which strongly interacts with the thylakoids, acts as both the photoreceptor and the mediator of the reduction of the amount of energy transferred from the phycobilisomes to the photosystems. These are novel roles for a soluble carotenoid protein.
Carotenoids, the most widely occurring pigments in nature, are essential to microbial, animal, and plant life. These lipophilic macromolecules play diverse roles, functioning as colorants, precursors of visual pigments, and antioxidants. In plant and algal photosynthesis, carotenoids are known to have a dual function in chlorophyll–membrane complexes: harvesting light and photoprotection. The photoprotective mechanisms include (1) reducing the amount of energy funneled to photochemical reaction centers (by screening or thermal energy dissipation), (2) quenching triplet chlorophyll to prevent the formation of singlet oxygen, and (3) directly quenching singlet oxygen.
In cyanobacteria, carotenoids are also associated with proteins devoid of chlorophyll. These water-soluble carotenoid binding proteins were first described by Holt and Krogmann (1981)
The structure of the Arthrospira maxima OCP has been determined to 2.1 Å resolution (Kerfeld et al., 2003
A role for the OCP under stress conditions has been proposed. Indeed, microarray studies indicate that the OCP transcript levels increase by >600% upon transfer to high light (Hihara et al., 2001
By harvesting solar energy and converting it into chemical energy, plants, algae, and cyanobacteria provide food and oxygen that are essential for life on earth. However, excess light can be lethal for photosynthetic organisms, because harmful reactive oxygen species are generated in the photochemical reaction centers when energy absorption exceeds the rate of carbon fixation. To survive, photosynthetic organisms have evolved several protective processes. In plants and algae, dissipation of the excess absorbed energy as heat in the light-harvesting chlorophyll antenna (LHCII) of photosystem II (PSII) decreases the amount of energy funneled to the photochemical centers (for reviews, see Demmig-Adams, 1990
Cyanobacteria do not have the integral membrane chlorophyll-containing light-harvesting complex, LHCII. Instead, light is captured by a membrane extrinsic complex, the phycobilisome, which is attached to the outer surface of thylakoid membranes (Gantt and Conti, 1966
Cyanobacteria have two well-characterized mechanisms that are associated with fluorescence quenching: state transitions and photoinhibition. In photoinhibition, high light intensities induce PSII fluorescence quenching and the irreversible inactivation of PSII caused by damage and degradation of the D1 protein, an essential constituent of PSII (for reviews, see Prasil et al., 1992
It has long been assumed that cyanobacteria do not use an antenna-related NPQ mechanism to decrease the amount of energy funneled to reaction center II (Campbell et al., 1998
In addition, results revealing the existence of a distinct blue light–induced NPQ mechanism, proposed to be associated with the phycobilisome, were first described in 2000 (El Bissati et al., 2000 Here, we report that the carotenoid protein OCP is essential for this phycobilisome-associated NPQ mechanism in Synechocystis PCC 6803. The OCP appears to act as both the photoreceptor and the mediator of a photoprotective energy dissipation mechanism, which decreases the amount of energy arriving at both photosystems from the phycobilisome.
Construction of OCP Mutants To determine the function and cellular location of the OCP, we constructed three mutants of Synechocystis PCC 6803: a mutant lacking the OCP protein, in which the ocp gene (slr1963) was inactivated ( OCP); a mutant containing an OCP–green fluorescent protein fusion under the control of the ocp promoter (OCP-GFP); and a mutant in which the gene slr1964 was inactivated (Slr1964), used as the control strain. An antibiotic resistance cassette was introduced into the HincII site in slr1963 (OCP) or into the ClaI site in slr1964 (Slr1964 and OCP-GFP) by double homologous recombination (Figure 1A
). To confirm the insertion sites and the complete segregation of the mutants, PCR analysis was performed. The amplification of the genomic region containing slr1963, isolated from OCP, Slr1964, and OCP-GFP, with the oligonucleotides car1 and car6 gave fragments of 4.0 kb (OCP and Slr1964) and 4.8 kb (OCP-GFP) (Figure 1B). No traces of the 2-kb fragment, observed in the wild type, were detected in the mutants, indicating complete segregation. The correct position of the antibiotic cassette in OCP and Slr1964 was controlled by PCR amplification of slr1963 by the oligonucleotides car1 and car2 (Figure 1B). In the OCP mutant, the 3-kb PCR fragment indicated that slr1963 was interrupted by the antibiotic cassette, whereas this gene was not interrupted in Slr1964 (0.9-kb PCR fragment). To check the position of the antibiotic cassette in this mutant, the 4-kb PCR fragment obtained by amplification with the oligonucleotides car1 and car6 was digested by the restriction enzyme HincII. As expected, digestion of the Slr1964 PCR fragment gave four fragments of 2 kb, 800 bp, 750 bp, and 440 bp, confirming that in this mutant slr1964 is interrupted by the antibiotic cassette (Figure 1C).
