菓子野 康浩

<jats:p><jats:italic>Prasiola crispa</jats:italic>, an aerial green alga, exhibits remarkable adaptability to the extreme conditions of Antarctica by forming layered colonies capable of utilizing far-red light for photosynthesis. Despite a recent report on the structure of <jats:italic>P. crispa</jats:italic>’s unique light-harvesting chlorophyll (Chl)-binding protein complex (Pc-frLHC), which facilitates far-red light absorption and uphill excitation energy transfer to photosystem II, the specific genes encoding the subunits of Pc-frLHC have not yet been identified. Here, we report a draft genome sequence of <jats:italic>P. crispa</jats:italic> strain 4113, originally isolated from soil samples on Ongul Island, Antarctica. We obtained a 92 Mbp sequence distributed in 1,045 scaffolds comprising 10,244 genes, reflecting 87.1% of the core eukaryotic gene set. Notably, 26 genes associated with the light-harvesting Chl <jats:italic>a</jats:italic>/<jats:italic>b</jats:italic> binding complex (LHC) were identified, including four Pc-frLHC genes, with similarity to a noncanonical Lhca gene with four transmembrane helices, such as Ot_Lhca6 in <jats:italic>Ostreococcus tauri</jats:italic> and Cr_LHCA2 in <jats:italic>Chlamydomonas reinhardtii</jats:italic>. A comparative analysis revealed that Pc-frLHC shares homology with certain Lhca genes found in <jats:italic>Coccomyxa</jats:italic> and <jats:italic>Trebouxia</jats:italic> species. This similarity indicates that Pc-frLHC has evolved from an ancestral Lhca gene with four transmembrane helices and branched out within the Trebouxiaceae family. Furthermore, RNA-seq analysis conducted during the initiation of Pc-frLHC gene induction under red light illumination indicated that Pc-frLHC genes were induced independently from other genes associated with photosystems or LHCs. Instead, the genes of transcription factors, helicases, chaperones, heat shock proteins, and components of blue light receptors were identified to coexpress with Pc-frLHC. Those kinds of information could provide insights into the expression mechanisms of Pc-frLHC and its evolutional development.</jats:p>

In the practical scale of cyanobacterial cultivation, the golden algae Poterioochromonas malhamensis is a well-known predator that causes devastating damage to the culture, referred to as pond crash. The establishment and maintenance of monoculture conditions are ideal for large-scale cultures. However, this is a difficult challenge because microbial contamination is unavoidable in practical-scale culture facilities. In the present study, we unexpectedly observed the pond crash phenomenon during the pilot-scale cultivation of Synechococcus elongatus PCC 7942 using a 100-L photobioreactor. This was due to the contamination with P. malhamensis, which probably originated from residual fouling. Interestingly, we found that S.elongatus PCC 7942 can alter its morphological structure when subjected to continuous grazing pressure from predators, resulting in cells that were more than 100 times longer than those of the wild-type strain. These hyper-elongated S.elongatus PCC 7942 cells had mutations in the genes encoding FtsZ or Ftn2 which are involved in bacterial cell division. Importantly, the elongated phenotype remained stable during cultivation, enabling S.elongatus PCC 7942 to thrive and resist grazing. The cultivation of the elongated S.elongatus PCC 7942 mutant strain in a 100-L pilot-scale photobioreactor under non-sterile conditions resulted in increased cyanobacterial biomass without encountering pond crash. This study demonstrates an efficient strategy for cyanobacterial cell culture in practical-scale bioreactors without the need for extensive decontamination or sterilization of the growth medium and culture facility, which can contribute to economically viable cultivation and bioprocessing of microalgae.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in signal transduction at the neuromuscular junction (NMJ). Movement of the nAChR extracellular domain following agonist binding induces conformational changes in the extracellular domain, which in turn affects the transmembrane domain and opens the ion channel. It is known that the surrounding environment, such as the presence of specific lipids and proteins, affects nAChR function. Diffracted X-ray tracking (DXT) facilitates measurement of the intermolecular motions of receptors on the cell membranes of living cells, including all the components involved in receptor function. In this study, the intramolecular motion of the extracellular domain of native nAChR proteins in living myotube cells was analyzed using DXT for the first time. We revealed that the motion of the extracellular domain in the presence of an agonist (e.g., carbamylcholine, CCh) was restricted by an antagonist (i.e., alpha-bungarotoxin, BGT).

