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Research ArticleArticle
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Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures

Denis Jallet, Adrien Thurotte, Ryan L. Leverenz, François Perreau, Cheryl A. Kerfeld, Diana Kirilovsky
Denis Jallet
Commissariat à l’Energie Atomique, Institut de Biologie et Technologies de Saclay, 91191 Gif sur Yvette, France (D.J., A.T., D.K.)
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Adrien Thurotte
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Ryan L. Leverenz
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François Perreau
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Cheryl A. Kerfeld
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Diana Kirilovsky
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  • For correspondence: diana.kirilovsky@cea.fr

Published February 2014. DOI: https://doi.org/10.1104/pp.113.229997

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Abstract

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.

The cyanobacterial Orange Carotenoid Protein (OCP) is a photoactive soluble protein of 35 kD that binds a ketocarotenoid, 3′-hydroxyechinenone (hECN). It is present in the majority of phycobilisome (PB)-containing cyanobacterial strains (Kirilovsky and Kerfeld, 2012, 2013). The PBs are light-harvesting extramembrane complexes formed by a core from which rods radiate. The core and rods are constituted of water-soluble blue and red phycobiliproteins, which covalently attach bilins (for review, see Glazer, 1984; Grossman et al., 1993; MacColl, 1998; Tandeau de Marsac, 2003; Adir, 2005). The OCP was first described by Holt and Krogmann (1981), and its structure was determined in 2003 (Kerfeld et al., 2003). However, its function was discovered only in 2006 (Wilson et al., 2006) and its photoactivity in 2008 (Wilson et al., 2008). The OCP is essential in a photoprotective mechanism that decreases the energy arriving at the reaction centers under high irradiance. Strong light induces thermal dissipation of the energy absorbed by the PBs, resulting in a decrease of PB fluorescence emission and of energy transfer from the PBs to the reaction centers (Wilson et al., 2006). This process, which is light intensity dependent, is induced by blue or green light but not by orange or red light (Rakhimberdieva et al., 2004; Wilson et al., 2006). The absorption of strong blue-green light by the OCP induces changes in the conformation of the carotenoid, converting the inactive orange dark form (OCPo) into an active red form (OCPr; Wilson et al., 2008). In OCPo, the hECN is in an all-trans-configuration (Kerfeld et al., 2003; Polívka et al., 2005). In OCPr, the apparent conjugation length of the carotenoid increases, resulting in a less distorted, more planar structure (Wilson et al., 2008). Fourier transform infrared spectra showed that conformational changes in the protein are also induced (Wilson et al., 2008) that are essential for the induction of the photoprotective mechanism. Only OCPr is able to bind to the core of PBs and to induce thermal energy dissipation (Wilson et al., 2008; Punginelli et al., 2009; Gorbunov et al., 2011; Gwizdala et al., 2011). Since the photoactivation of the OCP has a very low quantum yield (0.03; Wilson et al., 2008), the concentration of activated protein is zero in darkness and very low under low-light conditions (Wilson et al., 2008; Gorbunov et al., 2011). Thus, the photoprotective mechanism functions only under high-light conditions.

The crystal structures of the Arthrospira maxima OCP and of the Synechocystis PCC 6803 (hereafter Synechocystis) OCP were solved in 2003 and 2010, respectively (Kerfeld et al., 2003; Wilson et al., 2010). These structures, assumed to correspond to the dark OCPo form, are essentially identical. The OCP consists of an all-α-helical N-terminal domain (residues 1–165), unique to cyanobacteria, and an α-helical/β-sheet C-terminal domain that is a member of the Nuclear Transport Factor2 superfamily (residues 191–320; Synechocystis numbering). Both domains are joined by a linker (residues 166–190; Synechocystis numbering) that appears to be flexible. The hECN molecule spans the N- and C-terminal domains of the protein, with its carbonyl end embedded in and hydrogen bonded to two absolutely conserved residues (Tyr-201 and Trp-288) in the C-terminal domain. The carotenoid is almost entirely buried; only 3.4% of the 3′ hECN is solvent exposed (Kerfeld et al., 2003). Synechocystis OCP can also bind with high-affinity echinenone (ECN) and zeaxanthin. While the ECN OCP is photoactive, the zeaxanthin OCP is photoinactive (Punginelli et al., 2009), indicating the importance of the carotenoid carbonyl group for photoactivity. The largest interface through which the two domains interact and through which the carotenoid passes is stabilized by a small number of hydrogen bonds, including one formed between Arg-155 and Glu-244 (Wilson et al., 2010). This salt bridge stabilizes the closed structure of OCPo. Upon illumination, protein conformational changes cause the breakage of this bond and the opening of the protein (Wilson et al., 2012). Arg-155, which becomes more exposed upon the separation of the two domains, is essential for the OCPr binding to the PBs (Wilson et al., 2012).

After exposure to high irradiance, when the light intensity decreases, recovery of full antenna capacity and fluorescence requires another protein, the Fluorescence Recovery Protein (FRP; Boulay et al., 2010). The active form of this soluble 13-kD protein is a dimer (Sutter et al., 2013). It interacts with the OCPr C-terminal domain (Boulay et al., 2010; Sutter et al., 2013). This accelerates the red-to-orange OCP conversion and helps the OCP to detach from the PB (Boulay et al., 2010; Gwizdala et al., 2011).

Genes encoding the full-length OCP are found in the vast majority of cyanobacteria but not in all; 90 of 127 genomes recently surveyed contain at least one gene for a full-length OCP (Kirilovsky and Kerfeld, 2013). The genomes of Synechococcus elongatus and Thermosynechococcus elongatus, two cyanobacterial strains used as model organisms in photosynthesis and stress studies, do not contain a full-length ocp gene. These strains also lack FRP and β-carotene ketolase (involved in ketocarotenoid synthesis). As a consequence, these strains lack the OCP-related photoprotective mechanism and are more sensitive to fluctuating light intensities (Boulay et al., 2008).

The core of the hemidiscoidal PBs of Synechocystis, the model organism routinely used for the study of the OCP-related photoprotective mechanism, consists of three cylinders, each one formed by four trimers of allophycocyanin (APC; Fig. 1; for review, see Glazer, 1984; Bryant, 1991; Grossman et al., 1993; MacColl, 1998; Adir, 2005). The APC trimers are predominantly assembled from a two-subunit heterodimer, αAPC-βAPC, which binds two phycocyanobilins, one in each subunit. Of the 12 total APC trimers in the PB core, eight are trimers of αAPC-βAPC. These trimers have a maximal emission at 660 nm (APC660). The upper cylinder contains only APC660 trimers. In contrast, each basal cylinder contains only two APC660 trimers. Each basal cylinder also contains the following: (1) a trimer in which one αAPC subunit is replaced by a special αAPC-like subunit called ApcD, and (2) a trimer in which one β-subunit is replaced by ApcF, a βAPC-like subunit, and one α-subunit is replaced by the N-terminal domain of ApcE, an αAPC-like domain (Fig. 1). The trimers containing one or two of these special subunits have a maximal emission at 680 nm (APC680). In each cylinder, the two external trimers are stabilized by an 8.7-kD linker protein.

Figure 1.
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Figure 1.

Schematic orthogonal projections of the various PB cores. In the PBs containing three or five cylinders, the top complete cylinder is formed by four αAPC-βAPC trimers emitting at 660 nm. Each of the basal cylinders of three types of PBs contains two αAPC-βAPC trimers emitting at 660 nm and two trimers emitting at 683 nm. In one of them, one αAPC is replaced by ApcD, and in the other one, αAPC-βAPC is replaced by the dimer ApcF-ApcE. In the five cylinder PBs, two additional semicylinders formed by two αAPC-βAPC trimers are present. In all the cylinders, the two external trimers include an 8.7-kD linker protein (ApcC).

