Zsuzsanna Deák

The 9-kDa phosphoprotein of photosystem II. Generation and characterisation of Chlamydomonas mutants lacking PSII-H and a site-directed mutant lacking the phosphorylation site.

The chloroplast gene psbH encodes a 9-10 kDa thylakoid membrane protein (PSII-H) that is associated with photosystem II and is subject to light-dependent phosphorylation at a threonine residue located on the stromal side of the membrane. The function of PSII-H is not known, neither is it clear what regulatory role phosphorylation may play in the control of PSII activity. Using particle gun-mediated transformation, we have created chloroplast transformants of Chlamydomonas reinhardtii in which the synthesis of PSII-H is prevented by the disruption of psbH, or in which the phosphorylatable threonine is replaced by alanine through site-directed mutagenesis of the gene. The mutants lacking PSII-H have a photosystem II-deficient phenotype, with no detectable functioning PSII complex present in whole cells or isolated thylakoid membranes. In contrast, the alanine mutant (T3A) grows photoautotrophically, and PSII activity is comparable to wild-type cells as determined by various biochemical and biophysical assays.

In Vivo Target Sites of Nitric Oxide in Photosynthetic Electron Transport as Studied by Chlorophyll Fluorescence in Pea Leaves

The role of nitric oxide (NO) in photosynthesis is poorly understood as indicated by a number of studies in this field with often conflicting results. As various NO donors may be the primary source of discrepancies, the aim of this study was to apply a set of NO donors and its scavengers, and examine the effect of exogenous NO on photosynthetic electron transport in vivo as determined by chlorophyll fluorescence of pea (Pisum sativum) leaves. Sodium nitroprusside-induced changes were shown to be mediated partly by cyanide, and S-nitroso-N-acetylpenicillinamine provided low yields of NO. However, the effects of S-nitrosoglutathione are inferred exclusively by NO, which made it an ideal choice for this study. QA− reoxidation kinetics show that NO slows down electron transfer between QA and QB, and inhibits charge recombination reactions of QA− with the S2 state of the water-oxidizing complex in photosystem II. Consistent with these results, chlorophyll fluorescence induction suggests that NO also inhibits steady-state photochemical and nonphotochemical quenching processes. NO also appears to modulate reaction-center-associated nonphotochemical quenching.

Thermoluminescence and flash-oxygen characterization of the IC2 deletion mutant of Synechocystis sp. PCC 6803 lacking the photosystem II 33 kDa protein

Abstract The psbO gene product of Photosystem II (PS II), the so-called 33 kDa extrinsic protein, is believed to be closely associated with the catalytic Mn cluster responsible for light-induced water oxidation. However, this protein is not absolutely required for water-splitting and its precise role remains to be clarified. We have used flash-induced thermoluminescence and oxygen evolution measurements to characterize the process of water oxidation in the IC2 mutant of Synechocystis sp. PCC 6803 from which the psbO gene had been deleted by Mayes et al. (Mayes, S.R., Cook, K.M., Self, S.J., Zhang, Z. and Barber J, (1991) Biochim. Biophys. Acta 1060, 1–12). The thermoluminescence results show that the extent of charge stabilization in the S 2 Q A and S 2 Q B states is reduced in the IC2 mutant to about 25–30% of that observed in the wild-type, suggesting that functional oxygen evolution occurs in a proportion of the psbO -less mutant cells. The stability of the S 2 Q A , but not that of the S 2 Q B , charge pair is markedly increased in the mutant. This points to a structural change of the PS II reaction center complex in the absence of the psbO gene product which affects the redox properties of the Q A and Q B acceptors to a different extent. The flash-induced oscillation of the B thermoluminescence band, arising from the S 2 Q B and S 3 Q B charge recombinations, is largely dampened in the mutant. This indicates that the ability of the water-oxidizing complex to reach its higher oxidation states, S 3 and S 4 , is limited when the psbO gene product is absent. In agreement with the thermoluminescence results, flash-induced oxygen evolution shows a decreased yield and largely dampened oscillation pattern in the mutant. These results indicate that although the psbO gene product is not an absolute requirement for water oxidation its absence disturbs the redox cycling of the water-oxidizing complex and retards the formation of its higher S states. The rapid loss of thermoluminescence intensity during strong illumination of the mutated organism confirms its high susceptibility to photoinhibition. This effect is most likely the consequence of the limited rate of electron donation from the psbO -less water-oxidizing complex to the PS II reaction centre where the accumulation of highly oxidizing species may damage their pigment and protein surroundings.