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Å resolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment–protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

<title>Abstract</title><italic>Acaryochloris marina</italic> is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of <italic>A. marina</italic> photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll <italic>d</italic> and its epimer chlorophyll <italic>d</italic>′. The primary electron acceptor is pheophytin <italic>a</italic>, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.

Photosynthetic light-harvesting complexes (LHCs) play a pivotal role in collecting solar energy for photochemical reactions in photosynthesis. One of the major LHCs are fucoxanthin chlorophyll a/c-binding proteins (FCPs) present in diatoms, a group of organisms having important contribution to the global carbon cycle. Here, we report a 2.40-Å resolution structure of the diatom photosystem I (PSI)-FCPI supercomplex by cryo-electron microscopy. The supercomplex is composed of 16 different FCPI subunits surrounding a monomeric PSI core. Each FCPI subunit showed different protein structures with different pigment contents and binding sites, and they form a complicated pigment-protein network together with the PSI core to harvest and transfer the light energy efficiently. In addition, two unique, previously unidentified subunits were found in the PSI core. The structure provides numerous insights into not only the light-harvesting strategy in diatom PSI-FCPI but also evolutionary dynamics of light harvesters among oxyphototrophs.

Funding; the NIBB Collaborative Research Program for the Okazaki Large Spectrograph [no 15-604]

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of β-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via bphA4 and bphA3 gene products (BphA4 and BphA3, respectively), and converted to benzoic acid by bphB, bphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3, bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3, bphA1, bphA2, bphB, bphC and bphD. Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3, bphA1, bphA2, bphB and bphC suggested that petH, which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

© 2019 Elsevier Ltd To evaluate biodiesel production from microalgae, a flotation experiment was conducted using the living cell culture fluid of the diatom Chaetoceros gracilis using a venturi tube type microbubble generator. We compared the separation performance of three different culture periods: 1, 2, and 3 weeks from the start of cultivation. After 1 week, the cells were in the logarithmic growth phase, while after 2 and 3 weeks, cell growth had reached the stationary phase. The amounts of triacylglycerol (TAG) in the foam on the surface of the fluid tank were measured. TAG increased during the first 20 min after the start of circulation without additional coagulants and pH adjustment. The disruption of cells was achieved simultaneously. The amounts of TAG in the culture fluids at weeks 2 and 3 were higher than those at week 1. C. gracilis cells in the stationary phase accumulated large amounts of TAG and were easy to disrupt by pressure fluctuation in the venturi tube. These results provide insight into the fracture strength and buoyance of cells for efficiently separating the cells from large volumes of culture fluid.

Light-harvesting antenna systems in photosynthetic organisms harvest solar energy and transfer it to the photosynthetic reaction centres to initiate charge-separation and electron-transfer reactions. Diatoms are one of the important groups of oxyphototrophs and possess fucoxanthin chlorophyll a/c-binding proteins (FCPs) as light harvesters. The organization and association pattern of FCP with the photosystem II (PSII) core are unknown. Here we solved the structure of PSII-FCPII supercomplexes isolated from a diatom, Chaetoceros gracilis, by single-particle cryoelectron microscopy. The PSII-FCPII forms a homodimer. In each monomer, two FCP homotetramers and three FCP monomers are associated with one PSII core. The structure reveals a highly complicated protein-pigment network that is different from the green-type light-harvesting apparatus. Comparing these two systems allows the identification of energy transfer and quenching pathways. These findings provide structural insights into not only excitation-energy transfer mechanisms in the diatom PSII-FCPII, but also changes of light harvesters between the red- and green-lineage oxyphototrophs during evolution.