The C-terminal part of Synechocystis ApcE contains three interconnected repeated domains of about 120 residues (called Rep domains) that are similar to the conserved domains of rod linkers. Each Rep domain interacts with an APC trimer situated in different cylinders, which stabilizes the core of PB (Zhao et al., 1992; Shen et al., 1993; Ajlani et al., 1995; Ajlani and Vernotte, 1998). The ApcE protein also determines the number of APC cylinders that form the PB core (Capuano et al., 1991, 1993). Indeed, there are PBs containing only the two basal cylinders, as in S. elongatus (ex S. elongatus PCC 7942) and Synechococcus PCC 6301. In these strains, the approximately 72-kD ApcE possesses only two Rep domains. There also exist pentacylindrical cores in which, in addition to the three cylinders existing in Synechocystis PBs, there are two other cylinders, each formed by two APC660 trimers, for example in Anabaena variabilis, Anabaena PCC 7120, and Mastigocladus laminosus (Glauser et al., 1992; Ducret et al., 1998). In the pentacylindrical PBs, ApcE (approximately 125 kD) contains four Rep domains (Capuano et al., 1993). Finally, ApcE is also involved in the interaction between the PB and the thylakoids.

The bicylindric and tricylindric cores are surrounded by six rods formed generally by three hexamers of the blue phycocyanin (PC) or two PC hexamers and one hexamer containing phycoerythrin or phycoerythrincyanonin. The rods and the hexamers are stabilized by nonchromophorylated linker proteins. A linker protein, LRC also stabilizes the binding of the rods to the core. The pentacylindric PBs can contain up to eight rods. The quantity and length of rods and the presence of phycoerythrin or phycoerythrocyanin at the periphery of the rods depends on environmental conditions like light intensity or quality (Kipe-Nolt et al., 1982; Glauser et al., 1992).

The OCP probably binds to one of the APC660 trimers (Tian et al., 2011, 2012; Jallet et al., 2012), and the presence of the rods stabilizes this binding to Synechocystis PBs (Gwizdala et al., 2011). The different structures of PBs in other strains could affect the binding of the OCP. Thus, we undertook a study about the relationship between the structure of PBs and OCP binding in preparation for introducing the OCP-related photoprotective mechanism into S. elongatus using Synechocystis genes. In this study, we used the in vitro reconstitution system developed by Gwizdala et al. (2011) with three different types of isolated PBs: Arthrospira platensis PCC 7345 (hereafter Arthrospira) PBs, having a tricylindrical core like Synechocystis PBs; Anabaena variabilis (hereafter Anabaena) PBs, having a pentacylindrical core; and S. elongatus PCC 7942 (hereafter Synechococcus) PBs, having a bicylindrical core. We also used two different OCPs, the Synechocystis OCP and the Arthrospira OCP. Each OCP was isolated from mutant Synechocystis cells overexpressing one or the other ocp gene with a C-terminal His tag.

RESULTS

PB Isolation and Characterization

To isolate PBs Synechocystis, Arthrospira, Anabaena, and Synechococcus cells were broken in a highly concentrated potassium phosphate buffer (0.8–1 m). Triton X-100 was then used to solubilize membranes and release PBs in the aqueous phase. This phase was collected and deposited on Suc gradients. After ultracentrifugation, fully assembled PBs concentrated in a well-defined dark blue band at the bottom 0.75 m Suc layer (for details, see “Materials and Methods”).

The protein composition of the isolated PBs was analyzed by SDS-PAGE. The major phycobiliproteins, α/β-subunits of PC or APC, appeared as intense bands in the region between 16 and 20 kD (Fig. 2, A and B). Bands for ApcD and ApcF completely overlapped them. The rod-to-core and rod linkers (LRC and LR) were distributed from 27 to 35 kD (Fig. 2, A and B). The molecular mass of these proteins differed slightly in the four strains, as described previously in the literature (Lundell et al., 1981; Ducret et al., 1996; Nomsawai et al., 1999; Piven et al., 2005). ApcE had a higher molecular mass, between 75 and 125 kD, depending on PB architecture. In Anabaena, where PB cores contain five APC cylinders, the molecular mass of ApcE was 120 kD. The molecular mass of ApcE was only 95 kD in PBs containing three APC cylinders (Synechocystis and Arthrospira) and 75 kD in PBs containing two APC cylinders (Synechococcus; Fig. 2, A and B). The small rod (10 kD) and core (8.7 kD) linkers were barely detected on our SDS-PAGE gel.

Figure 2.
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Figure 2.

Composition analysis of the various PBs. A, Polypeptide composition of the isolated Anabaena PBs (Ana), Synechococcus PBs (Sus), and Synechocystis PBs (Sis). L, Ladder. B, Polypeptide composition of the isolated Synechocystis PBs, Arthrospira PBs (Art), and Anabaena PBs. C and D, Room-temperature absorption spectra of the PBs isolated from Synechocystis (solid orange line) and Arthrospira (dashed blue line; C) or Synechococcus (solid pink line) and Anabaena (dashed green line; D). Spectra are normalized at the maximum of absorbance around 620 nm. a.u., Absorbance units. [See online article for color version of this figure.]

Absorption spectra were recorded to obtain further insights into the relative quantities of PC and APC in the isolated PBs. PC, which is the most abundant phycobiliprotein in the PB, has a maximum of A620 for Synechocystis, Synechococcus, and Anabaena PBs (Fig. 2, C and D). The absorbance band was larger and the maximum shifted to 615 nm in Arthrospira PBs (Fig. 2C), owing to the fact that some PC α-subunits bind phycobiliviolin instead of phycocyanobilin (Babu et al., 1991). A more or less pronounced shoulder could be seen at 650 nm, related to APC absorbance. The PC-to-APC ratio was estimated by fitting the observed spectra to a combination of PC and APC absorbance spectra. As expected, the PC-to-APC ratio was higher in Synechococcus PBs (approximately 4.6) and lower in Anabaena PBs (approximately 1.3) than in Synechocystis PBs (approximately 3). Such differences correlate well with the PB core architectures, Synechococcus containing less and Anabaena containing more APC cylinders than Synechocystis. Arthrospira PBs seemed to contain less PC than Synechocystis PBs. The ratio of PC to APC in Arthrospira PBs was approximately 2, as already described (Nomsawai et al., 1999). In Anabaena and Arthrospira PBs, the rods seemed to contain only two PC hexamers, as observed previously (Ducret et al., 1996; Nomsawai et al., 1999).

Fluorescence emission spectra were used to confirm functional energy transfer in the PBs. In intact PBs at room temperature, excitation flows from PC to APC, which in turn equilibrates with terminal emitters (ApcD, ApcF, and ApcE). That results in a fluorescence peak with maximum at around 670 nm (at room temperature) when the PC is preferentially excited (excitation light at 590 nm; Fig. 3, A and C). At 77 K, energy back flow becomes less probable, so excitation arriving to the terminal emitters gets trapped. A fluorescence peak appeared at 683 to 684 nm for Arthrospira, Anabaena, and Synechocystis PBs (Fig. 3, B and D), which was slightly blue shifted in Synechococcus PBs (681 nm; Fig. 3D). Disconnected PC resulted in a small band at 645 nm, especially in Synechococcus PBs (Fig. 3, C and D). Altogether, these data indicated that the isolated PBs were well assembled and functionally connected.

