Photosystem II Activity of Wild Type Synechocystis PCC 6803 and Its Mutants with Different Plastoquinone Pool Redox States.

To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox(-) mutant with naturally reduced PQ is characterized by slower QA(-) reoxidation and O2 evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (Fv/Fm) as compared to the wild type and SDH(-) mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox(-) mutant after dark adaptation. While light adaptation increases Fv/Fm in all tested strains, indicating PSII activation, by far the greatest increase in Fv/Fm and O2 evolution rates is observed in the Ox(-) mutant. Continuous illumination of Ox(-) mutant cells with low-intensity blue light, that accelerates QA(-) reoxidation, also increases Fv/Fm and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH(-) mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm.

Functions of chromophore-containing domain in the large linker LCM- polypeptide of phycobilisome.

In the large linker ArcE polypeptide of the phycobilisome (PBS) from the cyanobacterium Synechocystis sp. PCC 6803, the chromophore-containing 26-kDa domain was deleted with consequent disturbance of the main PBS functions. Phycobilisomes in mutant cells staying in contact with photosystem I cannot transfer energy to the photosystem II. Under the bright light conditions, the interaction of PBSs with the photoprotective orange carotenoid protein in the mutant was lost and the implementation of transition states 1 and 2 of the pigment apparatus was significantly reduced.

Association of High Light-Inducible HliA/HliB Stress Proteins with Photosystem 1 Trimers and Monomers of the Cyanobacterium Synechocystis PCC 6803.

Hlip (high light-inducible proteins) are important for protection of the photosynthetic apparatus of cyanobacteria from light stress. However, the interaction of these proteins with chlorophyll-protein complexes of thylakoids remains unclear. The association of HliA/HliB stress proteins with photosystem 1 (PS1) complexes of the cyanobacterium Synechocystis PCC 6803 was studied to understand their function. Western blotting demonstrated that stress-induced HliA/HliB proteins are associated with PS1 trimers in wild-type cells grown under moderate light condition (40 µmol photons/m(2) per sec). The content of these proteins increased 1.7-fold after light stress (150 µmol photons/m(2) per sec) for 1 h. In the absence of PS1 trimers (ΔpsaL mutant), the HliA/HliB proteins are associated with PS1 monomers and the PS2 complex. HliA/HliB proteins are associated with PS1 monomers but not with PS1 trimers in Synechocystis PS2-deficient mutant grown at 5 µmol photons/m(2) per sec; the content of Hli proteins associated with PS1 monomers increased 1.2-fold after light stress. The HliA/HliB proteins were not detected in wild-type cells of cyanobacteria grown in glucose-supplemented medium at 5 µmol photons/m(2) per sec, but light stress induces the synthesis of stress proteins associated with PS1 trimers. Thus, for the first time, the association of HliA/HliB proteins not only with PS1 trimers, but also with PS1 monomers is shown, which suggests a universal role of these proteins in the protection of the photosynthetic apparatus from excess light.

Photosystem activity and state transitions of the photosynthetic apparatus in cyanobacterium Synechocystis PCC 6803 mutants with different redox state of the plastoquinone pool.

To better understand how photosystem (PS) activity is regulated during state transitions in cyanobacteria, we studied photosynthetic parameters of photosystem II (PSII) and photosystem I (PSI) in Synechocystis PCC 6803 wild type (WT) and its mutants deficient in oxidases (Ox(-)) or succinate dehydrogenase (SDH(-)). Dark-adapted Ox(-) mutant, lacking the oxidation agents, is expected to have a reduced PQ pool, while in SDH(-) mutant the PQ pool after dark adaptation will be more oxidized due to partial inhibition of the respiratory chain electron carriers. In this work, we tested the hypothesis that control of balance between linear and cyclic electron transport by the redox state of the PQ pool will affect PSII photosynthetic activity during state transition. We found that the PQ pool was reduced in Ox(-) mutant, but oxidized in SDH(-) mutant after prolonged dark adaptation, indicating different states of the photosynthetic apparatus in these mutants. Analysis of variable fluorescence and 77K fluorescence spectra revealed that the WT and SDH(-) mutant were in State 1 after dark adaptation, while the Ox(-) mutant was in State 2. State 2 was characterized by ~1.5 time lower photochemical activity of PSII, as well as high rate of P700 reduction and the low level of P700 oxidation, indicating high activity of cyclic electron transfer around PSI. Illumination with continuous light 1 (440 nm) along with flashes of light 2 (620 nm) allowed oxidation of the PQ pool in the Ox(-) mutant, thus promoting it to State 1, but it did not affect PSII activity in dark adapted WT and SDH(-) mutant. State 1 in the Ox(-) mutant was characterized by high variable fluorescence and P700(+) levels typical for WT and the SDH(-) mutant, indicating acceleration of linear electron transport. Thus, we show that PSII of cyanobacteria has a higher photosynthetic activity in State 1, while it is partially inactivated in State 2. This process is controlled by the redox state of PQ in cyanobacteria through enhancement/inhibition of electron transport on the acceptor side of PSII.

