Roles of ApcD and orange carotenoid protein in photoinduction of electron transport upon dark-light transition in the Synechocystis PCC 6803 mutant deficient in flavodiiron protein Flv1.

Flavodiiron proteins Flv1/Flv3 accept electrons from photosystem (PS) I. In this work we investigated light adaptation mechanisms of Flv1-deficient mutant of Synechocystis PCC 6803, incapable to form the Flv1/Flv3 heterodimer. First seconds of dark-light transition were studied by parallel measurements of light-induced changes in chlorophyll fluorescence, P700 redox transformations, fluorescence emission at 77 K, and OCP-dependent fluorescence quenching. During the period of Calvin cycle activation upon dark-light transition, the linear electron transport (LET) in wild type is supported by the Flv1/Flv3 heterodimer, whereas in Δflv1 mutant activation of LET upon illumination is preceded by cyclic electron flow that maintains State 2. The State 2-State 1 transition and Orange Carotenoid Protein (OCP)-dependent non-photochemical quenching occur independently of each other, begin in about 10 s after the illumination of the cells and are accompanied by a short-term re-reduction of the PSI reaction center (P700+). ApcD is important for the State 2-State 1 transition in the Δflv1 mutant, but S-M rise in chlorophyll fluorescence was not completely inhibited in Δflv1/ΔapcD mutant. LET in Δflv1 mutant starts earlier than the S-M rise in chlorophyll fluorescence, and the oxidation of plastoquinol (PQH2) pool promotes the activation of PSII, transient re-reduction of P700+ and transition to State 1. An attempt to induce state transition in the wild type under high intensity light using methyl viologen, highly oxidizing P700 and PQH2, was unsuccessful, showing that oxidation of intersystem electron-transport carriers might be insufficient for the induction of State 2-State 1 transition in wild type of Synechocystis under high light.

State of the phycobilisome determines effective absorption cross-section of Photosystem II in Synechocystis sp. PCC 6803.

Quenching of excess excitation energy is necessary for the photoprotection of light-harvesting complexes. In cyanobacteria, quenching of phycobilisome (PBS) excitation energy is induced by the Orange Carotenoid Protein (OCP), which becomes photoactivated under high light conditions. A decrease in energy transfer efficiency from the PBSs to the reaction centers decreases photosystem II (PS II) activity. However, quantitative analysis of OCP-induced photoprotection in vivo is complicated by similar effects of both photochemical and non-photochemical quenching on the quantum yield of the PBS fluorescence overlapping with the emission of chlorophyll. In the present study, we have analyzed chlorophyll a fluorescence induction to estimate the effective cross-section of PS II and compared the effects of reversible OCP-dependent quenching of PBS fluorescence with reduction of PBS content upon nitrogen starvation or mutations of key PBS components. This approach allowed us to estimate the dependency of the rate constant of PS II primary electron acceptor reduction on the amount of PBSs in the cell. We found that OCP-dependent quenching triggered by blue light affects approximately half of PBSs coupled to PS II, indicating that under normal conditions, the concentration of OCP is not sufficient for quenching of all PBSs coupled to PS II.

Deficiency in flavodiiron protein Flv3 promotes cyclic electron flow and state transition under high light in the cyanobacterium Synechocystis sp. PCC 6803.

Photosynthetic organisms adjust their activity to changes in irradiance by different ways, including the operation of cyclic electron flow around photosystem I (PSI) and state transitions that redistribute amounts of light energy absorbed by PSI and PSII. In dark-acclimated wild type cells of Synechocystis PCC 6803, linear electron transport was activated after the first 500 ms of illumination, while cyclic electron flow around PSI was long predominant in the mutant deficient in flavodiiron protein Flv3. Chlorophyll P700 oxidation associated with activation of linear electron flow extended in the Flv3- mutant to several tens of seconds and included a P700+ re-reduction phase. Parallel monitoring of chlorophyll fluorescence and the redox state of P700 indicated that, at low light intensity both in wild type and in the Flv3- mutant, the transient re-reduction step coincided in time with S-M fluorescence rise, which reflected state 2-state 1 transition (Kana et al., 2012). Despite variations in the initial redox state of plastoquinone pool, the oxidases-deficient mutant, succinate dehydrogenase-deficient mutant, and wild type cells did not show the S-M rise under high-intensity light until additional Flv3- mutation was introduced in these strains. Thus, the lack of available electron acceptor for PSI was the main cause for the appearance of S-M fluorescence rise under high light. It is concluded that the lack of Flv3 protein promotes cyclic electron flow around PSI and facilitates the subsequent state 2-state 1 transition in the absence of strict relation to the dark-operated pathways of plastoquinone reduction or oxidation.