Phenotype Conferred by OCP under High Light IntensitiesWe compared the behavior of OCP and wild-type cells under high-intensity white light. When fluorescence was measured with a PAM fluorometer during saturating light illumination, a faster quenching of fluorescence was observed in wild-type cells than in OCP cells. Figure 2
shows room temperature fluorescence traces in Synechocystis wild-type and OCP cells illuminated by different light intensities. Dark-adapted wild-type and OCP cells presented a characteristic low level of maximal fluorescence (Fm). Upon illumination with low intensities of white light, a maximal level of Fm was reached in both strains (Figure 2A). This increase of fluorescence is related to a state 1 transition induced by the oxidation of the plastoquinone pool upon illumination. Subsequently, exposure of cells to saturating white light intensities induced fluorescence quenching. A very fast quenching of the maximal fluorescence (Fm') was observed in wild-type cells, especially during the first minutes of high light illumination. The steady state (Fs) and minimal (Fo) fluorescence levels also decreased (Figure 2). In OCP cells, the quenching of Fm' was slower than in the wild type, and no quenching of Fo was detected (Figure 2).
When dark-adapted cells were exposed directly to high intensities of white light, a slight increase of Fm' was initially detected, probably attributable to oxidation of the plastoquinone pool and partial state 1 transition, followed by fluorescence quenching (Figures 2B and 2C). The quenching generated in OCP cells was not reversible in darkness (Figure 2C). By contrast, the large Fm' and Fo decrease observed in wild-type cells was almost completely reversible in the dark in the presence of chloramphenicol, a protein synthesis inhibitor; this finding strongly suggested that the observed quenching was not related to D1 protein damage, which is associated with photoinhibition (Figure 2B). These results implied that, in wild-type cells, an induction of a reversible antenna-associated NPQ occurs in high intensities of white light that could protect PSII from photoinhibition. By contrast, in OCP, the decrease of maximal fluorescence appeared to correlate with photoinhibition and not with NPQ.
Indeed, when the sensitivity of
Blue-Green Light–Induced Quenching and the OCP The unexpected fluorescence characteristics of the OCP mutant prompted us to investigate the possible relationship between the OCP and the blue-green light–induced NPQ reported previously (El Bissati et al., 2000 ). Blue-green light, depending on its intensity, may be involved in state transitions or in NPQ (El Bissati et al., 2000). Illumination of dark-adapted wild-type and OCP cells by dim blue-green light (which preferentially excites PSI) increased fluorescence levels, indicating a transition to state I induced by the oxidation of the plastoquinone pool (Figure 4
). Subsequently, exposure of dim light–adapted cells to high blue-green light intensities induced a quenching of all fluorescence levels in the wild type but not in OCP (Figures 4A and 4B), even under very high light intensities. Similar characterization of the mutant in which the slr1964 gene, located immediately downstream of the ocp gene, was disrupted (Slr1964) confirmed that the inhibition of NPQ is attributable to the absence of the OCP and not to the lack of transcription of slr1964 or to other polar effects of the mutation (Figure 4C).
Transition to state II was not affected in OCP. Illumination by orange light (which preferentially excites PSII) of wild-type and OCP cells in state I (adapted to low intensities of blue-green light) induced a decrease of the Fm' level, characteristic of state II transition, in both strains (Figures 4D and 4E). Collectively, these data demonstrate that absence of the OCP inhibits the blue-green light–induced NPQ and does not affect state transitions.
Is the OCP-Associated NPQ Related to Phycobilisomes or to Chlorophyll Antennae?
OCP-Associated NPQ in Phycobilisome and PSII-Deficient Mutants To substantiate the hypothesis that the OCP is specifically involved in a phycobilisome-dependent NPQ mechanism, additional mutants were characterized: (1) lacking phycocyanin (CK, cpc operon) (B. Ughy and G. Ajlani, unpublished data); (2) lacking phycocyanin and OCP (CK-OCP) (this work); (3) lacking allophycocyanin (AB, apcAB) (Ajlani et al., 1995); (4) lacking phycobilisome (PAL, apcAB, apcE, PC–) (Ajlani and Vernotte, 1998); (5) lacking PSII but retaining intact phycobilisome (CP47 [this work] and CP43-psbD; psbDI/C/DII– [Yu and Vermaas, 1990]); (6) lacking both the OCP and PSII (CP47-OCP) (this work); and (7) lacking IsiA (IsiA) (this work). The construction of the new mutants used in this study is described in Methods.