Functional Characterization and Quantification of the Alternative PsbA Copies in Thermosynechococcus elongatus and Their Role in Photoprotection

The D1 protein (PsbA) of photosystem II (PSII) from Thermosynechococcus elongatus is encoded by a psbA gene family that is typical of cyanobacteria. Although the transcription of these three genes has been studied previously (Kós, P. B., Deák, Z., Cheregi, O., and Vass, I. (2008) Biochim. Biophys. Acta 1777, 74–83), the protein quantification had not been possible due to the high sequence identity between the three PsbA copies. The successful establishment of a method to quantify the PsbA proteins on the basis of reverse phase-LC-electrospray mass ionization-MS/MS (RP-LC-ESI-MS/MS) enables an accurate comparison of transcript and protein level for the first time ever. Upon high light incubation, about 70% PsbA3 could be detected, which closely corresponds to the transcript level. It was impossible to detect any PsbA2 under all tested conditions. The construction of knock-out mutants enabled for the first time a detailed characterization of both whole cells and also isolated PSII complexes. PSII complexes of the ΔpsbA1/psbA2 mutant contained only copy PsbA3, whereas only PsbA1 could be detected in PSII complexes from the ΔpsbA3 mutant. In whole cells as well as in isolated complexes, a shift of the free energy between the redox pairs in the PsbA3 complexes in comparison with PsbA1 could be detected by thermoluminescence and delayed fluorescence measurements. This change is assigned to a shift of the redox potential of pheophytin toward more positive values. Coincidentally, no differences in the QA-QB electron transfer could be observed in flash-induced fluorescence decay or prompt fluorescence measurements. In conclusion, PsbA3 complexes yield a better protection against photoinhibition due to a higher probability of the harmless dissipation of excess energy.

Charge equilibrium between the water-oxidizing complex and the electron donor tyrosine-D in Photosystem II

Abstract The decay kinetics for the S2 and S3 states of the water-oxidizing complex have been measured with an unmodulated Joliot-type oxygen electrode in isolated spinach thylakoids. The S2 and S3 states decay biphasically (Vermaas, W.F.J., Renger, G. and Dohnt, G. (1985) Biochim. Biophys. Acta 764, 194–202) with half-decay times of 1–1.5 s and 30–35 s at room temperature. The proportion of the fast phase is negligible in preilluminated thylakoids but increases during dark adaptation to 22–24% for both S2 and S3. This process, t 1 2 ≈ 10 min , is accompanied with the conversion of the S0 state to S1 in about 25% of the centers. Chemical reduction of tyrosine-D+, which gives rise to the EPR Signals IIslow, by dichlorphenolindophenol/ascorbate increases the proportion of the fast decaying phase of S2 and S3 to about 70–80%. The decay of S2 is accompanied by the accumulation of S1 and the decay of S3 results in a transient increase of S2. These data led us to conclude that the fast phase in the S2 and S3 decay is correlated with one-electron donation from tyrosine-D to the water-oxidizing complex located within the same center. This process results in the S3D → S2D+ (→ S1D+) and S2D → S1D+ univalent sequences of deactivating reactions. The electron transfer from tyrosine-D to the S2 and S3 states is strongly temperature-dependent and shows 0.46 and 0.49 eV activation energy, respectively, over the +8 to +37°C temperature range. The deactivation process which is reflected by the slower phase of S2 and S3 decay has an activation energy of 0.65 and 0.76 eV, respectively. An extension of the Kok model of oxygen evolution is also presented taking into account the effect of fast electron donation from tyrosine-D to the water-oxidizing complex.

Pure forms of the singlet oxygen sensors TEMP and TEMPD do not inhibit Photosystem II.

In a recent article (Hakala-Yatkin and Tyystjärvi BBA 1807 (2011) 243-250) it was reported that the singlet oxygen spin traps 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidone (TEMPD) inhibit Photosystem II (PSII), the water oxidizing enzyme. O₂ evolution, chlorophyll fluorescence and thermoluminescence were measured and were shown to be greatly affected by these chemicals. This work casts doubts over an earlier body of work in which these chemicals were used as spin traps for monitoring ¹O₂ production when PSII was inhibited by high light intensities. Here we show that these spin probes hardly affect PSII. We show that the commercial batches of TEMPD and TEMP used by Hakala-Yatkin and Tyystjärvi contained impurities and/or derivatives that inhibited PSII and caused the specific effects on fluorescence. Earlier work that used pure spin traps to measure ¹O₂ during photoinhibition, thus remains valid. However, concern must be expressed towards using these spin traps without proper controls.