The biodiesel fuel ethyl oleate (fatty acid ethyl ester, FAEE) was produced using Novozym® 435 (immobilized lipase) by transesterification of a mixture of ethanol and triolein in a fixed-bed reactor operated in circulating batch mode. The miscibility of the mixtures during transesterification was predicted thermodynamically and the conversion ratio of triolein was studied as a function of reaction time. The yield of ethyl oleate increased when the feed molar composition of ethanol was increased. The glycerin by-product could be removed from the enzyme particles by the circulating feed mixture. The inactivation of Novozym® 435 by glycerin was also examined in the circulating batch mode experiments, and an effective reaction model was proposed to describe the inactivation of Novozym® 435 and ethanol effect during the transesterification reaction by using the modified Michaelis-Menten equation.

As a method for efficiently recovering marine organisms, which is expected as a resource for next generation biofuels, crystallizing concentration which can be considered as application of melt crystallization was studied. An operation of forming an ice phase from the outer wall surface to the central portion in the crystallizer was carried out by a cooling jacket with circulating solution. As the operation conditions, the stirring rotation number, the cooling temperature, and the freezing rate were varied to clarify the influence on the concentration ratio of Chaetoceros gracilis in the solution, the dilution ratio of Chaetoceros gracilis in ice, and the solid-liquid distribution coefficient of Chaetoceros gracilis measured experimentally in this study. Furthermore, it is suggested that the solid-liquid distribution coefficients are correlated by two kinds of solid-liquid distribution coefficients models, and concentration by high-speed freezing is also efficient as a result of comparison of the two models.

Background: Diatoms have attracted interest as biofuel producers. Here, the contents of lipids and photosynthetic pigments were analyzed in a marine centric diatom, Chaetoceros gracilis. This diatom can be genetically engineered using our previously reported transformation technique and has a potential to produce valuable materials photosynthetically. Sustainable culture conditions for cost-effective production of biological materials under autotrophic conditions with atmospheric carbon dioxide were investigated in the laboratory. A large-scale, open-air culture was also performed. Results: Cell population doubling time was ~10 h under continuous illumination without CO2 enrichment, and large amounts of triacylglycerols (TAG) and fucoxanthin accumulated under a wide range of salinity and nutrient conditions, reaching ~200 and 18.5 mg/L, respectively. It was also shown that C. gracilis produced high amounts of TAG without the need for nitrogen or silica deprivation, which is frequently imposed to induce lipid production in many other microalgae. Furthermore, C. gracilis was confirmed to be highly tolerant to changes in environmental conditions, such as salinity. The diatom grew well and produced abundant lipids when using sewage water or liquid fertilizer derived from cattle feces without augmented carbon dioxide. High growth rates (doubling time &lt 20 h) were obtained in a large-scale, open-air culture, in which light irradiance and temperature fluctuated and were largely different from laboratory conditions. Conclusions: The ability of this microalga to accumulate TAG without nutrient deprivation, which incurs added labor, high costs, and complicates scalability, is important for low-cost industrial applications. Furthermore, its high tolerance to changes in environmental conditions and high growth rates observed in large-scale, open-air culture implied scalability of this diatom for industrial applications. Therefore, C. gracilis would have great potential as a biofactory.

