The role of ApcD and ApcF in energy transfer from phycobilisomes to PS I and PS II in a cyanobacterium

The role of the phycobilisome core components, ApcD and ApcF, in transferring energy from the phycobilisome to PS I and PS II in the cyanobacterium Synechocystis sp. PCC6803 has been investigated. The genes encoding these proteins have been disrupted in the genomes of wild type Synechocystis sp. PCC6803 and a PS II deficient mutant, PsbD1CD2^-, by inserting antibiotic resistance genes into their coding regions. Data from fluorescence emission spectra and pigment content analysis for these inactivation mutants is presented. These data suggest that both ApcD and ApcF are involved in the energy transfer route to PS II and PS I. In both cases, the energy transfer may to the reaction centres may be via the chromophore of ApcE (the L cm) or anchor polypeptide). The major route of energy transfer to both kinds of reaction centre appears to involve ApcF rather than ApcD. When both ApcF and ApcD are absent, the phycobilisomes are unable to transfer energy to either reaction centre. We suggest a model for the pathways of energy transfer from the phycobilisomes to PS I and PS II.     

Site of non-photochemical quenching of the phycobilisome by orange carotenoid protein in the cyanobacterium Synechocystis sp. PCC 6803

In cyanobacteria, the thermal dissipation of excess absorbed energy at the level of the phycobilisome (PBS)-antenna is triggered by absorption of strong blue-green light by the photoactive orange carotenoid protein (OCP). This process known as non-photochemical quenching, whose molecular mechanism remains in many respects unclear, is revealed in vivo as a decrease in phycobilisome fluorescence. In vitro reconstituted system on the interaction of the OCP and the PBS isolated from the cyanobacterium Synechocystis sp. PCC 6803 presents evidence that the OCP is not only a photosensor, but also an effecter that makes direct contacts with the PBS and causes dissipation of absorbed energy. To localize the site(s) of quenching, we have analyzed the role of chromophorylated polypeptides of the PBS using PBS-deficient mutants in conjunction with in vitro systems of assembled PBS and of isolated components of the PBS core. The results demonstrated that L(CM), the core-membrane linker protein and terminal emitter of the PBS, could act as the docking site for OCP in vitro. The ApcD and ApcF terminal emitters of the PBS core are not directly subjected to quenching. The data suggests that there could be close contact between the phycocyanobilin chromophore of L(CM) and the 3'-hydroxyechinenone chromophore present in OCP and that L(CM) could be involved in OCP-induced quenching. According to the reduced average life-time of the PBS-fluorescence and linear dependence of fluorescence intensity of the PBS on OCP concentration, the quenching has mostly dynamic character. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.     

Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes

Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE(-) mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with--presumably--allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

蓝细菌别藻蓝蛋白ApcE与ApcF不形成复合物

进行了Anabaena sp.PCC7120的别藻蓝蛋白ApcE与ApcF的体内重组的光谱和apcE和apcF基因的细菌双杂交的研究。在提纯前PCB-ApcE/PCB-ApcF的吸收及荧光光谱峰与PCB-ApcF的峰位置一致,而提纯后PCB-ApcE/PCB-ApcF的吸收及荧光光谱峰与PCB-ApcE的峰位置一致,表明ApcE与ApcF不能形成复合物。pBT-apcF与pTRG-apcE的转化细胞能在无3氨基-1,2,4三氮唑（3-AT）的非选择性培养基上生长,而不能在有3AT的选择性培养基上生长,说明在转化过程中未产生能抗3-AT的HIS3报告基因,进一步证实ApcE与ApcF间没有相互作用。     

Bright near-infrared fluorescence bio-labeling with a biliprotein triad

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700–770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650–700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE⁻ mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with - presumably - allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism

Plants and algae have developed multiple protective mechanisms to survive under high light conditions. Thermal dissipation of excitation energy in the membrane-bound chlorophyll-antenna of photosystem II (PSII) decreases the energy arriving at the reaction center and thus reduces the generation of toxic photo-oxidative species. This process results in a decrease of PSII-related fluorescence emission, known as non-photochemical quenching (NPQ). It has always been assumed that cyanobacteria, the progenitor of the chloroplast, lacked an equivalent photoprotective mechanism. Recently, however, evidence has been presented for the existence of at least three distinct mechanisms for dissipating excess absorbed energy in cyanobacteria. One of these mechanisms, characterized by a blue-light-induced fluorescence quenching, is related to the phycobilisomes, the extramembranal antenna of cyanobacterial PSII. In this photoprotective mechanism the soluble carotenoid-binding protein (OCP) encoded by the slr1963 gene in Synechocystis sp. PCC 6803, of previously unknown function, plays an essential role. The amount of energy transferred from the phycobilisomes to the photosystems is reduced and the OCP acts as the photoreceptor and as the mediator of this antenna-related process. These are novel roles for a soluble carotenoid protein.     

