Photosynthesis of the Cyanidioschyzon merolae cells in blue, red, and white light.

Photosynthesis and respiration rates, pigment contents, CO2 compensation point, and carbonic anhydrase activity in Cyanidioschizon merolae cultivated in blue, red, and white light were measured. At the same light quality as during the growth, the photosynthesis of cells in blue light was significantly lowered, while under red light only slightly decreased as compared with white control. In white light, the quality of light during growth had no effect on the rate of photosynthesis at low O2 and high CO2 concentration, whereas their atmospheric level caused only slight decrease. Blue light reduced markedly photosynthesis rate of cells grown in white and red light, whereas the effect of red light was not so great. Only cells grown in the blue light showed increased respiration rate following the period of both the darkness and illumination. Cells grown in red light had the greatest amount of chlorophyll a, zeaxanthin, and ß-carotene, while those in blue light had more phycocyanin. The dependence on O2 concentration of the CO2 compensation point and the rate of photosynthesis indicate that this alga possessed photorespiration. Differences in the rate of photosynthesis at different light qualities are discussed in relation to the content of pigments and transferred light energy together with the possible influence of related processes. Our data showed that blue and red light regulate photosynthesis in C. merolae for adjusting its metabolism to unfavorable for photosynthesis light conditions.

Deletion of psbQ' gene in Cyanidioschyzon merolae reveals the function of extrinsic PsbQ' in PSII.

KEY MESSAGE: We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ' subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ' subunit in the PSII complex. We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ' extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ' protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ' is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments.

KEY MESSAGE: We have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism. Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae-an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi: 10.1007/s00709-015-0936-9 ). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae-a new model organism.

Differences in photosynthetic responses of NADP-ME type C4 species to high light.

MAIN CONCLUSION: Three species chosen as representatives of NADP-ME C4 subtype exhibit different sensitivity toward photoinhibition, and great photochemical differences were found to exist between the species. These characteristics might be due to the imbalance in the excitation energy between the photosystems present in M and BS cells, and also due to that between species caused by the penetration of light inside the leaves. Such regulation in the distribution of light intensity between M and BS cells shows that co-operation between both the metabolic systems determines effective photosynthesis and reduces the harmful effects of high light on the degradation of PSII through the production of reactive oxygen species (ROS). We have investigated several physiological parameters of NADP-ME-type C4 species (e.g., Zea mays, Echinochloa crus-galli, and Digitaria sanguinalis) grown under moderate light intensity (200 µmol photons m-2 s-1) and, subsequently, exposed to excess light intensity (HL, 1600 µmol photons m-2 s-1). Our main interest was to understand why these species, grown under identical conditions, differ in their responses toward high light, and what is the physiological significance of these differences. Among the investigated species, Echinochloa crus-galli is best adapted to HL treatment. High resistance of the photosynthetic apparatus of E. crus-galli to HL was accompanied by an elevated level of phosphorylation of PSII proteins, and higher values of photochemical quenching, ATP/ADP ratio, activity of PSI and PSII complexes, as well as integrity of the thylakoid membranes. It was also shown that the non-radiative dissipation of energy in the studied plants was not dependent on carotenoid contents and, thus, other photoprotective mechanisms might have been engaged under HL stress conditions. The activity of the enzymes superoxide dismutase and ascorbate peroxidase as well as the content of malondialdehyde and H2O2 suggests that antioxidant defense is not responsible for the differences observed in the tolerance of NADP-ME species toward HL stress. We concluded that the chloroplasts of the examined NADP-ME species showed different sensitivity to short-term high light irradiance, suggesting a role of other factors excluding light factors, thus influencing the response of thylakoid proteins. We also observed that HL affects the mesophyll chloroplasts first hand and, subsequently, the bundle sheath chloroplasts.

Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae.

The binding affinity of the two substrate-water molecules to the water-oxidizing Mn4CaO5 catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S2 and S3 states by the exchange of bound ¹6O-substrate against ¹8O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: kf=52 ± 8s⁻¹ and ks=1.9 ± 0.3s⁻¹ in the S2 state, and kf=42 ± 2s⁻¹ and kslow=1.2 ± 0.3s⁻¹ in S3, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate-water site in the S2 state, which confirms beyond doubt that both substrate-water molecules are already bound in the S2 state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S2→S3 transition. Implications for recent models for water-oxidation are briefly discussed.

A reaction center-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga, Cyanidioschyzon merolae.

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. Although we now have a detailed structural model of photosystem II (PSII) from cyanobacteria at an atomic resolution, no corresponding structure of the eukaryotic PSII complex has been published to date. Here we report the isolation and characterization of a highly active and robust dimeric PSII complex from C. merolae. We show that this complex is highly stable across a range of extreme light, temperature, and pH conditions. By measuring fluorescence quenching properties of the isolated C. merolae PSII complex, we provide the first direct evidence of pH-dependent non-photochemical quenching in the red algal PSII reaction center. This type of quenching, together with high zeaxanthin content, appears to underlie photoprotection mechanisms that are efficiently employed by this robust natural water-splitting complex under excess irradiance. In order to provide structural details of this eukaryotic form of PSII, we have employed electron microscopy and single particle analyses to obtain a 17 Å map of the C. merolae PSII dimer in which we locate the position of the protein mass corresponding to the additional extrinsic protein stabilizing the oxygen-evolving complex, PsbQ'. We conclude that this lumenal subunit is present in the vicinity of the CP43 protein, close to the membrane plane.