In CK (lacking phycocyanin), the PSII antenna is composed of the phycobilisome core containing allophycocyanin. In this mutant, blue-green light still induced a reversible fluorescence decrease (Figure 6A
). The NPQ was smaller and the recovery faster than in the wild type. This slight fluorescence quenching was completely inhibited in the absence of the OCP (Figure 6B). In PAL, devoid of phycobiliproteins, and in
In the CP47 mutant, which lacks PSII reaction centers and contains only traces of unconnected CP43 (Eaton-Rye and Vermaas, 1991), fluorescence at room temperature is almost entirely emitted by the phycobilisomes. As expected, because of the lack of active PSII, no variable fluorescence was detected in this mutant. In dark-adapted CP47 cells, blue-green light induced a large amount of fluorescence quenching that recovered in the dark in the presence of a protein synthesis inhibitor (Figures 7A
and 7B). The quenching of the 660-nm emission fluorescence band was even larger than that observed in wild-type cells (Figure 7A).
The fluorescence quenching increased with blue-green light intensity in wild-type and CP47 cells (Figures 7C and 7D). Blue-green light up to 160 µmol·m–2·s–1 induced the transition to state I in dark-adapted, low-fluorescence wild-type cells (data not shown) and did not induce any quenching in the CP47 mutant (Figure 7D). Above this intensity, fluorescence quenching was induced, and it increased with light intensity (Figures 7C and 7D). Green light also induced fluorescence quenching with high efficiency in dark-adapted and low-light-adapted cells (Figures 7C, 7E, and 7F). By contrast, orange-red light did not induce any NPQ (Figures 7E and 7F). Similar results were obtained with the CP43-PsbD mutant, lacking both interior chlorophyll antennas (CP43 and CP47) and PSII (Yu and Vermaas, 1990) (data not shown). In the CP47-OCP double mutant, lacking both the OCP and the PSII core, even very high intensities of blue-green light were unable to induce fluorescence quenching (Figure 7D). We also examined the relationship between the OCP-associated NPQ and the NPQ mediated by the IsiA chlorophyll binding protein. Figure 7H shows that in a mutant lacking the IsiA protein, blue-green light induced NPQ similar to that induced in the wild type, demonstrating that this protein is not involved in the OCP-mediated process.
Thus, the blue-green light–induced NPQ associated with OCP can occur in the absence of the chlorophyll core antenna (CP43, CP47, and IsiA) as well as in the absence of reaction center II. Moreover, the kinetics of Fm' quenching induced by strong blue-green light were similar in the absence or presence of DCMU, a PSII electron transport inhibitor, or nigericin, an uncoupler (Figure 7G). These chemicals had no observable effect on the recovery kinetics (Figure 7G). This finding indicates that the cyanobacterial NPQ induced by blue-green light was not related to the redox state of the plastoquinone pool or to
The OCP Is Associated with Thylakoids and Phycobilisomes
Cells containing the OCP-GFP fusion protein were also studied by immunogold labeling with analysis by electron microscopy. Visualization of the anti-GFP antibodies showed that most of the OCP-GFP fusion proteins were localized in the interthylakoid cytoplasmic region, the phycobilisome side of the membranes (Figures 8B and 8C, panel 1). The density of the gold particles in the cytoplasmic interthylakoid region (which represents 65% of the cellular surface) was significantly higher (average = 46.33 ± 3.93 particles/µm2) than in the rest of the cytoplasm (average = 17.06 ± 1.78 particles/µm2; P < 10–6). Thus, 82% of the gold particles were in the thylakoid region. In the absence of the primary antibody, no labeling was observed (Figure 8C, panel 2). Evidence for the interaction between the OCP and the thylakoids was corroborated by the presence of the OCP-GFP fusion protein in phycobilisome-associated (MP) and phycobilisome-free (M) membrane preparations (Figure 9 ). The membrane fractions were obtained by centrifugation of cells broken in a phosphate/citrate buffer (to obtain MP) or in a MES buffer (to obtain M). The GFP fluorescence emission spectra of different fractions are shown in Figures 9A and 9B. When the soluble proteins were separated from the membrane fractions by centrifugation, most of the OCP-GFP coprecipitated with the membrane fractions (Figure 9A). The GFP fluorescence emission in the M fraction was slightly smaller than that in the MP fraction, whereas in both supernatants, the GFP emission was very small, being smaller in the phosphate/citrate than in the MES supernatant. Isolation of the MP and M fractions in a sucrose gradient gave similar results (data not shown). When the MP fraction was resuspended in a low-phosphate buffer to dissociate the phycobilisomes from the membrane, most of the OCP-GFP remained attached to the membrane (Figure 9B). By contrast, allophycocyanin and phycocyanin emissions were prominent in the MP fraction but small in the M fraction (data not shown). These results were supported by protein separation by SDS-PAGE and detection of the OCP-GFP fusion protein by an anti-GFP antibody. This antibody reacted with a 65-kD polypeptide corresponding to the expected molecular mass of the whole fusion protein. The antibody binding was greater in the MP and M fractions (Figure 9D) than in the supernatant fractions (Figure 9C). Thus, our preliminary localization results suggest that there is a relatively strong interaction between the OCP and the thylakoids, with almost all of the OCP-GFP in the MP fraction.