Features of temporal behavior of fluorescence recovery in Synechocystis sp. PCC6803.

Under high photon flux density of solar radiation, the photosynthetic apparatus can be damaged. To prevent this photodestruction, cyanobacteria developed special mechanisms of non-photochemical quenching (NPQ) of excitation energy in phycobilisomes. In Synechocystis, NPQ is triggered by the orange carotenoid protein (OCP), which is sensitive to blue-green illumination allowing it to bind to the phycobilisome reducing the flow of energy to the photosystems. Consequent decoupling of OCP and recovery of phycobilisome fluorescence in vivo is controlled by the so called fluorescence recovery protein (FRP). In this work, the role of the phycobilisome core components, apcD and apcF, in non-photochemical quenching and subsequent fluorescence recovery in the phycobilisomes of the cyanobacterium Synechocystis sp. PCC6803 has been investigated. Using a single photon counting technique, we have registered fluorescence decay spectra with picosecond time resolution during fluorescence recovery. In order to estimate the activation energy for the photocycle, spectroscopic studies in dependency on the temperature from 5 to 45 °C have been performed. It was found that fluorescence quenching and recovery were strongly temperature dependent for all strains exhibiting characteristic non-linear time courses. The rise of the fluorescence intensity during fluorescence recovery after NPQ can be completely described by the increase of the phycobilisome core fluorescence lifetime. It was shown that fluorescence recovery of apcD- and apcF-deficient mutants is characterized by a significantly lower activation energy barrier compared to wild type. This phenomenon indicates that apcD and apcF gene products may be required for proper interaction of FRP and OCP coupled to the phycobilisome core. In addition, we found that the rate of fluorescence recovery decreases with an increase of the non-photochemical quenching amplitude, probably due to depletion of substrate for the enzymatic reaction catalyzed by FRP.

Effect of APCD and APCF subunits depletion on phycobilisome fluorescence of the cyanobacterium Synechocystis PCC 6803.

Long-wavelength allophycocyanin (APC) subunits in cyanobacteria (APCD, APCE, and APCF) are required for phycobilisome (PBS) assembly, stability, and energy transfer to photosystems. Here we studied fluorescence properties of PBS in vivo, using Synechocystis PCC 6803 mutant cells deficient in both photosystems and/or long-wavelength APC subunits. At room temperature, an absence of APCD and APCF subunits resulted in ∼2-fold decrease of long-wavelength APC (APC680) fluorescence. In 77K fluorescence spectra, we observed only a slight shift of long-wavelength emission. However, 77K fluorescence of a PSI/PSII/APCF-less mutant was also characterized by increased emission from short-wavelength APC, which suggested the importance of this subunit in energy transfer from APC660 to APC680. Under blue-green actinic light, all mutants showed significant non-photochemical fluorescence quenching of up to 80% of the initial dark fluorescence level. Based on the mutants' quenching spectra, we determined quenching to originate from the pool of short-wavelength APC, while the spectral data alone was not sufficient to make unambiguous conclusion on the involvement of long-wavelength APC in non-photochemical quenching. Using a model of quenching center formation, we determined interaction rates between PBS and orange carotenoid protein (OCP) in vivo. Absence of APCD or APCF subunits had no effect on the rates of quenching center formation confirming the data obtained for isolated OCP-PBS complexes. Thus, although APCD and APCF subunits were required for energy transfer in PBS in vivo, their absence did not affect rates of OCP-PBS binding.

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry.

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures - from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 µmolphotonsm⁻²s⁻¹. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Investigation of OCP-triggered dissipation of excitation energy in PSI/PSII-less Synechocystis sp. PCC 6803 mutant using non-linear laser fluorimetry.

In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet-singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3'hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center.

Regulation of cyclic electron transport through photosystem I in cyanobacterium Synechocystis sp. PCC 6803 mutants deficient in respiratory dehydrogenases.

The rate of PSI mediated cyclic electron transport was studied in wild type and mutant cells of Synechocystis sp. PCC 6803 deficient in NDH-1 (M55) or succinate dehydrogenase (SDH(-)) that are responsible for the dark reduction of the plastoquinone pool. Kinetics of P700 photooxidation and P700(+) dark reduction in the presence of 5·10(-5) M 3-(3,4-dichlorophenyl)-1,1-dimethylurea have been registered as light induced absorbance changes at 810 nm resulting from illumination of cells with 730-nm actinic light for 1 sec. It is shown that in the absence of dehydrogenases the rate of dark reduction of P700(+) in both mutants did not decrease but even increased in NDH-1-less mutant cells as compared with the rate in wild type cells. Dibromothymoquinone drastically reduced the rate of P700(+) dark reduction both in wild type and in mutant cells. Thus, the cyclic electron transfer from ferredoxin through the plastoquinone pool to P700(+), which is independent from dehydrogenases, takes place in all the types of cells. Preillumination of cells of wild type and both mutants for 30 min or anaerobic conditions resulted in delay of P700 photooxidation and acceleration of P700(+) dark reduction, while the level of photosynthesis and respiration terminal acceptors (NAD(P)(+) and oxygen) decreased. It appears that the rate of P700 photooxidation and P700(+) dark reduction in cyclic electron transport in Synechocystis wild type and mutant cells is determined by the level of NADP+ and oxygen in stroma. A possible approach to evaluation of the levels of these acceptors in vivo is proposed, based on kinetic curve parameters of P700 photoconversions induced by 730-nm light with 1-sec duration.