Factors affecting light harvesting in the red alga Cyanidioschyzon merolae.

The phycobilisome antennas, which contain phycobilin pigments instead of chlorophyll, are crucial for the photosynthetic activity of Cyanidioschyzon merolae cells, which thrive in an acidic and hot water environment. The accessible light intensity and quality, temperature, acidity, and other factors in this environment are quite different from those in the air available for terrestrial plants. Under these conditions, adaptation to the intensity and quality of light, as well as temperature, which are key factors in photosynthesis of higher plants, also affects this process in Cyanidioschyzon merolae cells. Adaptation to varying light conditions requires fast remodeling and re-tuning of their light-harvesting antennas (phycobilisomes) at multiple levels, from regulation of gene expression to structural reorganization of protein-pigment complexes. This review presents selected data on the structure of phycobilisomes, the genetic engineering of the constituent proteins, and the latest results and opinions on the adaptation of phycobilisomes to light intensity and quality, and temperature to photosynthetic activities. We pay special attention to the latest results of the C. merolae research.

How Light Modulates the Growth of Cyanidioschyzon merolae Cells by Changing the Function of Phycobilisomes.

The aim of this study was to examine how light intensity and quality affect the photosynthetic apparatus of Cyanidioschyzon merolae cells by modulating the structure and function of phycobilisomes. Cells were grown in equal amounts of white, blue, red, and yellow light of low (LL) and high (HL) intensity. Biochemical characterization, fluorescence emission, and oxygen exchange were used to investigate selected cellular physiological parameters. It was found that the allophycocyanin content was sensitive only to light intensity, whereas the phycocynin content was also sensitive to light quality. Furthermore, the concentration of the PSI core protein was not affected by the intensity or quality of the growth light, but the concentration of the PSII core D1 protein was. Finally, the amount of ATP and ADP was lower in HL than LL. In our opinion, both light intensity and quality are main factors that play an important regulatory role in acclimatization/adaptation of C. merolae to environmental changes, and this is achieved by balancing the amounts of thylakoid membrane and phycobilisome proteins, the energy level, and the photosynthetic and respiratory activity. This understanding contributes to the development of a mix of cultivation techniques and genetic changes for a future large-scale synthesis of desirable biomolecules.

Cross-linking of dimeric CitS and GltS transport proteins.

CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na+-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.

Projection structure by single-particle electron microscopy of secondary transport proteins GltT, CitS, and GltS.

The structure of three secondary transporter proteins, GltT of Bacillus stearothermophilus, CitS of Klebsiella pneumoniae, and GltS of Escherichia coli, was studied. The proteins were purified to homogeneity in detergent solution by Ni(2+)-NTA affinity chromatography, and the complexes were determined by BN-PAGE to be trimeric, dimeric, and dimeric for GltT, CitS, and GltS, respectively. The subunit stoichiometry correlated with the binding affinity of the Ni(2+)-NTA resin for the protein complexes. Projection maps of negatively stained transporter particles were obtained by single-particle electron microscopy. Processing of the GltT particles revealed a projection map possessing 3-fold rotational symmetry, in good agreement with the trimer observed in the crystal structure of a homologous protein, Glt(Ph) of Pyrococcus horikoshii. The CitS protein showed up in two main views: as a kidney-shaped particle and a biscuit-shaped particle, both with a long axis of 160 A. The latter has a width of 84 A, the former of 92 A. Symmetry considerations identify the biscuit shape as a top view and the kidney shape as a side view from within the membrane. Combining the two images shows that the CitS dimer is a protein with a strong curvature at one side of the membrane and, at the opposite side, an indentation in the middle at the subunit interface. The GltS protein was shaped like CitS with dimensions of 145 A x 84 A. The shapes and dimensions of the CitS and GltS particles are consistent with a similar structure of these two unrelated proteins.

PEG-mediated, Stable, Nuclear and Chloroplast Transformation of Cyanidioschizon merolae.

The ability to achieve nuclear or chloroplast transformation in plants has been a long standing goal, especially in microalgae research. Over past years there has been only little success, but transient and stable nuclear transformation has been achieved in multiple species. Our newly developed method allows for relatively simple transformation of Cyanidioschizon merolae in both nuclear and chloroplast genome by means of homologous recombination between the genome and a transformation vector. The use of chloramphenicol resistance gene as the selectable marker allows for plate-based efficient selection of mutant colonies. Overall, the method allows the generation of mutant strains within 6 months.

Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae.

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Structure and function of photosystem I and its application in biomimetic solar-to-fuel systems.

Photosystem I (PSI) is one of the most efficient biological macromolecular complexes that converts solar energy into condensed energy of chemical bonds. Despite high structural complexity, PSI operates with a quantum yield close to 1.0 and to date, no man-made synthetic system approached this remarkable efficiency. This review highlights recent developments in dissecting molecular structure and function of the prokaryotic and eukaryotic PSI. It also overviews progress in the application of this complex as a natural photocathode for production of hydrogen within the biomimetic solar-to-fuel nanodevices.