Yasuhiro Kashino

<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.

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.

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 terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and Antarctica. N. commune has a large amount of extracellular polysaccharides (EPS) that hold moisture and protect the cells and at the same time accumulate light-blocking substances which is believed to play an important part in adaptation to a severe environment. To evaluate the photoadaptation processes in N. commune and clarify the role(s) of EPS under ambient environmental condition at Antarctica, separation of cells from EPS is necessary. High yield is a prerequisite for the use of only small amount of natural N. commune from Antarctica. For this purpose, we developed a separation method by improving the Percoll density gradient centrifugation method using an EPS-coated field-grown Nostoc population. We established the most suitable condition to separate naked cells from EPS at high yield retaining high photosynthetic activity. The method is composed of centrifugation of cell homogenated N. commune in 10% (v/v) Percoll to separate cells efficiently from EPS followed by fractionating centrifugation to remove impurities using the gradient of Percoll (80% and 50%, v/v).

In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to dehydration in a chlorolichen (Ramalina yasudae) and its photobiont (Trebouxia sp.) was studied. Responses to dehydration in the isolated Trebouxia sp. were different from R. yasudae. That is, (1) the photosystem II (PSII) reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained active in Trebouxia sp. (2) Dehydration-induced quenching of PSII fluorescence was smaller in the Trebouxia sp. compared to that in R. yasudae. (3) The isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae in the dehydrated condition. We analyzed about the water extract of R. yasudae and found out that arabitol which contained large amount in water extract accelerated quenching of PSII fluorescence of Trebouxia in the dehydrated state. The phenomenon was confirmed by the analysis of picosecond time-resolved fluorescence spectra. In other words, it indicates that accumulated arabitol in lichen body play a important role against dehydrated tolerance of Trebouxia.