Figure 3.
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Figure 3.

Room temperature (A and C) and 77 K (B and D) fluorescence emission spectra of the isolated PBs. A and B, Synechocystis PBs (solid lines) are compared with Arthrospira PBs (dashed lines). C and D, Synechococcus PBs (solid lines) are compared with Anabaena PBs (dashed lines). Spectra are normalized to the maximum of emission. Excitation was at 590 nm. a.u., Absorbance units.

Quenching in Vitro Using Synechocystis OCP

To test whether Synechocystis OCP can interact with the different PBs and trigger their fluorescence quenching, an in vitro reconstitution system was employed (Gwizdala et al., 2011). Synechocystis OCP was isolated from a Synechocystis mutant strain overexpressing the ocp gene and lacking the β-carotene hydroxylase (CrtR). In this strain, the OCP binds only ECN (not zeaxanthin or 3′ hECN). The ECN-binding OCP isolated from the ΔCrtR strain is photoactive and induces a large blue light-induced fluorescence quenching in cells (Punginelli et al., 2009; Wilson et al., 2011).

Reconstitution experiments require a minimum of 0.8 m phosphate to maintain PB integrity. However, it was already demonstrated that such high phosphate concentrations hinder OCP photoactivation (Gwizdala et al., 2011). In the experiments described here, the OCP was converted to the red form before being mixed with the PB solution. OCP conversion from its orange/inactive form (OCPo) to its red/active one (OCPr) was triggered with strong white light in Tris-HCl buffer at 4°C. OCPr was then added to a PB solution in 0.8 m potassium phosphate buffer under continuous blue-green illumination (900 µmol photons m−2 s−1), and the fluorescence quenching was followed using a pulse amplitude modulated (PAM) fluorometer (Gwizdala et al., 2011). After Synechocystis OCPr addition, the Synechocystis PB fluorescence decreased strongly and rapidly (95% in 50 s; Fig. 4). Almost no effect was observed for the other PBs. For Anabaena PBs, only a slight quenching (20%) could be seen, which became even smaller for Synechococcus and Arthrospira PBs (approximately 3%; Fig. 4). This indicates that Synechocystis OCP interacts poorly with PBs from other strains or that it cannot trigger their fluorescence quenching under conditions in which it strongly interacts with Synechocystis PBs (0.8 m phosphate).

Figure 4.
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Figure 4.

Fluorescence quenching induced by Synechocystis OCP in vitro. The PBs (0.012 µm) were illuminated with blue-green light (900 µmol m−2 s−1) in the presence of an excess of preconverted Synechocystis OCPr (0.48 µm; 40 per PB) at 23°C and 0.8 m potassium phosphate. Fluorescence decrease was measured using a PAM fluorometer for Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds). [See online article for color version of this figure.]

Arthrospira OCP Purification

The next question addressed was whether all OCPs are specific to their cognate PBs. Since Arthrospira OCP is also well characterized and its structure is known, we selected it to test for OCP-PB specificity. No method is known that allows introducing modifications in the Arthrospira genome, precluding the production of His-tagged OCP in Arthrospira cells. Thus, a region containing the Arthrospira ocp and frp genes was cloned into the pPSBA2 plasmid (Lagarde et al., 2000). A sequence encoding for six His residues was added at the 3′ end of the Arthrospira ocp gene, which was under the control of the strong psbA2 promoter. Synechocystis wild-type and ΔCrtR cells were then transformed using the resulting plasmid. In the mutants obtained, the endogenous Synechocystis ocp gene (slr1963) was interrupted by introduction of a spectinomycin/streptomycin resistance cassette. This last step led to the oApOCPWT and oApOCPΔCrtR Synechocystis mutants, producing Arthrospira OCP. Additional details are described in “Materials and Methods.”

Western-blot analysis of cell extracts using a primary antibody directed against A. maxima OCP permitted estimation of the OCP content in mutant and wild-type Synechocystis cells (Fig. 5A). The western blot revealed a 35-kD band corresponding to the OCP. The band was much stronger in oApOCPWT cells than in wild-type Synechocystis cells, indicating a large accumulation of Arthrospira OCP in this mutant. By contrast, almost no OCP was detected in the oApOCPΔCrtR strain. For comparison, it is shown that in oSynOCPΔCrtR (a strain overaccumulating Synechocystis OCP), the Synechocystis OCP content was largely higher than in ΔCrtR. Thus, Arthrospira OCP is unable to accumulate in a strain lacking hECN, while Synechocystis OCP is not affected by the lack of this carotenoid (Wilson et al., 2011).

Figure 5.
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Figure 5.

Isolation of Arthrospira OCP. A, Immunoblot detection using a primary antibody directed against Arthrospira OCP on whole cell extracts of wild-type (lane 1), oApOCPWT (lane 2), ΔCrtR (lane 3), oApOCPΔCrtR (lane 4), and oSynOCPΔCrtR (lane 5) cells. Three micrograms of chlorophyll was deposited per well. B, Absorbance spectra of Arthrospira OCPo (orange solid line) and OCPr (red dashed line) isolated from oApOCPWT cells. [See online article for color version of this figure.]

Arthrospira OCP was purified from oApOCPWT cells using a protocol similar to that developed for Synechocystis OCP isolation (Wilson et al., 2008; see “Materials and Methods”). In the dark, the absorbance spectrum of the isolated Arthrospira OCP showed peaks at 467 and 496 nm as well as a shoulder around 440 nm (Fig. 5B). This spectrum is identical to those already published for A. maxima (Holt and Krogmann, 1981; Polívka et al., 2005) and Synechocystis OCP (Wilson et al., 2008). After illumination, OCPo converted to its red form. Its absorbance spectrum matched the one of Synechocystis OCPr (Fig. 5B). The carotenoid content of the isolated Arthrospira OCP was analyzed by HPLC. hECN was detected in 81% of OCPs, while the 19% remaining OCPs bound ECN (Supplemental Fig. S1).

Arthrospira OCP photoactivation kinetics were then studied. OCPr accumulation under illumination results in an increase of the 550-nm absorbance (Fig. 5B) that can be monitored over time. Figure 6A compares the photoactivation kinetics of hECN-Arthrospira and ECN-Synechocystis OCPs at 9°C and 23°C. At both temperatures, Arthrospira OCPo converted faster to OCPr (t1/2 approximately 37 and 10 s, respectively) than Synechocystis OCPo (t1/2 approximately 62 and 14 s, respectively). In addition, with recovery to the orange form being nonnegligible at 23°C, only 62.9% of the Arthrospira OCP and 54.5% of the Synechocystis OCP were in the red form at equilibrium. A similar difference in the kinetics of photoconversion was observed when ECN-Arthrospira OCP was compared with the ECN-Synechocystis OCP (Supplemental Fig. S2), thus indicating that the differences in kinetics were not due to differences in the bound carotenoid. These experiments were made in 40 mm Tris-HCl and did not mimic the buffer conditions applied for quenching reconstitution (i.e. high phosphate concentration; Gwizdala et al., 2011). Indeed, high phosphate concentration also affected the accumulation of Arthrospira OCPr as in the case of Synechocystis OCPr (Gwizdala et al., 2011). Only 24% of Arthrospira OCP was converted to the red form at 0.8 m phosphate and 5.3% at 1.4 m phosphate at 23°C (Fig. 6B).

Figure 6.
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Figure 6.