The accessory electron donor tyrosine-D of Photosystem II is slowly reduced in the dark during low-temperature storage of isolated thylakoids

Storage of thylakoids for several months at 203 K in the dark changes the flash pattern of oxygen evolution by gradually shifting the first oxygen maximum from the third flash, where it is usually observed, to the fourth flash. This effect is accompanied with the increase of a fast phase (t12 = 1.5 s) in the decay of the S2 and S3 states of the water-oxidizing complex in Photosystem II. In parallel to the changes in the oxygen flash pattern the EPR signal from the stable tyrosine-D+ radical (Signal IIslow) completely disappears with a half-time of approx. 13 weeks. The normal oxygen yield sequence, showing the first maximum at the third flash, as well as the original amplitude of Signal IIslow can be restored by a single flash or by continuous illumination at room temperature. These data show that tyrosine-D+ is reduced by an endogenous redox component in Photosystem II during dark storage of thylakoids at 203 K. In parallel with the reduction of tyrosine-D+ we observed the oxidation of high potential cytochrome b-559, and it is proposed that at low temperature an electron can be transferred from high-potential cytochrome b-559 to tyrosine-D+ in a slow reaction in most of the centers.

Bacterial symbionts enhance photo-fermentative hydrogen evolution of Chlamydomonas algae

The green algae Chlamydomonas sp. MACC-549 and Chlamydomonas reinhardtii cc124 were investigated for their hydrogen-evolution capability in mixed algal–bacterial cultures. Stable bacterial contaminations were identified during the cultivation of Chlamydomonas sp. 549. The bacterial symbionts belonged to various genera, mostly Brevundimonas, Rhodococcus, and Leifsonia, each of which enhanced the algal hydrogen production. This phenomenon was not limited to natural associations. Increased algal hydrogen evolution was achieved by simple artificial algal–bacterial communities as well. Algal–bacterial cocultures were designed and tested in hydrogen evolution experiments. The highest hydrogen yields were obtained when hydrogenase-deficient Escherichia coli was used as a symbiotic bacterium (Chlamydomonas sp. 549 generated 1196.06 ± 4.42 μL H2 L−1, while C. reinhardtii cc124 produced 5800.54 ± 65.73 μL H2 L−1). The results showed that oxygen elimination is the most crucial factor for algal hydrogen production and that efficient bacterial respiration is essential for the activation of algal Fe-hydrogenase. The algae-based hydrogen evolution method described represents a novel combination of fermentative and photolytic hydrogen generation processes. Active photosynthesis was maintained during the entire hydrogen evolution process, which contributes to the sustainability of hydrogen production.

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity.

BACKGROUND AND AIMS Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca(2+) and Cl(-) as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl(-) and Ca(2+), but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. METHODS Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT-PCR and time-resolved chlorophyll fluorescence were used. KEY RESULTS Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. CONCLUSIONS In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.

Symbiodinium sp cells produce light-induced intra- and extracellular singlet oxygen, which mediates photodamage of the photosynthetic apparatus and has the potential to interact with the animal host in coral symbiosis

Coral bleaching is an important environmental phenomenon, whose mechanism has not yet been clarified. The involvement of reactive oxygen species (ROS) has been implicated, but direct evidence of what species are involved, their location and their mechanisms of production remains unknown. Histidine-mediated chemical trapping and singlet oxygen sensor green (SOSG) were used to detect intra- and extracellular singlet oxygen ((1) O2 ) in Symbiodinium cultures. Inhibition of the Calvin-Benson cycle by thermal stress or high light promotes intracellular (1) O2 formation. Histidine addition, which decreases the amount of intracellular (1) O2 , provides partial protection against photosystem II photoinactivation and chlorophyll (Chl) bleaching. (1) O2 production also occurs in cell-free medium of Symbiodinium cultures, an effect that is enhanced under heat and light stress and can be attributed to the excretion of (1) O2 -sensitizing metabolites from the cells. Confocal microscopy imaging using SOSG showed most extracellular (1) O2 around the cell surface, but it is also produced across the medium distant from the cells. We demonstrate, for the first time, both intra- and extracellular (1) O2 production in Symbiodinium cultures. Intracellular (1) O2 is associated with photosystem II photodamage and pigment bleaching, whereas extracellular (1) O2 has the potential to mediate the breakdown of symbiotic interaction between zooxanthellae and their animal host during coral bleaching.