Biology subjects

Nagao, R.

Publications and source records attributed to Nagao, R..

13 recordsLinked to original sources

bioRxiv2026

Lhcf9 is a novel negative regulator of non-photochemical quenching in the diatom Chaetoceros gracilis

Photosynthetic organisms in aquatic environments experience rapid fluctuations in light intensity, light quality, and carbon availability, requiring tight regulation of photosynthetic energy conversion. In marine diatoms, non-photochemical quenching (NPQ), particularly energy-dependent quenching (qE), plays a central role in dissipating excess excitation energy as heat. However, excessive NPQ can reduce photosynthetic efficiency under light-limiting or carbon-rich conditions, and how this trade-off is regulated remains poorly understood. Here, we identify CgLhcf9, a previously uncharacterized light-harvesting complex (LHC) protein, as a negative regulator of qE-type NPQ in the centric diatom Chaetoceros gracilis. Expression of CgLhcf9 is induced under low red-light and high CO2 conditions and strongly suppressed by blue light, indicating regulation by both light quality and carbon availability. Functional analyses using CgLhcf9 knockout and overexpression lines reveal that CgLhcf9 suppresses qE: NPQ induction is enhanced in the absence of CgLhcf9, whereas its accumulation downregulates NPQ without affecting other established qE effectors, including Lhcx1 or xanthophyll cycle pigments. Notably, CgLhcf9 accumulation improves cellular growth under light-limiting conditions. These results identify CgLhcf9 as a novel LHC-type regulator that fine-tunes photosynthetic energy dissipation in response to environmental signals. Our findings establish a regulatory mechanism that balances photoprotection, electron transport, and carbon fixation, advancing our understanding of how marine diatoms optimize photosynthesis under fluctuating light and CO2 conditions. Significance statementPhotosynthetic microalgae must balance light-driven electron transport with carbon fixation to maximize growth under fluctuating light and CO2 conditions. While non-photochemical quenching (NPQ) protects photosystems from excess light, excessive NPQ can limit photosynthetic efficiency when light or carbon is limiting. Here, we identify the antenna protein CgLhcf9 as a negative regulator of energy-dependent NPQ in the marine diatom Chaetoceros gracilis. CgLhcf9 integrates light-quality and CO2 signals to suppress NPQ without altering canonical quenching effectors, thereby improving growth under light-limiting conditions. This study reveals a regulatory role for a light-harvesting complex protein in tuning the balance between photoprotection and photosynthetic efficiency, providing insight into how marine diatoms coordinate electron transport and carbon fixation in dynamic environments.

plant biology↗

bioRxiv2026

Structural basis for impaired oxygen evolution in extrinsic-protein-reconstituted photosystem II

Photosystem II (PSII) is a membrane-bound pigment-protein complex in oxygenic photosynthesis that catalyzes water splitting and oxygen evolution. Here, we present the X-ray crystallographic structure of a PsbO/V/U-reconstituted PSII from Thermosynechococcus vulcanus at 2.0 [A] resolution, revealing proper rebinding of the three extrinsic subunits, PsbO, PsbV, and PsbU. The overall geometry of the Mn4CaO5 cluster is largely preserved, although a subtle shortening of the Mn2-O2 bond suggests a minor local rearrangement. Structural analysis identifies perturbations that may underlie the reduced oxygen-evolving activity, including altered bicarbonate-binding interactions on the electron acceptor side and the loss of water molecules W658 and W660 in the O1 channel, disrupting a hydrogen-bond network critical for water delivery. In contrast, the Cl-1 and O4 channels remain intact. These findings suggest that disrupted water delivery and electron transport, together with minor rearrangements within the Mn4CaO5 cluster, may contribute to the decreased activity of the reconstituted PSII.

plant biology↗

bioRxiv2025

Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes from Euglena gracilis strain Z

Euglena gracilis, a phototrophic flagellate, possesses light-harvesting complexes (LHCs) with a pigment composition distinct from that of land plants and green algae, despite notable similarities in LHC polypeptide sequences to green algae. Here, we purified photosystem I-LHCI (PSI-LHCI) supercomplexes from E. gracilis strain Z and characterized their biochemical and spectroscopic properties. The purified complex exhibited a unique pigment profile, notably including diadinoxanthin that is typically found in red-lineage organisms, setting E. gracilis apart from green-lineage organisms. The absorption spectrum displayed the Qy band of chlorophyll a at 675{square}nm, while the 77-K fluorescence-emission spectrum revealed a prominent peak at 732{square}nm, closely resembling those in land plants. These features suggest that long-wavelength chlorophylls bound to LHCI may be evolutionarily conserved. Nevertheless, the absence of neoxanthin, lutein, and violaxanthin further differentiates the Euglena LHCIs from other oxyphototrophs. Together, these results illuminate the evolutionary diversification of PSI-LHCI supercomplexes and offer insights into the unique pigment-binding features of the Euglena light-harvesting system.