Light-driven photoconversion and dark recovery of Arthrospira OCP. Arthrospira OCPo (1.8 µm) was illuminated using strong white light (5,000 µmol m−2 s−1), and its A550 was recorded over time. A, In 40 mm Tris-HCl, pH 8, Synechocystis OCP (closed symbols) and Arthrospira OCP (open symbols) were compared at 23°C (squares) or 9°C (circles). Data were normalized to the final percentage of the red form in each condition. B, Arthrospira OCP in 40 mm Tris-HCl (blue circles), 0.8 m potassium phosphate (red triangles), or 1.4 m potassium phosphate (green squares) during its photoconversion at 23°C. Data were normalized to the final percentage of the red form at 9°C. C, After Arthrospira OCP photoconversion, the light source was turned off, and A550 evolution was followed at 9°C with (blue squares) or without (red circles) FRP addition (one per two OCPs). Recovery was also followed at 23°C (black triangles). Data were normalized to the initial A550. [See online article for color version of this figure.]

Recovery from OCPr to OCPo was also studied (Fig. 6C). At 9°C, in 40 mm Tris-HCl, Arthrospira OCPr recovers very slowly, while at 23°C, it reverts to OCPo rapidly (t1/2 approximately 22 s), similar to Synechocystis OCP (Wilson et al., 2008). Addition of the FRP from Synechocystis largely accelerated the conversion of OCPr to OCPo at 9°C (t1/2 from 15 min to 60 s; Fig. 6C), indicating that Arthrospira OCP is able to interact with Synechocystis FRP.

Quenching in Vitro Using Arthrospira OCP

The decrease of fluorescence yield induced by strong blue-green light in wild-type, oApOCPWT, oApOCPΔCrtR, and oSynOCPΔCrtR Synechocystis cells is compared in Figure 7A. As expected, due to the low concentration of the OCP in the oApOCPΔCrtR strain (Fig. 5A), almost no fluorescence quenching was observed in this strain. In contrast, in the oApOCPWT strain, strong blue-green light induced a huge fluorescence quenching as in the oSynOCPΔCrtR strain (60% drop in about 50 s). In vitro, when Synechocystis PBs were illuminated in the presence of Arthrospira OCPr at 0.8 m phosphate, Arthrospira OCP rapidly quenched almost all PB fluorescence with kinetics similar to that of Synechocystis OCP (Fig. 7B). Thus, Arthrospira OCP is able to interact with Synechocystis PBs in vivo and in vitro and to quench their fluorescence.

Figure 7.
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Figure 7.

Fluorescence quenching induced by Arthrospira OCP in vitro and in vivo. A, Fluorescence quenching triggered by strong blue-green light (1,400 µmol photons m−2 s−1) in Synechocystis wild-type (orange open circles), oSynOCPΔCrtR (cyan open triangles), oApOCPWT (brown closed squares), and oApOCPΔCrtR cells (purple closed circles) cells at 33°C. Fm′, Maximum PSII fluorescence in the light-adapted state. B, Isolated Synechocystis PBs were illuminated with blue-green light (900 µmol m−2 s−1) in the presence of an excess of preconverted Synechocystis OCPr (orange triangles) or Arthrospira OCPr (blue squares; 0.48 µm; 40 per PB) at 0.8 m potassium phosphate, 23°C. C, Fluorescence quenching induced by strong blue-green light (900 µmol m−2 s−1) at 0.8 m potassium phosphate in the absence (open symbols) or the presence (closed symbols) of an excess of Arthrospira OCPr in Synechocystis PBs (orange circles), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds). D, Fluorescence recovery in darkness of “quenched” PBs. The light was turned off after 300 s of illumination. Symbols are as in C. [See online article for color version of this figure.]

Figure 7C reveals that Arthrospira OCPr was also able to induce a large quenching of Synechococcus, Arthrospira, and Anabaena PB fluorescence in 0.8 m potassium phosphate buffer; in contrast, Synechocystis OCPr was inactive (Fig. 4). Illumination of PBs in the absence of OCP did not induce any fluorescence quenching (Fig. 7C). In Anabaena PBs, a fast and large-magnitude fluorescence decrease occurred (68% decrease in 10 s) followed by a slow regain phase related to dislodging of the OCP from the PB and partial reconversion of OCPr to OCPo in the solution (9–300 s; final quenching, 57.2%). Similar profiles were observed with Synechococcus PBs (40.7% decrease after 8 s, 17.7% at 300 s) and Arthrospira PBs (40.8% after 34 s, 22.75% at 300 s). Thus, Arthrospira OCP seems to be less specific than Synechocystis OCP. Moreover, it seems to interact more strongly with Synechocystis and Anabaena PBs than with Arthrospira PBs. This was also illustrated by the faster fluorescence recovery in the dark for Synechococcus and Arthrospira PBs than for Anabaena and Synechocystis PBs (Fig. 7D).

The differences between Arthrospira and Synechocystis OCP are not due to the fact that one attaches hECN and the other ECN. Results similar to that shown in Figure 4 were obtained when hECN-Synechocystis OCP, isolated from wild-type Synechocystis cells, was used in the in vitro quenching experiments (Supplemental Fig. S3A). A similar observation was made when employing ECN-Arthrospira OCP isolated from oApOCPΔCrtR instead of the 3′ hECN binding OCP from oApOCPWT cells (Supplemental Fig. S3B). The observed differences in PB binding must be due to differences in the protein, not the pigment, of the two OCPs.

Effect of Increasing Phosphate Concentration on the Induction of Fluorescence Quenching

Increasing phosphate concentration strengthens the Synechocystis OCP binding to Synechocystis whole PBs and PB cores (Gwizdala et al., 2011). The OCP is unable to induce fluorescence quenching of isolated cores of PBs at 0.5 m phosphate, but it induces a large quenching at 0.8 m phosphate (Gwizdala et al., 2011). Although at 0.5 m phosphate, OCP is able to induce the total quenching of whole Synechocystis PBs, the rate of quenching increases with the concentration of phosphate (Supplemental Fig. S4). We tested if Synechocystis OCP is able to bind to other PBs at concentrations higher than 0.8 m phosphate. Figure 8A shows the percentage of fluorescence quenching induced for Arthrospira PBs in potassium phosphate buffers from 0.8 to 1.6 m. An optimum appeared at 1.4 m for Arthrospira PBs, where fluorescence yield dropped by 62.5% after 300 s (instead of 2.5% at 0.8 m). Figure 8B compares the fluorescence quenching induced by Synechocystis OCPr in Arthrospira, Synechococcus, and Anabaena PBs at 1.4 m phosphate. At this concentration, Synechocystis OCP induced a large fluorescence quenching in Anabaena PBs (75.1%), Arthrospira PBs (60.9%), and Synechococcus PBs (59.3%). Nevertheless, the fluorescence quenching induced was still smaller than in Synechocystis PBs (88.6%; Fig. 8B), suggesting that Synechocystis OCP is still more specific for Synechocystis PBs even at 1.4 m phosphate. Note that for Arthrospira and Synechococcus PBs, a minimum fluorescence yield was reached after about 15 s, followed by a slow recovery even under illumination. Part of the OCPr reverted spontaneously to OCPo, and the binding strength to these PBs was not sufficient to compensate. Changing buffer concentrations showed that Synechocystis OCP is able to trigger fluorescence quenching in any PB when the binding is sufficiently strong.

Figure 8.
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Figure 8.