Reduction of photosystem I reaction center by recombinant DrgA protein in isolated thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803.

To study the function of soluble NAD(P)H:quinone oxidoreductase of the cyanobacterium Synechocystis sp. PCC 6803 encoded by drgA gene, recombinant DrgA protein carrying 12 histidine residues on the C-terminal end was expressed in Escherichia coli and purified. Recombinant DrgA is a flavoprotein that exhibits quinone reductase and nitroreductase activities with NAD(P)H as the electron donor. Using EPR spectroscopy, it was demonstrated that addition of recombinant DrgA protein and NADPH to DCMU-treated isolated thylakoid membranes of the cyanobacterium increased the dark re-reduction rate of the photosystem I reaction center (P700(+)). Thus, DrgA can participate in electron transfer from NADPH to the electron transport chain of the Synechocystis sp. PCC 6803 thylakoid membrane.

Effects of dark adaptation on light-induced electron transport through photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

Kinetics of the redox reactions in the reaction center (P700) of photosystem I (PSI) of the cyanobacterium Synechocystis sp. PCC 6803 have been studied by EPR spectroscopy. The redox kinetics were recorded based on accumulation of the EPRI signal when the final signal was the sum of individual signals produced in response to illumination of the cells. After prolonged (more than 3 sec) dark intervals between illuminations, the kinetic curve of the EPR signal from P700+ was multiphasic. After a sharp increase in the signal amplitude at the beginning of illumination (phase I), the amplitude rapidly (for 0.1-0.2 sec) decreased (phase II). Then the signal amplitude gradually increased (phase III) until the steady rate of electron transfer was established. With short-term (1 sec) dark intervals between the flashes and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the kinetics of the light-induced increase in the EPR signal from P700+ were monophasic. Inhibition with iodoacetamide of electron transport on the acceptor side of PSI under anaerobic conditions or an increase in the amount of respiration substrates on addition of glucose into a suspension of DCMU-treated wild-type cells increased the level of P700 reduction in phase III. The findings suggest that the kinetic curve of the EPR signal from P700+ is determined by both the electron entrance onto P700+ on the donor side of PSI and activity of electron acceptors of PSI.

Reduction of photosystem I reaction center in DrgA mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking soluble NAD(P)H:quinone oxidoreductase.

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.

Role of NAD(P)H:quinone oxidoreductase encoded by drgA gene in reduction of exogenous quinones in cyanobacterium Synechocystis sp. PCC 6803 cells.

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).

Functional analysis of the Na+/H+ antiporter encoding genes of the cyanobacterium Synechocystis PCC 6803.

The role of putative Na+/H+ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na+/H+ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na+/H+ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na+/H+ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na+/H+ antiporters was studied. No induction of any Na+/H+ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na+/H+ antiporter encoding genes showed induction. These results might indicate that some of Na+/H+ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.

Resistance to nitrophenolic herbicides and metronidazole in the cyanobacterium Synechocystis sp. PCC 6803 as a result of the inactivation of a nitroreductase-like protein encoded by drgA gene.

Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole.

Cloning and sequencing of mutantpsbB genes of the cyanobacteriumSynechocystis PCC 6803.

Ten strains from a collection of mutants ofSynechocystis 6803 defective in Photosystem II (PS II) function were transformed with chromosomal DNA of wild-type and mutant cells. Cross hybridization data allowed to identify four groups of PS II-mutants. Highly efficient transformation was observed between different mutant groups, but not within the groups. Restoration of photosynthetic activity of the mutant cells was also achieved by transformation with different parts of a 5.6 kbBam HI fragment of wild typeSynechocystis DNA containing thepsbB gene. Each group of mutants was transformed to photoautotrophic growth by specific subfragments of thepsbB gene. DNA fragments of four selected mutant strains hybridizing with thepsbB gene were isolated and sequenced. The mutations were identified as a single nucleotide insertion or substitution leading to stop codon formation in two of the mutants, as a deletion of 12 nucleotides, or as a nucleotide substitution resulting in an amino acid substitution in the other two mutants. Deletion of 12 nucleotides in mutant strain PMB1 and stop codon formation in strain NF16 affect membrane-spanning regions of the gene product, the CP 47 protein.