The OCP was very sensitive to a membrane protease activated during membrane solubilization (20 min, 4 or 40°C) in the various sample buffers used for protein separation by gel electrophoresis. Typically, we observed a band of 50 kD instead of the expected 65 kD, which corresponds to the whole fusion protein (Figure 9D). Only solubilization of the MP and M fractions at 95°C for 3 min prevented the proteolysis (no trace of the 50-kD band was detected). By contrast, the OCP-GFP detected in the soluble protein fraction was not proteolyzed under any conditions. As noted above, a 16-kD RCP corresponding to the N terminus of the OCP was isolated from cells concomitantly with OCP. The 50-kD band may correspond to the GFP fused to the C-terminal domain of proteolyzed OCP. The biological significance of this derivative form, if any, is unknown, but in several cyanobacterial genomes (Gloeobacter violaceus, Nostoc punctiforme, and Nostoc PCC 7120), in addition to the full-length OCP gene there are other genes corresponding to RCP-like proteins (the N-terminal domain of the OCP) (Kerfeld, 2004b ). Moreover, in Thermosynechoccocus elongatus, there are single-copy open reading frames for the N- and C-terminal domains of the OCP. These data suggest that the N-terminal RCP may have a distinct function and/or that different combinations of N- and C-terminal domains result in different OCPs that may be tailored for different (photoprotective) roles.
OCP Provides Photoprotection through NPQ The results presented in this work demonstrate that the OCP is specifically involved in NPQ that appears to be associated with a photoprotective energy-dissipation mechanism. In the absence of the OCP, the NPQ induced by strong white or blue-green light in Synechocystis PCC 6803 cells is completely inhibited, and the cells are more sensitive to high light intensities. The observation that the effective antenna size was smaller in the cells in the quenched state and that the oxygen-evolving activity decreased faster in the OCP mutant under high-light conditions strongly supports the hypothesis that the OCP-related mechanism dissipates the excess absorbed energy, thereby decreasing the amount of energy arriving at the photochemical centers.
OCP-Related NPQ Is Associated with Phycobilisomes
This NPQ also occurs in a Synechocystis PCC 6803 mutant lacking phycocyanin (CK) in which only phycobilisome cores are present. This finding suggests that the OCP interacts with a component of the phycobilisome core. The Lcm, the linker-membrane protein that acts as the terminal energy acceptor in the phycobilisome, is a good candidate for this role. The faster recovery from the quenched state in CK compared with the wild type implies a weaker interaction between the OCP and the phycobilisome and/or the involvement of the movement of the phycobilisome relative to the membrane, as suggested by Joshua et al. (2005)
Under Fe-starvation conditions, a large reversible quenching of Fo and Fm levels, suggested to be mediated by the IsiA protein, is also induced by blue light (Cadoret et al., 2004
NPQ Appears to Be Induced via Activation of the OCP by Light Absorption
Recently, two independent studies showed that in two different Synechocystis PCC 6803 mutants lacking PSII, blue-green light induces a quenching of phycobilisome emission (Rakhimberdieva et al., 2004
Working Models for OCP Activity Our results do not permit conclusions to be drawn on the mechanism by which energy is dissipated. There are several possibilities. The OCP could be the light sensor, a mediator of quenching, the quencher itself, or play a combination of these roles. The activated OCP, through interaction with the core of the phycobilisome, could cause an alteration of the phycobilisome structure, leading to the quenched state. Alternatively, the carotenoid of the OCP could interact directly with a phycobilin chromophore (most probably the terminal acceptor) and dissipate the absorbed energy. This study demonstrated that the OCP is specifically involved in a phycobilisome-associated NPQ and not in other mechanisms affecting the levels of fluorescence (e.g., state transitions or D1 damage). This mechanism, induced by both blue-green light and saturating intensities of white light, likely protects PSII from high light exposure. We report here a novel function for a soluble carotenoid protein as a mediator of a photoprotective response. Our results reveal the role of the OCP and confirm the hypothesis that in cyanobacteria, as in higher plants, there exists a mechanism involving energy dissipation in the antenna (phycobilisomes). The molecular mechanism of this novel process awaits elucidation. We propose a working model in which the OCP acts as a photoreceptor that responds to blue-green light and subsequently induces energy dissipation (and fluorescence quenching) through interaction with the phycobilisome core. Further testing of this hypothesis is likely to open new frontiers in carotenoid function.