The postsynaptic density (PSD) is a protein complex that is critical for synaptic transmission. Ultrastructural changes in the PSD are therefore likely to modify synaptic functions. In this study, we investigated the ultrastructural changes in the PSD in the hippocampal CA1 stratum radiatum following neuronal excitation. Oxygen-glucose deprivation-induced PSD thickening in hippocampal slice cultures was blocked by the N-methyl- d-aspartate (NMDA) receptor antagonist MK801. To gain more insight into the mechanisms underlying NMDA receptor-mediated PSD thickening, we assessed the area, length, and thickness of the PSD after NMDA treatment. The PSDs thickened with just 2. min of NMDA receptor stimulation, and this treatment was considered sublethal. When N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, cathepsins, and the proteasome, was applied, NMDA-induced PSD thickening was abolished. Furthermore, the calcium-induced calcium release inhibitor, ryanodine, reduced NMDA receptor-mediated PSD thickening. These results suggest that NMDA receptor activation induces PSD thickening by proteolysis through intracellular calcium increase, including that induced by calcium.

The major light-harvesting pigment protein complex (fucoxanthin-chlorophyll-binding protein complex FCP) was purified from a marine centric diatom, Chaetoceros gracilis, by mild solubilization followed by sucrose density gradient centrifugation, and then characterized. The dynamic light scattering measurement showed unimodality, indicating that the complex was highly purified. The amount of chlorophyll a (Chl a) bound to the purified FCP accounted for more than 60 % of total cellular Chl a. The complex was composed of three abundant polypeptides, although there are nearly 30 FCP-related genes. The two major components were identified as Fcp3 (Lhcf3)- and Fcp4 (Lhcf4)-equivalent proteins based on their internal amino acid sequences and a two-dimensional isoelectric focusing electrophoresis analysis developed in this work. Compared with the thylakoids, the FCP complex showed higher contents of fucoxanthin and chlorophyll c but lower contents of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Fluorescence excitation spectra analyses indicated that light harvesting, rather than photosystem protection, is the major function of the purified FCP complex, which is associated with more than 60 % of total cellular Chl a. These findings suggest that the huge amount of Chl bound to the FCP complex composed of Lhcf3, Lhcf4, and an unidentified minor protein has a light-harvesting function to allow efficient photosynthesis under the dim-light conditions in the ocean.

Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ∼400 positive transgenic clones per 108 recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami-Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl2-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (δP). The PSII-mediated oxygen-evolving activity of the δP cells was more susceptible to depletion of CaCl2 than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and δP cells under CaCl2-depleted conditions, and was more evident for the δP mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.

A divinyl chlorophyll (DV-Chl) a harboring mutant of Synechocystis sp. PCC 6803, in which chlorophyll species is replaced from monovinyl(normal)-Chl a to DV-Chl a, was characterized. The efficiency of light utilization for photosynthesis was decreased in the mutant. Absorption spectra at 77 K and their fourth derivative analyses revealed that peaks of each chlorophyll forms were blue-shifted by 1-2 nm, suggesting lowered stability of chlorophylls at their binding sites. This was also true both in PSI and PSII complexes. On the other hand, fluorescence emission spectra measured at 77 K were not different between wild type and the mutant. This indicates that the mode of interaction between chlorophyll and its binding pockets responsible for emitting fluorescence at 77 K is not altered in the mutant. P700 difference spectra of thylakoid membranes and PSI complexes showed that the spectrum in Soret region was red-shifted by 7 nm in the mutant. This is a characteristic feature of DV-Chl a. Microenvironments of iron-sulfur center of a terminal electron acceptor of PSI complex, P430, were practically the same as that of wild type.

A highly efficient nuclear transformation method was established for the pennate diatom Phaeodactylum tricornutum using an electroporation system that drives multi-sequence pulses to introduce foreign DNAs into the cells. By optimizing pulse conditions, the diatom cells can be transformed without removing rigid silicabased cell walls, and high transformation efficiency (about 4,500 per 108 cells) is achieved.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression. © 2013 The Author.