Effect of potassium (K) phosphate concentration on PB fluorescence quenching. PBs (0.012 µm) were illuminated with blue-green light (900 µmol m−2 s−1) in the presence of preconverted OCPr (0.48 µm; 40 per PB) at 23°C. A, Percentage of Arthrospira PB fluorescence quenching reached after 5 min of illumination in the presence of Synechocystis OCPr (black bars) or Arthrospira OCPr (white bars) in increasing potassium phosphate concentrations from 0.8 to 1.6 m. B, Fluorescence decrease induced by Synechocystis OCPr in Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds) at 1.4 m potassium phosphate. C, Fluorescence decrease induced by Arthrospira OCPr in Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), or Anabaena PBs (green diamonds) at 1.4 m potassium phosphate. [See online article for color version of this figure.]

Increasing phosphate concentration also strongly influenced Arthrospira PB fluorescence quenching by Arthrospira OCP (Fig. 8, A and C). Between 0.8 and 1.2 m phosphate, a maximum fluorescence quenching was observed after 15 to 35 s, followed by a partial fluorescence recovery as shown in Figure 7C. At higher phosphate concentrations, the fluorescence quenching increased to 85.5% and the recovery phase almost disappeared, suggesting an irreversible OCP binding. At 1.4 m phosphate, Arthrospira OCPr induced 94%, 78.5%, and 92% fluorescence in Synechocystis PBs, Synechococcus PBs, and Anabaena PBs, respectively (Fig. 8C).

Comparison of Blue Light-Induced Fluorescence Quenching in Arthrospira and Synechocystis in Vivo

Figure 9A shows that strong blue-green light induced a slightly faster and larger fluorescence quenching in Synechocystis cells than in Arthrospira cells. This can be ascribed to a lower concentration of OCP in Arthrospira cells or to a weaker interaction between the OCP and Arthrospira PBs in vivo. We first tested the quantity of OCP present in Arthrospira and Synechocystis cells and membrane-bound PB complexes isolated from both strains. It was already shown that all the OCP present in the cells is attached to these complexes (Wilson et al., 2006). Western-blot analysis showed that OCP is present in similar concentrations in both strains (Fig. 9B), suggesting a weaker OCP interaction or a less effective induction of fluorescence quenching in Arthrospira PBs. On the other hand, absorbance spectra of whole cells presented a much larger chlorophyll (absorbance at 680 nm)-to-phycobiliprotein (absorbance at 615–655 nm) ratio in Arthrospira cells than in Synechocystis cells (Fig. 9C). This indicated a lower PBs concentration in Arthrospira and, as a consequence, a higher OCP-to-PB ratio. This is also suggested by SDS-PAGE, in which the bands in the 15- to 20-kD region were stronger in Synechocystis than in Arthrospira cells and membrane-bound PB complexes (Fig. 9B). Since the ratio of OCP to PBs is higher in Arthrospira cells than in Synechocystis cells, a larger fluorescence quenching was expected in Arthrospira cells. This was not observed.

Figure 9.
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Figure 9.

Comparison of OCP-related fluorescence quenching in Arthrospira and Synechocystis cells. A, Fluorescence quenching triggered by strong blue-green light (1,400 µmol photons m−2 s−1) in Synechocystis wild-type (red closed squares) and Arthrospira (black open circles) cells at 33°C. Fm′, Maximum PSII fluorescence in the light-adapted state. B, Coomassie blue-stained gel electrophoresis (top) and immunoblot detection (bottom) of the OCP protein in Synechocystis wild-type cells (lane 1), Arthrospira cells (lane 2), and membrane-PB fractions (prepared as described by Wilson et al. [2006]) obtained from Arthrospira (lane 3) or Synechocystis (lane 4). Each lane contains 2 µg of chlorophyll. C, Room temperature absorbance spectra of Synechocystis wild-type (solid red line) and Arthrospira (dashed black line) cells. Cells were diluted to 3 µg chlorophyll mL−1. a.u., Absorbance units. [See online article for color version of this figure.]

DISCUSSION

This study was aimed at determining whether the OCP isolated from a given cyanobacterial strain can bind to PBs from a different strain, including those that lack the OCP, and can induce their fluorescence quenching. Using an in vitro reconstitution system, the combinations between various OCPrs (from Synechocystis and Arthrospira) and PBs (from Synechocystis, Arthrospira, Synechococcus, and Anabaena) were tested. The results obtained demonstrated that different OCPs are not equivalent in their capacity to induce PB fluorescence quenching or in their specificity. At 0.8 m phosphate, Synechocystis OCP induced a large fluorescence quenching only in Synechocystis PBs (Fig. 4), and Arthrospira OCP was able to induce a rather large quenching in all types of PBs (Fig. 7C). At higher phosphate concentrations, Synechocystis OCP was able to induce fluorescence quenching of all PBs; however, the amplitude of quenching was always smaller than that induced by the Arthrospira OCP (Fig. 8A). It was previously shown that PB fluorescence quenching and OCP binding to PBs are correlated and that the absence of fluorescence quenching is due to a lack of binding between OCP and PBs (Gwizdala et al., 2011). Thus, the results described in this work strongly suggest that Arthrospira OCP binds more strongly than Synechocystis OCP to all tested PBs. However, each strain’s OCP is able to quench the fluorescence of all PBs once bound to it.

Influence of OCP Primary Structures on Carotenoid Binding

The other distinctive difference between the two OCPs resides in their capacity to bind ECN instead of hECN; Synechocystis OCP binds both carotenoids, but Arthrospira OCP cannot stabilize the ECN binding. The crystal structures of Synechocystis OCPo (1.65 Å resolution; Wilson et al., 2010) and of A. maxima OCPo (2.1 Å resolution; Kerfeld et al., 2003) are known. The latter is 100% identical in primary structure to the Arthrospira OCP used in this study. The carotenoid structure and the secondary and tertiary protein structures of Synechocystis and Arthrospira OCPs are nearly identical (Wilson et al., 2010). The primary structures of the two OCPs investigated in this study are 83% identical. By analyzing the amino acids that differ between Arthrospira and Synechocystis OCPs, we can develop hypotheses about the underlying basis of the differences observed in carotenoid binding and in their ability to induce fluorescence quenching (probably due to changes in PB binding specificity) in these two OCPs.

In wild-type cells, both Synechocystis and Arthrospira OCPs bind hECN. When the Synechocystis ocp gene is overexpressed in a Synechocystis mutant lacking zeaxanthin and hECN, cells contain a high concentration of OCP binding only ECN (Punginelli et al., 2009; Wilson et al., 2010). In contrast, overexpression of the Arthrospira ocp gene in the same Synechocystis mutant results in cells in which almost no Arthrospira OCP is accumulated (Fig. 5A). Likewise, in a wild-type background, the carotenoid preferentially bound differs: Synechocystis OCP isolated from the overexpressing strain contained more ECN (50%–82% depending on the preparation) than hECN (11%–32%; Punginelli et al., 2009; Wilson et al., 2010). This is probably related to the higher concentration of ECN than that of hECN in cells. In contrast, Arthrospira OCP overexpressed in wild-type Synechocystis cells binds preferentially hECN over ECN (81% versus 19%; Supplemental Fig. S1), strongly suggesting that Arthrospira OCP is not able (or is less able) to stably bind ECN. The interaction of the carotenoid hydroxyl group with the protein seems to be essential for the stabilization of carotenoid binding in Arthrospira OCP. We have demonstrated that in Synechocystis OCP, the interaction of Tyr-44 and Trp-110 with the hydroxyl ring of the carotenoid is important for photoactivity (Wilson et al., 2010). In addition, the interaction between Trp-110 and the carotenoid seems to be important to stabilize ECN binding. When Tyr-44 was replaced by Ser, the OCP was present in high concentrations in the overexpressing Y44S-OCP cells and still bound 70% ECN, despite increased solvent accessibility of the carotenoid (Wilson et al., 2010). However, when Trp-110 was replaced by Ser or Phe, the mutated OCPs bound only 20% of ECN and the OCP concentration in the cells was reduced (Wilson et al., 2010). The replacement of Trp by Phe did not affect photoactivity but destabilized ECN binding, indicating that the interaction between Trp-110 and the carotenoid is essential for carotenoid binding. Due to the high sequence identity among amino acids forming the carotenoid-binding pocket and their similar orientation in the structures, it is difficult to explain why Arthrospira OCP cannot stably bind ECN. We can hypothesize that small changes in the positions of amino acid side chains could disturb the interaction between Trp-110 and the carotenoid ring. In the Arthrospira OCP structure, a conserved water molecule (H2O 452) is hydrogen bonded directly to the 3′-OH group of 3′ hECN (Kerfeld et al., 2003). It is possible that this bond is partially responsible for the stabilization of 3′ hECN in Arthrospira OCP. We cannot discard that subtle differences in solvent accessibility and the number of water molecules in the carotenoid pocket could also destabilize the Π-Π interactions between the carotenoid ring and Trp-110 or other hydrophobic carotenoid-protein interactions, rendering the ECN binding more or less favorable. Finally, nothing is known about the mechanism of carotenoid binding to the OCP, and it is possible that nonconserved amino acids outside the carotenoid-binding pocket of OCPo could be involved in this process.