Phycobilisomes from the mutant cyanobacterium Synechocystis sp. PCC 6803 missing chromophore domain of ApcE

Phycobilisome (PBS) is a giant photosynthetic antenna associated with the thylakoid membranes of cyanobacteria and red algae. PBS consists of two domains: central core and peripheral rods assembled of disc-shaped phycobiliprotein aggregates and linker polypeptides. The study of the PBS architecture is hindered due to the lack of the data on the structure of the large ApcE-linker also called L_CM. ApcE participates in the PBS core stabilization, PBS anchoring to the photosynthetic membrane, transfer of the light energy to chlorophyll, and, very probably, the interaction with the orange carotenoid protein (OCP) during the non-photochemical PBS quenching. We have constructed the cyanobacterium Synechocystis sp. PCC 6803 mutant lacking 235 N-terminal amino acids of the chromophorylated PBL_CM domain of ApcE. The altered fluorescence characteristics of the mutant PBSs indicate that the energy transfer to the terminal emitters within the mutant PBS is largely disturbed. The PBSs of the mutant become unable to attach to the thylakoid membrane, which correlates with the identified absence of the energy transfer from the PBSs to the photosystem II. At the same time, the energy transfer from the PBS to the photosystem I was registered in the mutant cells and seems to occur due to the small cylindrical CpcG2-PBSs formation in addition to the conventional PBSs. In contrast to the wild type Synechocystis, the OCP-mediated non-photochemical PBS quenching was not registered in the mutant cells. Thus, the PBL_CM domain takes part in formation of the OCP binding site in the PBS.     

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

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 (OCP^o) into an active red form (OCP^r; Wilson et al., 2008). In OCP^o, the hECN is in an all-trans-configuration (Kerfeld et al., 2003; Polívka et al., 2005). In OCP^r, 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 OCP^r 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 OCP^o 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 OCP^o. 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 OCP^r 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 OCP^r 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.Open in a separate windowFigure 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, L^RC 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.     

In Vitro Reconstitution of the Cyanobacterial Photoprotective Mechanism Mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.     

Synechocystis sp. PCC 6803 mutant lacking both photosystems exhibits strong carotenoid-induced quenching of phycobilisome fluorescence

Blue light induced quenching in a Synechocystis sp. PCC 6803 strain lacking both photosystems is only related to allophycocyanin fluorescence. A fivefold decrease in the fluorescence level in two bands near 660 and 680 nm is attributed to different allophycocyanin forms in the phycobilisome core. Some low-heat sensitive component inactivated at 53 °C is involved in the quenching process. Enormous allophycocyanin fluorescence in the absence of the photosystems reveals a dark stage in this quenching. Thus, we present evidence that light activation of the carotenoid-binding protein and formation of a quenching center within the phycobilisome core in vivo are discrete events in a multistep process.     

Far-red light photoacclimation (FaRLiP) in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7335. II.Characterization of phycobiliproteins produced during acclimation to far-red light

Phycobilisomes (PBS) are antenna complexes that harvest light for photosystem (PS) I and PS II in cyanobacteria and some algae. A process known as far-red light photoacclimation (FaRLiP) occurs when some cyanobacteria are grown in far-red light (FRL). They synthesize chlorophylls d and f and remodel PS I, PS II, and PBS using subunits paralogous to those produced in white light. The FaRLiP strain, Leptolyngbya sp. JSC-1, replaces hemidiscoidal PBS with pentacylindrical cores, which are produced when cells are grown in red or white light, with PBS with bicylindrical cores when cells are grown in FRL. This study shows that the PBS of another FaRLiP strain, Synechococcus sp. PCC 7335, are not remodeled in cells grown in FRL. Instead, cells grown in FRL produce bicylindrical cores that uniquely contain the paralogous allophycocyanin subunits encoded in the FaRLiP cluster, and these bicylindrical cores coexist with red-light-type PBS with tricylindrical cores. The bicylindrical cores have absorption maxima at 650 and 711 nm and a low-temperature fluorescence emission maximum at 730 nm. They contain ApcE2:ApcF:ApcD3:ApcD2:ApcD5:ApcB2 in the approximate ratio 2:2:4:6:12:22, and a structural model is proposed. Time course experiments showed that bicylindrical cores were detectable about 48 h after cells were transferred from RL to FRL and that synthesis of red-light-type PBS continued throughout a 21-day growth period. When considered in comparison with results for other FaRLiP cyanobacteria, the results here show that acclimation responses to FRL can differ considerably among FaRLiP cyanobacteria.     

A soluble carotenoid protein involved in phycobilisome-related energy dissipation in cyanobacteria

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 DeltapH-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.     

Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins

In response to iron deficiency, cyanobacteria synthesize the iron stress-induced chlorophyll binding protein IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light-induced nonphotochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacterial cell cultures, strong blue light is known to induce a mechanism that dissipates excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism, the soluble Orange Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. By contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light-induced nonphotochemical quenching involves the phycobilisome OCP-related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the stress generated by iron starvation, initially by increasing the size of the photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes.     

Mechanism of the down regulation of photosynthesis by blue light in the Cyanobacterium synechocystis sp. PCC 6803

Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.     

Induction of Nonphotochemical Energy Dissipation and Absorbance Changes in Leaves (Evidence for Changes in the State of the Light-Harvesting System of Photosystem II in Vivo)

Simultaneous measurements of nonphotochemical quenching of chlorophyll fluorescence and absorbance changes in the 400- to 560-nm region have been made following illumination of dark-adapted leaves of the epiphytic bromeliad Guzmania monostachia. During the first illumination, an absorbance change at 505 nm occurred with a half-time of 45 s as the leaf zeaxanthin content rose to 14% of total leaf carotenoid. Selective light scattering at 535 nm occurred with a half-time of 30 s. During a second illumination, following a 5-min dark period, quenching and the 535-nm absorbance change occurred more rapidly, reaching a maximum extent within 30 s. Nonphotochemical quenching of chlorophyll fluorescence was found to be linearly correlated to the 535-nm absorbance change throughout. Examination of the spectra of chlorophyll fluorescence emission at 77 K for leaves sampled at intervals during this regime showed selective quenching in the light-harvesting complexes of photosystem II (LHCII). The quenching spectrum of the reversible component of quenching had a maximum at 700 nm, indicating quenching in aggregated LHCII, whereas the irreversible component represented a quenching of 680-nm fluorescence from unaggregated LHCII. It is suggested that this latter process, which is associated with the 505-nm absorbance change and zeaxanthin formation, is indicating a change in state of the LHCII complexes that is necessary to amplify or activate reversible pH-dependent energy dissipation, which is monitored by the 535-nm absorbance change. Both of the major forms of nonphotochemical energy dissipation in vivo are therefore part of the same physiological photoprotective process and both result from alterations in the LHCII system.     

Low-temperature energy transfer in LHC-II trimers from the Chl a/b light-harvesting antenna of photosystem II.

Temperature dependence in electronic energy transfer steps within light-harvesting antenna trimers from photosystem II was investigated by studying Chl a pump-probe anisotropy decays at several wavelengths from 675 to 682 nm. The anisotropy lifetime is markedly sensitive to temperature at the longest wavelengths (680-682 nm), increasing by factors of 5 to 6 as the trimers are cooled from room temperature to 13 K. The temperature dependence is muted at 677 and 675 nm. This behavior is modeled using simulations of temperature-broadened Chl a absorption and fluorescence spectra in spectral overlap calculations of Förster energy transfer rates. In this model, the 680 nm anisotropy decays are dominated by uphill energy transfers from 680 nm Chl a pigments at the red edge of the LHC-II spectrum; the 675 nm anisotropy decays reflect a statistical average of uphill and downhill energy transfers from 676-nm pigments. The measured temperature dependence is consistent with essentially uncorrelated inhomogeneous broadening of donor and acceptor Chl a pigments.     

Organization and role of the long-wave chlorophylls in the photosystem I of the Cyanobacterium spirulina.

The data on the organization and function of the photosystem I pigment-protein complexes of the cyanobacterium Spirulina and the characteristics of pigment antenna of the photosystem I monomeric and trimeric core complexes are presented and discussed. We proved that the photosystem I complexes in the cyanobacterial membrane pre-exist mainly as trimers, though both types of complexes contribute to the photosynthetic electron transport. In contrast to monomers, the antenna of the photosystem I trimeric complexes of Spirulina contains the extreme long-wave chlorophyll form absorbing at 735 nm and emitting at 760 nm (77 K). The intensity of fluorescence at 760 nm depends strongly on the P700 redox state: it is maximum with the reduced P700 and strongly decreased with the oxidized P700 which is the most efficient quencher of fluorescence at 760 nm. The energy absorbed by the extreme long-wave chlorophyll form is active in the photooxidation of P700 in the trimeric complex. The data obtained indicate that the long-wave form of chlorophyll originates from interaction of the chlorophyll molecules localized on monomeric subunits forming the photosystem I trimer. Kinetic analysis of the P700 photooxidation and light-induced quenching of fluorescence at 760 nm (77 K) allows the suggestion that the excess energy absorbed by the antenna monomeric subunits within the trimer migrates via the extreme long-wave chlorophyll to the P700 cation radical and is quenched, which prevents the photodestruction of the pigment-protein complex.