In this study, gene sequences coding for the light-harvesting (LH) 2 polypeptides from a thermophilic purple sulfur bacterium Thermochromatium tepidum are reported and characterization of the LH2 complex is described. Three sets of pucBA genes have been identified, and the gene products have been analyzed by electrophoresis and reversed-phase chromatography. The result shows that all of the genes are expressed but the distribution of the expression is not uniform. The gene products undergo post-translational modification, where two of the β-polypeptides appear to be N-terminally methylated. Absorption spectrum of the purified LH2 complex exhibits Q y transitions at 800 and 854 nm in dodecyl β-maltopyranoside solution, and the circular dichroism spectrum shows a "molischianum"-like characteristic. No spectral change was observed for the LH2 when the bacterium was cultured under different conditions of light intensity. In lauryl dimethylamine N-oxide (LDAO) solution, significant changes in the absorption spectrum were observed. The B850 peak decreased and blue-shifted with increasing the LDAO concentration, whereas the B800 intensity increased without change in the peak position. The spectral changes can be partially or almost completely reversed by addition of metal ions, and the divalent cations seem to be more effective. The results indicate that ionic interactions may exist between LH2, detergent molecules and metal ions. Possible mechanisms involved in the detergent- and cation-induced spectral changes are discussed. © 2011 Springer Science+Business Media B.V.

陸生ラン色細菌のイシクラゲ(Nostoc commune)は南極や日本にも生育する汎存種である.イシクラゲの細胞の周りに存在する細胞外多糖(EPS)は,水分保持や細胞の保護に役立つと共に遮光性物質を蓄積しており,これが苛酷な環境への適応に重要な役割を果たしていると考えられている.南極産の貴重なイシクラゲを材料にしてこの仮説を検証するためには,EPSを除去した細胞を高収率で回収する必要がある.そこで,これまでに報告のあるEPS除去方法を改良し,Percollを用いた遠心分離で細胞の回収率向上を図った. その結果, イシクラゲ破砕懸濁液に10%濃度のPercollを混合して遠心することでEPSを効率良く除去できることが分かった.更に細胞画分を80% と50% のPercoll に上層して遠心することで他の不純物の除去が達成され,少量のイシクラゲを生理学実験に用いるのに適した細胞単離法が確立された.

Thylakoid membranes contain two types of quinones, benzoquinone (plastoquinone) and naphthoquinone, which are involved in photosynthetic electron transfer. Unlike the benzoquinone, the chemical species of naphthoquinone present (phylloquinone, menaquinone-4 and 5′- monohydroxyphylloquinone) varies depending on the oxygenic photosynthetic organisms. The green alga Chlamydomonas reinhardtii has been used as a model organism to study the function of the naphthoquinone bound to PSI. However, the level of phylloquinone and the presence of other naphthoquinones in this organism remain unknown. In the present study, we found that 5′-monohydroxyphylloquinone is the predominant naphthoquinone in cell and thylakoid extracts based on the retention time during reverse phase HPLC, absorption and mass spectrometry measurements. It was shown that 5′-monohydroxyphylloquinone is enriched 2.5-fold in the PSI complex as compared with thylakoid membranes but that it is absent from PSI-deficient mutant cells. We also found a small amount of phylloquinone in the cells and in the PSI complex and estimated that accumulated 5′-monohydroxyphylloquinone and phylloquinone account for approximately 90 and 10, respectively, of the total naphthoquinone content. The ratio of these two naphthoquinones remained nearly constant in the cells and in the PSI complexes from logarithmic and stationary cell growth stages. We conclude that both 5′- monohydroxyphylloquinone and phylloquinone stably co-exist as major and minor naphthoquinones in Chlamydomonas PSI. © 2011 The Author.