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803.

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (LCM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.

Radioprotective role of cyanobacterial phycobilisomes.

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called "Great Oxygenation Event" that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.

Photoinduction of electron transport on the acceptor side of PSI in Synechocystis PCC 6803 mutant deficient in flavodiiron proteins Flv1 and Flv3.

After transferring the dark-acclimated cyanobacteria to light, flavodiiron proteins Flv1/Flv3 serve as a main electron acceptor for PSI within the first seconds because Calvin cycle enzymes are inactive in the dark. Synechocystis PCC 6803 mutant Δflv1/Δflv3 devoid of Flv1 and Flv3 retained the PSI chlorophyll P700 in the reduced state over 10 s (Helman et al., 2003; Allahverdiyeva et al., 2013). Study of P700 oxidoreduction transients in dark-acclimated Δflv1/Δflv3 mutant under the action of successive white light pulses separated by dark intervals of various durations indicated that the delayed oxidation of P700 was determined by light activation of electron transport on the acceptor side of PSI. We show that the light-induced redox transients of chlorophyll P700 in dark-acclimated Δflv1/Δflv3 proceed within 2 min, as opposed to 1-3 s in the wild type, and comprise a series of kinetic stages. The release of rate-limiting steps was eliminated by iodoacetamide, an inhibitor of Calvin cycle enzymes. Conversely, the creation with methyl viologen of a bypass electron flow to O2 accelerated P700 oxidation and made its extent comparable to that in the wild-type cells. The lack of major sinks for linear electron flow in iodoacetamide-treated Δflv1/Δflv3 mutant, in which O2- and CO2-dependent electron flows were impaired, facilitated cyclic electron flow, which was evident from the decreased steady-state oxidation of P700 and from rapid dark reduction of P700 during and after illumination with far-red light. The results show that the photosynthetic induction in wild-type Synechocystis PCC 6803 is largely hidden due to the flavodiiron proteins whose operation circumvents the rate-limiting electron transport steps controlled by Calvin cycle reactions.

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 LCM. 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 PBLCM 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 PBLCM domain takes part in formation of the OCP binding site in the PBS.

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria.

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

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.

Photosystem 2 effective fluorescence cross-section of cyanobacterium Synechocystis sp. PCC6803 and its mutants.

The effective fluorescence cross-section of photosystem 2 (PS2) was defined by measurements of chlorophyll a fluorescence induction curves for the wild type of the unicellular cyanobacterium Synechocystis sp. PCC6803, C-phycocyanin deficient mutant (CK), and mutant that totally lacks phycobilisomes (PAL). It was shown that mutations lead to a strong decrease of the PS2 effective fluorescence cross-section. For instance, the effective fluorescence cross-section of PS2 for wild type, CK and PAL mutants excited at λ(ex)=655 nm were found to be 896, 220 and 83 Å(2) respectively. Here we present an estimation of energy transfer efficiency from phycobilisomes to the pigment-protein complexes of PS2. It was shown that the PS2 fluorescence enhancement coefficient reaches a maximum value of 10.7 due to the energy migration from phycobilisomes. The rate constant of energy migration was found to be equal to 1.04 × 10(10) s(-1).

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.

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems.

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems' activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions-P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L(CM) is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L(CM) may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium Synechocystis sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes.

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue-green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.

Protein-protein interactions in carotenoid triggered quenching of phycobilisome fluorescence in Synechocystis sp. PCC 6803.

An inquiry into the effect of temperature on carotenoid triggered quenching of phycobilisome (PBS) fluorescence in a photosystem II-deficient mutant of Synechocystis sp. results in identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of PBSs subject to quenching depends on the state of some membrane component.

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.

Carotenoid-induced quenching of the phycobilisome fluorescence in photosystem II-deficient mutant of Synechocystis sp.

Brief--10-second long--irradiation of a photosystem II-deficient mutant of cyanobacterium Synechocystis sp. PCC 6803 with intense blue or UV-B light causes an about 40% decrease of phycobilisome (PBS) fluorescence, slowly reversible in the dark. The registered action spectrum of PBS fluorescence quenching only shows bands at 500, 470 and 430 nm, typical of carotenoids, and an additional UV-B band; no peaks in the region of chlorophyll or PBS absorption have been found. We propose that quenching induced by carotenoids, possibly protein-bound or glycoside, reveals a new regulatory mechanism protecting photosynthetic apparatus of cyanobacteria against photodamage.