Comparison of OCP-PB Interactions of Both OCPs

No significant differences in photoconversion kinetics or fluorescence quenching kinetics and amplitude were observed for hECN-OCP versus ECN-OCP from Arthrospira (Supplemental Figs. S2 and S3). Thus, the differences observed in photoactivity and fluorescence quenching properties of ECN-OCP from Synechocystis versus hECN-OCP from Arthrospira most likely result from differences in protein structures between both OCPs. Many amino acid substitutions between Synechocystis OCP and Arthrospira OCP are localized on the outer surface of the N-terminal domain: several neutral amino acids in the N-terminal domain of Synechocystis OCP are replaced by charged amino acids in Arthrospira OCP (Fig. 10A). These substitutions include N14E, S29K, Q72R, R112E, A127E, Q130K, and T164D. If the OCP-PB interaction has an important electrostatic component, the additional charged residues could strengthen the interactions between Arthrospira OCP and PBs. In addition, the replacement of a negative charge in Synechocystis OCP (Asp-115) by a positive charge in Arthrospira OCP (Arg-115) could also affect electrostatic interactions with the PBs. Differences in OCP binding strength to PBs are probably the cause of differences in fluorescence quenching, as demonstrated previously (Gwizdala et al., 2011).

Figure 10.
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Figure 10.

Structural differences between Arthrospira OCP and Synechocystis OCP. A, Overview of the A. maxima OCP structure. Teal, N-terminal domain; green, linker region; red, C-terminal domain. 3′ hECN is shown in orange. The amino acids changing between Arthrospira OCP and Synechocystis OCP are represented using purple sticks, and blue sticks are used for the ones bearing charges (and for Tyr-171). B, Electrostatic surface maps of Arthrospira and Synechocystis OCPs. For details, see “Materials and Methods.” SB, Salt bridge between Arg-155 and Glu-244(246).

The OCP’s binding to the PB is known to involve a direct interaction between OCP’s Arg-155 residue and the APC protein (Wilson et al., 2012). Electrostatic surface plots of the region around Arg-155 of Arthrospira and Synechocystis OCPs are shown in Figure 9B. In each structure, Arg-155 (which forms a salt bridge with Glu-244/246 in the nonquenching form of the OCP) is buried near the carotenoid chromophore in a large, centralized surface depression at the interface of the C-terminal and N-terminal domains of the protein. In this interface, most amino acids are identical or conservatively substituted (i.e. Asp-146 versus Glu-146 or Lys-231 versus Arg-233). However, we note two nonconservative substitutions at the entrance to this surface depression: Ser-29 and Thr-164 (Synechocystis) to Lys-29 and Asp-164 (Arthrospira). While residue 29 is not highly conserved among OCP orthologs (Wilson et al., 2010), the positively charged Lys-29 of Arthrospira adds an additional positive charge in the surface depression leading to Arg-155. If the PB-OCP interaction involving Arg-155 is electrostatic in nature, the addition of a positive charge in this region could potentially enhance the binding interaction. The Thr-164 residue of Synechocystis is likewise notable, since residue 164 is highly conserved across orthologs (Wilson et al., 2010) and is most often negatively charged (as is the case with Asp-164 in Arthrospira). Given its proximity to Arg-155, the nature of its nonconservative substitution versus Asp-164 of Arthrospira, and its deviation from the majority of OCP sequences, we speculate that Thr-164 might be at least partly responsible for the unique specificity of Synechocystis OCP to Synechocystis PBs.

Finally, the flexible linker region between the C- and N-terminal domains could also lead to differences in activity of both OCPs if this region is involved in light-induced protein conformational changes or PB binding. The flexible linker is the least conserved region of the primary structure across OCP orthologs (Wilson et al., 2010) and possesses 59.5% identity in amino acid sequences between the OCPs investigated in this work. It is shorter in Synechocystsis OCP, lacking two amino acids (Ser-170 and Tyr-171), which could restrict the opening of the protein in the red form and subsequently affect the binding to APC trimers. These residue-specific hypotheses await further testing. Additional interpretation is hindered by the fact that no structural data are yet available concerning OCPr or OCPr-PB complexes.

Influence of PB Structure on the OCP-PB Interaction

In addition to differences in amino acid sequence between the Arthrospira and Synechocystis OCP, the PB structures also influenced the amplitude and kinetics of fluorescence quenching: a given OCP reacted differently with PBs isolated from different cyanobacterial strains. This is particularly true in 0.8 m phosphate, insufficient for a full stabilization of the OCP-PB complex when PBs other than Synechocystis PBs were used. In the past, this concentration of phosphate was considered optimal for in vitro fluorescence quenching induction (Gwizdala et al., 2011). Here, our results clearly demonstrated that Synechocystis PBs are quenched much more efficiently by both Synechocystis and Arthrospira OCPs than any other PB (Figs. 4 and 8). It could be assumed that OCP and PBs from the same cyanobacterial strain, having evolved concomitantly, developed a high affinity toward each other for an optimized response to high light. The fact that Arthrospira PBs are only poorly quenched by Arthrospira OCP (Fig. 7C) raises important questions. One possibility is that the Arthrospira PBs used here could have been partially disassembled or could have lost some components required for OCP binding during isolation. However, absorption spectra combined with 77 K fluorescence emission spectra indicated that they were assembled and functionally connected PBs (Figs. 2 and 3). No striking variations compared with Synechocystis PBs appeared in terms of peptide composition, as revealed by SDS-PAGE (Fig. 2A). Nevertheless, Tian et al. (2011, 2012) showed that although the mechanism inducing fluorescence quenching was identical in vivo and in vitro and that the amounts of quenching were similar, the rate of quenching was slightly slower in the in vitro system than in vivo. This indicated that even in the case of Synechocystis, slight variations in PB structure, not detectable in fluorescence spectra, could affect OCP binding. In the case of Arthrospira PBs, perhaps changes in the core of the PB are induced that affect OCP binding but not energy transfer. A second interpretation can be formulated in which Synechocystis PBs are simply more prone to quenching than Arthrospira PBs even in vivo. The reasons could lie in different overall structures between the PBs or more specific changes in the primary structure of one or more PB proteins. We showed that a similar concentration of OCP induced slightly slower and smaller fluorescence quenching in Arthrospira cells than in Synechocystis cells and that a larger OCP-to-PB ratio in Arthrospira cells did not lead to larger fluorescence quenching. Although our results suggest that Arthrospira PBs have a lower affinity for OCP than Synechocystis PBs in vivo, the effect seems to be much less pronounced than in vitro, since the difference in the amplitude of fluorescence quenching was small between both strains.