The psb30 (ycf12) gene is conserved in a wide variety of oxygenic-photosynthetic organisms except angiosperms and some marine cyanobacteria. Psb30 protein is found in cyanobacterial photosystem II (PSII) core complexes and is dispensable for PSII structure and function. The most recent three-dimensional structure of cyanobacterial PSII core complex has revealed that Psb30 is located in proximity of PsbJ, PsbK, and PsbZ. However Psb30 has not yet been detected in PSII complexes from eukaryotic photosynthetic organisms. Here we found the expression of the chloroplast psb30 gene in the green alga Chlamydomonas reinhardtii by immunoblotting and Psb30 is exclusively co-purifies with PSII core complex and is significantly reduced in PSII-deficient mutants. Partial disintegration of PSII core complex and subsequent fractionation of the resulting subcomplexes revealed that Psb30 is exclusively associated with PSII reaction center. We have generated chloroplast transformants in which the psb30 gene is disrupted and the resulting Delta Psb30 cells showed decreased oxygen evolution activity by 15%, grew photosynthetically under moderate light, and displayed increased sensitivity to high light relative to wild type. We conclude that Psb30 is a PSII reaction center subunit and is required for optimal PSII function under high light environments. (C) 2011 Elsevier B.V. All rights reserved.

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3'-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation. © 2010 Elsevier Inc.

Sll1252 was identified as a novel protein in photosystem II complexes from Synechocystis sp. PCC 6803. To investigate the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. Despite the homology of Sll1252 to YlmH, which functions in the cell division machinery in Streptococcus, the growth rate and cell morphology of the mutant were not affected in normal growth medium. Instead, it seems that cells lacking this polypeptide have increased sensitivity to Cl(-) depletion. The growth and oxygen evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells when Cl(-) and/or Ca(2+) was depleted from the medium. Recovery of photosystem II from photoinhibition was suppressed in the mutant. Despite the defects in photosystem II, in the light, the acceptor side of photosystem II was more reduced and the donor side of photosystem I was more oxidized compared with wild-type cells, suggesting that functional impairments were also present in cytochrome b(6)/fcomplexes. The amounts of cytochrome c(550) and cytochromef were smaller in the mutant in the Ca(2+)- and Cl(-)-depleted medium. Furthermore, the amount of IsiA protein was increased in the mutant, especially in the Cl(-)-depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. The amount of accompanying cytochrome c(550) in purified photosystem II complexes was also smaller in the mutant. Overall, the Sll1252 protein appears to be closely related to redox sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

A time-resolved fluorescence study of living lichen thalli at 5 K was conducted to clarify the dynamics and mechanism of the effective dissipation of excess light energy taking place in lichen under extreme drought conditions. The decay-associated spectra obtained from the experiment at 5 K were characterized by a drastically sharpened spectral band which could not be resolved by experiments at higher temperatures. The present results indicated the existence of two distinct dissipation components of excess light energy in desiccated lichen one is characterized as rapid fluorescence decay with a time constant of 27 ps in the far-red region that was absent in wet lichen thalli, and the other is recognized as accelerated fluorescence decay in the 685-700 nm spectral region. The former energy-dissipation component with extremely high quenching efficiency is most probably ascribed to the emergence of a rapid quenching state in the peripheral-antenna system of photosystem II (PS II) on desiccation. This is an extremely effective protection mechanism of PS II under desiccation, which lichens have developed to survive in the severely desiccated environments. The latter, which is less efficient at 5 K, might have a supplementary role and take place either in the core antenna of PS II or aggregated peripheral antenna of PS II. © Springer Science+Business Media B.V. 2011.

Snow algae inhabit unique environments such as alpine and high latitudes, and can grow and bloom with visualizing on snow or glacier during spring-summer. The chrysophytes Ochromonas smithii and Ochromonas itoi are dominant in yellow-colored snow patches in mountainous heavy snow areas from late May to early June. It is considered to be effective utilizing the xanthophyll cycle and holding sunscreen pigments as protective system for snow algae blooming in the vulnerable environment such as low temperature and nutrients, and strong light, however the study on the photoprotection of chrysophytes snow algae has not been shown. To dissolve how the chrysophytes snow algae can grow and bloom under such an extreme environment, we studied with the object of light which is one point of significance to this problem. We collected the yellow snows and measured photosynthetically active radiation at Mt. Gassan in May 2008 when the bloom occurred, then tried to establish unialgal cultures of O. smithii and O. itoi, and examined their photosynthetic properties by a PAM chlorophyll fluorometer and analyzed the pigment compositions before and after illumination with high-light intensities to investigate the working xanthophyll cycle. This experimental study using unialgal cultures revealed that both O. smithii and O. itoi utilize only the efficient violaxanthin cycle for photoprotection as a dissipation system of surplus energy under prolonged high-light stress, although they possess chlorophyll c with diadinoxanthin. © 2011 Tanabe et al.