Plasmid Construction OCP PlasmidThe ocp gene (slr1963) was amplified by PCR using genomic DNA of Synechocystis PCC 6803 as template. Two synthesized oligonucleotides were used as primers: car1, 5'-CTACGCGGATCCATTGACTCTGCCCGCGGAATTT-3', and car2, 5'-CGGCCGCTCGAGCAAAGTTGAGTAATTCTTTGGG-3'. The resulting PCR product was digested by EagI and AvaI restriction enzymes and then cloned in the polylinker EagI-XhoI restriction sites of pBluescript SK+ (Stratagene) plasmid. The slr1963 gene was interrupted by inserting a 2.2-kb DNA fragment (  cassette) containing the aadA gene from Tn7, conferring resistance to Sp/Sm, in the unique restriction site HincII.
OCP-GFP Plasmid
Transformation, Selection, and Genetic Analysis of Mutants
Genomic DNA was isolated from Synechocystis PCC 6803 essentially as described by Cai and Wolk (1990)
Culture Conditions
Photoinhibition
Fluorescence Measurements and Detection The fluorescence emission spectra were recorded on a Hitachi F-3010 fluorescence spectrophotometer. Excitation was done at 600 or 430 nm. The cells were at a concentration of 5 µg chlorophyll/mL. Phycoerythrin or fluorescein (1.75 µM) was added to facilitate normalization of the spectra. Phycoerythrin (isolated from Rhodella violacea) was added to obtain a phycoerythrin emission slightly higher than those of Synechocystis phycocyanin and allophycocyanin. To detect green fluorescence from the GFP-OCP, cyanobacteria were deposited on a glass slide and mounted for observation. Micrographs were taken with an Olympus VAN-Ox AH2 fluorescence microscope fitted with a color cooled charge-coupled device camera (Coolsnap; Roper Scientific).
MP and M Membrane Preparations
The OCP-GFP in the M, MP, and soluble fractions compared in protein gel blots and fluorescence spectra (Figure 9) was isolated and quantified as follows: 0.5 mL of broken cells (1 mg chlorophyll/mL) (in MES or P/C buffer) was centrifuged. The pellet containing the membrane fraction was resuspended in 100 µL of MES buffer. The supernatant was concentrated to 100 µL. We assumed that 1 µL of the concentrated supernatant corresponded to
Gel Electrophoresis and Protein Gel Blots
Electron Microscopy
Accession Numbers
We thank W. Vermaas for the gift of the psbD-psbC mutant, A. William Rutherford for stimulating discussions and critical reading of the manuscript, Bernard Lagoutte for helpful advice and discussions, Estelle Delphin for the low-temperature fluorescence spectra, and Krisztian Cser for the light saturation curves of oxygen evolution. This research was partially supported by European Union network INTRO2.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Ghada Ajlani (ghada.ajlani{at}cea.fr) and Diana Kirilovsky (diana.kirilovsky{at}cea.fr). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.040121. Received December 7, 2005; Revision received February 6, 2006. accepted February 17, 2006.
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ABSTRACT
INTRODUCTION
-helical N-terminal domain and a mixed 
max = 450 nm), but in the OCP, it appears orange (
30 to 40 µmol·m–2·s–1 under a CO2-enriched atmosphere. The selected mutants were grown in the presence of the appropriate antibiotics. The cells were maintained in the logarithmic phase of growth and were collected having optical densities of 0.6 to 0.8 at 800 nm. 