Electron microscopy on isolated complexes showed that Synechocystis, Arthrospira, Synechococcus, and Anabaena PBs have a common hemidiscoidal organization with PC rods radiating from an APC core (for review, see MacColl, 1998; Adir, 2005). The number of APC cylinders differs from strain to strain. This could be a factor influencing OCP-PB interactions, as OCP binds to APC upon photoactivation (Tian et al., 2011, 2012; Jallet et al., 2012). However, our results did not confirm the hypothesis of a greater OCP binding in PBs with more APC trimers. Synechococcus PBs have only two APC cylinders but behaved like Arthrospira PBs (with three cylinders) in terms of fluorescence quenching in vitro (Figs. 4, 7, and 8). The missing upper cylinder, containing α/β-subunits of APC plus a small linker polypeptide, seems not to be required for OCP attachment. Anabaena PBs were generally less quenched than Synechocystis PBs. Thus, the two extra APC cylinders present in these PBs seem not to result in an increased affinity for OCP. Our results strengthen the proposal that the OCP binds to a basal APC cylinder.

Many isolated phycobiliproteins in their trimeric or hexameric aggregation states have been crystallized and their x-ray structures determined (for review, see Adir, 2005). However, a global picture of fully assembled PBs is still lacking. For example, it is not known how PC rods and APC cores are biochemically connected. PC rods stabilize OCP binding to PBs (Gwizdala et al., 2011), and species-specific changes of this connection could lead to different affinities for the OCP. It was recently shown that a more or less pronounced cavity exists between the APC trimers (Marx and Adir, 2013). This could influence PC rod binding or OCP attachment, but a systematic crystallographic study is needed for verification. We have already observed that OCP is able to induce less fluorescence quenching when mixed with “old” PB preparations or reconstituted PBs, in which energy transfer to the terminal emitters was slightly decreased, suggesting a weaker connection between APC trimers (D. Jallet and D. Kirilovsky, unpublished data). Thus, the interaction between APC trimers seems to be important for OCP binding stabilization. This interaction could be modified in the PBs of the different cyanobacteria strains at least in the in vitro isolated preparations.

Finally, the OCP is thought to interact with α/βAPC subunits emitting at 660 nm (Tian et al., 2011, 2012; Jallet et al., 2012) or with the ApcE core membrane linker (Stadnichuk et al., 2012) or both (Kuzminov et al., 2012). αAPCs and βAPCs are extremely well conserved, particularly within the group of cyanobacteria employed for this study (between 77% and 90% identity in primary structures compared with Synechocystis). ApcE is less conserved (Capuano et al., 1993), especially in the N-terminal phycobiliprotein part (60%–64% identity in primary structures to Synechocystis within the group employed here), because it contains a quite variable loop region of unknown function. This could also influence OCP attachment. Once again, the lack of structural data on OCPr-PB complexes makes any further interpretation merely speculative.

CONCLUSION

This study results in four important conclusions about the relationship between OCP and PBs. (1) Structurally distinct OCPs from different strains exhibit unique PB interaction properties. Synechocystis OCP manifests a high specificity for its own PBs. Arthrospira OCP has a stronger affinity for all PBs relative to that of Synechocystis OCP and, therefore, appears to be less specific. Our results suggest a role for electrostatics in the interaction. (2) The structure of the PBs, probably the interactions between APC trimers forming the core cylinders, could have a big influence on OCP binding and its stabilization. (3) The upper APC cylinder is not necessary for OCP interaction with PBs, strongly suggesting that OCP binds to one of the basal APC cylinders. (4) Once OCP binds to the PB, it is able to quench the fluorescence of any type of PBs, even those isolated from strains lacking OCP. This is true for both OCPs investigated here. We also conclude that Arthrospira OCP would be a better candidate to introduce the photoprotective mechanism in S. elongatus and T. elongatus cells. These two cyanobacterial strains lack the OCP and are more sensitive to high-light conditions (Boulay et al., 2008).

MATERIALS AND METHODS

Culture Conditions

Synechocystis PCC 6803, Synechococcus elongatus PCC 7942, and Anabaena variabilis cells were grown photoautotrophically in a modified BG11 medium containing double amounts of sodium nitrate (Herdman et al., 1973). Cells were kept in a rotary shaker (120 rpm) at 30°C, under CO2 enrichment, illuminated by fluorescent white lamps giving a total intensity of about 90 µmol photons m−2 s−1. The Arthrospira platensis PCC 7345 culture conditions were identical, except for temperature. They were grown at 23°C. Cells were maintained in their logarithmic phase of growth.

oApOCP Plasmid Construction

A 1.7-kb DNA region containing the ocp and frp genes was amplified by PCR using genomic DNA of Arthrospira as a template. Two synthesized oligonucleotides containing the sequences for creating NdeI and HpaI restriction sites were employed for that purpose: F_AP_OFNdeI (5′-GACTTCCATATGCCATTCACCATTGACTCGGC-3′) and R_AP_OFHpaI (5′-GTAAGCGTTAACAGTCCAACTACTCAACCCGC-3′). The boldface nucleotides correspond to the restriction sites and to the nucleotides coding the histidines.

The resulting PCR product was cloned into pPSBA2 (Lagarde et al., 2000) into the NdeI and HpaI restriction sites of the plasmid. Nucleotides encoding for 6×His were added on the 3′ side of OCP by site-directed mutagenesis (Quickchange XL kit; Stratagene) using the mutagenic oligonucleotides F_AP_OFHis (5′-CACCACCACCACCACCACTAGAATAGAGTTCACCTAGAAATTATATAGG-3′) and R_AP_OFHis (5′-GTGGTGGTGGTGGTGGTGGCGCACCAAGTTCAACAACTCTTTGG-3′). A 1.3-kb kanamycin resistance cassette was finally inserted in the unique HpaI site, situated 48 bp downstream of the FRP stop codon.

Transformation, Selection, and Genetic Analysis of Mutants

The oApOCP plasmid construct was used to transform wild-type and ΔCrtR Synechocystis cells (lacking the β-carotene hydroxylase and unable to produce zeaxanthin or 3′ hECN), giving the oApOCPWT and oApOCPΔCrtR mutants, respectively. Selection was made at 33°C, under dim light (30 µmol photons m−2 s−1), on plates containing 40 µg mL−1 kanamycin. The endogenous slr1963 gene was then interrupted using the ΔOCP plasmid construct bearing a spectinomycin/streptomycin resistance cassette (Wilson et al., 2006). To confirm the complete segregation of the different mutants, PCR analysis and specific digestions by restriction enzymes were performed.