In high-latitude waters such as the Southern Ocean, the primary production of phytoplankton supports the ecosystem. To understand the photo-acclimation strategy of such phytoplankton within cold environments, the vertical distribution profile of photosynthetic pigments was analyzed in the Southern Ocean. Samples were taken along 110A degrees E during the austral summer, and along 150A degrees E and around the edge of the seasonal sea ice of the Antarctic Continent during the austral autumn. Pigment extraction methods were optimized for these samples. The standing crop of chlorophyll a was larger in the region along the edge of the seasonal sea ice than at sampling stations in open ocean areas. Chlorophyll concentration seemed to be dependent on the formation of thermo- and haloclines along the edge of the seasonal sea ice, but not in the open ocean where such clines are less pronounced. The marker pigments fucoxanthin and/or 19&apos;-hexanoyloxyfucoxanthin were dominant at most sampling stations throughout the water column, while other marker pigments such as alloxanthin were quite low. This indicated that diatoms and/or haptophytes were the major phytoplankton in this area. Comparison of the relative ratio of fucoxanthin with that of 19&apos;-hexanoyloxyfucoxanthin allowed some stations to be characterized as either diatom-dominant or haptophyte-dominant. The relative ratio of xanthophyll-cycle pigments (diadinoxanthin plus diatoxanthin) to chlorophyll a was high in surface waters and decreased gradually with depth. This suggests that near the ice edge during summer in the Southern Ocean, both diatoms and haptophytes acclimate to their light environments to protect their photosystems under high-light conditions.

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA(3) gene (WT*). The Delta Psb30 mutant appears more susceptible to photodamage, has a cytochrome b(559) that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the Delta Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b(559) into the low potential form. Structural reasons for such effects are discussed. (C) 2010 Elsevier B.V. All rights reserved.

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kutz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 mu mol photons . m(-2) . s(-1), respectively. The difference seemed to reflect the presence of high amounts of UV-absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30 degrees C-35 degrees C and 20 degrees C-25 degrees C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration-induced quenching of PSII fluorescence. Re-reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20 degrees C-25 degrees C and at a light intensity of 700 mu mol photons . m(-2) s(-1). Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ&apos;, PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 mu mol O(2) (mg Chl a)(-1) h(-1) in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl(2). This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 degrees C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells. (C) 2009 Elsevier B.V. All rights reserved.

地衣の共生光合成生物が真菌との共生によって得ている利益を乾燥耐性の面から明らかにし、地衣の優れた乾燥耐性機構について解明するため緑藻共生地衣、Ramalina yasudaeとその共生藻であるTrebouxiaの乾燥応答について比較したところ、以下のような違いが見られた。(1)R. yasudaeでは乾燥時に光化学系II(PSII)反応中心が完全に停止しているのに対し、Trebouxiaでは活性が一部残存する。(2)乾燥によって誘導されるPSIIにおける蛍光消光が、TrebouxiaではR. yasudaeに比べ小さい。(3)Trebouxiaは地衣に比べ乾燥時の光感受性が高く、光阻害を受けやすい。これらの違いはTrebouxiaの乾燥時における過剰光エネルギーの熱散逸機構が地衣体内で強化されていることを示している。そこで、地衣成分中に乾燥時の蛍光消光を促進する物質があるかを調べたところ、地衣の水溶性成分中に多量に含まれるアラビトールにその効果があることが分かった。アラビトール添加による乾燥時の蛍光減衰速度の増加は、ストリークカメラを用いたピコ秒時間分解蛍光スペクトル解析によって確かめられた。これらのことから、地衣体内に蓄積されたアラビトールがTrebouxiaの乾燥耐性に大きな役割を果たしていることが示唆された。

A stable and highly sensitive HPLC method for uranine has been developed. Because of unstableness of silica-based octadecyl-C18 columns at high pH condition. a reversed phase HPLC analysis under alkaline conditions has not necessarily taken as a usual method. However, the application for uranine seems to be advantageous, since the fluorescence yield of uranine is markedly enhanced at high pH condition. The detection limit of the HPLC system was 0.9 pg. The analytical consideration was also paid for the solid phase extraction (SPE) prior to the HPLC analysis with careful consideration of the recently revised pK(a) values of uranine. The recovery rate of uranine by SPE was found to depend on the sample volume and a few ml of seawater was applied to SPE in order to maintain the recovery rate during SPE. A combination of HPLC and SPE methods achieved detection of uranine at concentrations as low as 0.2 ng l(-1) (0.5 pM), which was comparable to the background concentration of uranine in coastal water off Japan. For the practical use of the detected tracer-uranine concentration values after substantial duration after release, the photodegradation of uranine in surface water was also evaluated in terms of incident solar radiation dose as an exponential rate constant of -0.135 mol photon(-1) m(2). (C) 2009 Elsevier B.V. All rights reserved.

Photosystem II (PS II) complexes are membrane protein complexes that are composed of &gt;20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is similar to 650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.

Ice algae are the major primary producers in seasonally ice-covered oceans during the cold season. Diurnal change in solar radiation is inevitable for ice algae, even beneath seasonal sea ice in lower-latitude regions. In this work, we focused on the photosynthetic response of ice algae under diurnally changing irradiance in Saroma-ko Lagoon, Japan. Photosynthetic properties were assessed by pulse-amplitude modulation (PAM) fluorometry. The species composition remained almost the same throughout the investigation. The maximum electron transport rate (rETR(max)), which indicates the capacity of photosynthetic electron transport, increased from sunrise until around noon and decreased toward sunset, with no sign of the afternoon depression commonly observed in other photosynthetic organisms. The level of non-photochemical quenching, which indicates photo-protection activity by dissipating excess light energy via thermal processes, changed with diurnal variations in irradiance. The pigment composition appeared constant, except for xanthophyll cycle pigments, which changed irrespective of irradiance. These results indicate that ice algae tune their photosynthetic system harmonically to achieve efficient photosynthesis under diurnally changing irradiance, while avoiding damage to photosystems. This regulation system may be essential for productive photosynthesis in ice algae. (C) 2009 Elsevier B.V. and NIPR. All rights reserved.

In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to air-drying and hypertonic treatments in a green-algal lichen (a chlorolichen, Ramalina yasudae Rsnen) and its green algal photobiont (freshly released and cultured Trebouxia sp.) were studied. Responses to dehydration in the isolated Trebouxia sp. were different from those in the lichen, R. yasudae, i.e. (i) the PSII reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained partially active in isolated Trebouxia sp (ii) dehydration-induced quenching of PSII fluorescence was less in the isolated Trebouxia sp. compared with that in R. yasudae, suggesting that a substance(s) or a mechanism(s) to dissipate absorbed light energy to heat was lost by the isolation of the photobiont and (iii) the air-dried isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae. These results support the idea that association of the photobionts with the mycobionts increases tolerance to photoinhibition under drying conditions.

Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and highlight (300 mu E m(-2) s(-1))-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl2, but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO2 (0.03%) or normal (100 mu E m(-2) s(-1), 3% CO2) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl2. In high-salt (0.55 m NaCl)-grown cells, the amount of ferredoxin-NADP(+) oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl2 were strong inhibitors of the FNR route.