Isolation of PBs

The protocol used derives from that described by Ajlani et al. (1995). After reaching optical density at 800 nm = 1, cyanobacteria cells were harvested through centrifugation at 54,00g, 23°C, for 6 min. They were washed twice using 0.8 m potassium phosphate buffer, pH 7.5, and their chlorophyll concentrations were then determined and adjusted to 1 mg mL−1. Protease inhibitors were added (1 mm EDTA, 1 mm caproic acid, and 1 mm phenylmethylsulfonyl fluoride) as well as DNase (50 µg mL−1) prior to breaking. Synechococcus, Anabaena, and Arthrospira cells were broken using a French press system (800 p.s.i.). Synechocystis cells were broken through vortexing in the presence of glass beads (diameter, 200 µm). The unbroken cells were removed by centrifugation at 2,000g, 23°C, for 5 min. The supernatant was incubated in the presence of 2% (v/v) Triton X-100 under dim stirring, at 23°C, during 2 h. For Synechocystis, Arthrospira, and Synechococcus PBs, the Triton X-100 phase and debris were removed by centrifugation at 20,000g, 23°C, for 20 min, and the dark blue supernatant was directly loaded onto a discontinuous Suc gradient. For Anabaena PBs, the Triton X-100-treated supernantant was centrifuged at 86,000g, 23°C, for 1 h. The dark-green supernatant was collected, and PBs were precipitated through centrifugation at 130,000g, 23°C, for 1 h. The dark blue pellet was resuspended using 1 m potassium phosphate buffer, pH 7.5, and deposited onto a Suc gradient. The Suc gradient for isolation of all PBs contained 0.25, 0.5, 0.75, and 1.5 m Suc layers in 1 m (final) potassium phosphate buffer, pH 7.5. The gradient was spun at 150,000g, 23°C, for 12 h. The different layers were collected, and their absorbance spectra were recorded.

Calculation of PB Concentrations

The calculation of PB concentrations was based on absorbance spectra. For Synechocystis PBs, 95% of the A620 comes from the absorption of PC (Yamanaka et al., 1978). The extinction coefficient of a PC hexamer is 2,370 mm−1 cm−1 (Glazer, 1984). We estimated that the extinction coefficient of Synechocystis PBs containing 18 hexamers of PC (six rods with three PC hexamers each) is 42,660 mm−1 cm−1. We estimated that PC contributes to 85% of the A620 in Arthrospira PBs and Anabaena PBs and to 95% in Synechococcus PBs (Supplemental Fig. S1). Taking into account the different architectures, this leads to extinction coefficients of 39,390 mm−1 cm−1 for Arthrospira PBs, 30,004 mm−1 cm−1 for Synechococcus PBs, and 52,520 mm−1 cm−1 for Anabaena PBs.

Purification of the Arthrospira and Synechocystis OCP

His-tagged Arthrospira OCP was purified from oApOCPWT or oApOCPΔCrtR Synechocystis mutants and Synechocystis OCP from oSynOCPΔCrtR as described (Wilson et al., 2008) using a Ni-ProBond resin column and a Whatman DE-52 column. The isolated OCP was dialyzed against 40 mm Tris-HCl, pH 8.0, and frozen at −80°C.

Protein Separation and Immunoblot Analysis

Proteins were analyzed by SDS-PAGE on 12% polyacrylamide/2 m urea gels in a Tris-MES system (Kashino et al., 2001). PB samples were concentrated by precipitation with 10% (v/v) TCA prior to loading (equal quantities in each lane). For whole cell extracts, 3 µg of chlorophyll was deposited per well. The gels were stained by Coomassie Brilliant Blue. The OCP protein was detected using a polyclonal antibody directed against Arthrospira maxima OCP.

Absorbance Measurements

The orange-to-red OCP conversion (and red-to-orange recovery) was monitored in a Specord S600 spectrophotometer (Analytikjena) and triggered using strong white light (5,000 µmol m−2 s−1). PB absorbance spectra were recorded in a Uvikon XL spectrophotometer (Secomam) at 23°C.

Fluorescence Measurements

PAM Fluorometer

Fluorescence yield quenching was monitored using a pulse amplitude fluorometer (101/102/103-PAM; Walz). Measurements were made in 1-cm-pathlength stirred cuvettes. Experiments carried out on whole cells were performed at a chlorophyll concentration of 3 µg mL−1 at 33°C. In vitro reconstitutions were handled with a PB concentration of 0.012 µm in potassium phosphate buffer (pH 7.5) concentrations ranging from 0.8 to 1.6 m at 23°C. Fluorescence quenching was induced in vivo by 1,400 µmol m−2 s−1 blue-green light (halogen white light filtered by a Corion cutoff 550-nm filter; 400–550 nm). In vitro blue-green light of 900 µmol m−2 s−1 was used for quenching.

Emission Spectra

Fluorescence emission spectra were monitored in a CARY Eclipse spectrophotometer (Varian). For studies at room temperature, samples were placed in a 1-cm stirred cuvette. For 77 K measurements, samples were collected in Pasteur pipettes and then frozen by immersion in liquid nitrogen. Excitation was made at 590 nm.

OCP Structural Modeling and Analysis

Prior to the calculation of electrostatic surface potentials, missing residues in Synechocystis OCP were modeled using Coot (Emsley and Cowtan, 2004), which allowed for the placement of previously missing portions of the flexible linker and the side chain of Thr-164 in this structure. Electrostatic surface potentials of chain A from 3MG1 and 1M98 were calculated using the Adaptive Possion-Boltzmann Solver plugin implemented in the PyMOL Molecular Graphics System (version 1.6; Schrödinger).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. HPLC analysis of carotenoids isolated from purified Arthrospira OCP (oApOCPWT mutant cells).

  • Supplemental Figure S2. Photoactivation kinetics of the 3′ hECN or ECN binding Arthrospira OCP.

  • Supplemental Figure S3. Effect of ECN or 3′ hECN binding on PB fluorescence quenching induced by Synechocystis and Arthrospira OCP in vitro.

  • Supplemental Figure S4. Effect of phosphate concentration on Synechocystis PB fluorescence quenching by Synechocystis OCPr.

Acknowledgments

We thank Sandrine Cot and Markus Sutter for technical assistance, Adjélé Wilson for technical assistance in OCP preparation and discussions and Dr. Ghada Ajlani for fruitful discussions.

Footnotes

  • The author 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.plantphysiol.org) is: Diana Kirilovsky (diana.kirilovsky{at}cea.fr).

  • www.plantphysiol.org/cgi/doi/10.1104/pp.113.229997

  • ↵1 This work was supported by the Agence Nationale de la Recherche (project CYANOPROTECT), the Centre National de la Recherche Scientifique, the Commissariat à l’Energie Atomique, and the HARVEST European Union FP7 Marie Curie Research Training Network, by fellowships from the Paris-Saclay University (to D.J.) and the IDEX Paris-Saclay (to A.T.), by the National Science Foundation Division of Molecular and Cellular Biosciences (grant no. 0851094 to R.L.L. and C.A.K.), by the National Science Foundation (grant no. MCB 0851094 to R.L.L. and C.A.K.), and by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (grant no. DE–FG02–91ER20021 to R.L.L. and C.A.K.).

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[W] The online version of this article contains Web-only data.

Glossary

hECN
3′-hydroxyechinenone
PB
phycobilisome
ECN
echinenone
PC
phycocyanin
t1/2
half-life
APC
allophycocyanin
  • Received October 7, 2013.
  • Accepted December 7, 2013.
  • Published December 13, 2013.

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Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures
Denis Jallet, Adrien Thurotte, Ryan L. Leverenz, François Perreau, Cheryl A. Kerfeld, Diana Kirilovsky
Plant Physiology Feb 2014, 164 (2) 790-804; DOI: 10.1104/pp.113.229997

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Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures
Denis Jallet, Adrien Thurotte, Ryan L. Leverenz, François Perreau, Cheryl A. Kerfeld, Diana Kirilovsky
Plant Physiology Feb 2014, 164 (2) 790-804; DOI: 10.1104/pp.113.229997
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