plant biology↗

bioRxiv2024

Biochemical evidence for the diversity of LHCI proteins in PSI-LHCI from the red alga Galdieria sulphuraria NIES-3638

Red algae are photosynthetic eukaryotes whose light-harvesting complexes (LHCs) associate with photosystem I (PSI). In this study, we examined characteristics of PSI-LHCI, PSI, and LHCI isolated from the red alga Galdieria sulphuraria NIES-3638. The PSI-LHCI supercomplexes were purified using anion-exchange chromatography followed by hydrophobic interaction chromatography, and finally by trehalose density gradient centrifugation. PSI and LHCI were similarly prepared following the dissociation of PSI-LHCI with Anzergent 3-16. Polypeptide analysis of PSI-LHCI revealed the presence of PSI and LHC proteins, along with a red-lineage chlorophyll a/b-binding-like protein (RedCAP), which is distinct from LHC proteins within the LHC protein superfamily. RedCAP, rather than LHC proteins, exhibited tight binding to PSI. Carotenoid analysis of LHCI identified zeaxanthin, {beta}-cryptoxanthin, and {beta}-carotene, with zeaxanthin particularly enriched, which is consistent with other red algal LHCIs. A Qy peak of chlorophyll a in the LHCI absorption spectrum was blue-shifted compared with those of PSI-LHCI and PSI, and a fluorescence emission peak was similarly shifted to shorter wavelengths. Based on these results, we discuss the diversity of LHC proteins, including RedCAP, in red algal PSI-LHCI supercomplexes.

plant biology↗

bioRxiv2024

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein-protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

plant biology↗

bioRxiv2024

Contributions of PD1 and PD2 to the difference spectrum in the Soret region in Photosystem II

Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by [~] 3 nm to [~] 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]{longleftrightarrow}[PD1PD2+] equilibrium or a more complex mechanism.

biophysics↗

bioRxiv2023

Structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and their structural variety in photosystem I-LHC (PSI-LHCI) supercomplexes has been shown. However, structural and evolutionary correlations of red-lineage LHCs are unknown. Here we determined a 1.92-[A] resolution cryo-electron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137) which is an important taxon in the Cyanidiophyceae, and subsequently investigated these correlations through structural comparisons and phylogenetic analysis. The PSI-LHCI structure shows five LHCI subunits together with a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

plant biology↗

bioRxiv2022

Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits are identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 is inserted at the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide novel insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.

plant biology↗

bioRxiv2022

Structural insights into an evolutionary turning-point of photosystem I from prokaryotes to eukaryotes

Photosystem I (PSI) contributes to light-conversion reactions; however, its oligomerization state is variable among photosynthetic organisms. Herein we present a 3.8-[A] resolution cryo-electron microscopic structure of tetrameric PSI isolated from a glaucophyte alga Cyanophora paradoxa. The PSI tetramer is organized in a dimer of dimers form with a C2 symmetry. Different from cyanobacterial PSI tetramer, two of the four monomers are rotated around 90{degrees}, resulting in a totally different pattern of monomer-monomer interactions. Excitation-energy transfer among chlorophylls differs significantly between Cyanophora and cyanobacterial PSI tetramers. These structural and spectroscopic features reveal characteristic interactions and energy transfer in the Cyanophora PSI tetramer, thus offering an attractive idea for the changes of PSI from prokaryotes to eukaryotes.

plant biology↗

bioRxiv2021

Structural basis for the absence of low-energy chlorophylls responsible for photoprotection from a primitive cyanobacterial PSI

Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-[A] resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

plant biology↗

bioRxiv2021

Molecular phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros gracilis and Lhcq/Lhcf diversity

Diatoms adapt to various aquatic light environments and play major roles in the global carbon cycle using their unique light-harvesting system, i.e., fucoxanthin chlorophyll a/c binding proteins (FCPs). Structural analyses of photosystem II (PSII)-FCPII and photosystem I (PSI)-FCPI complexes from the diatom Chaetoceros gracilis have revealed the localization and interactions of many FCPs; however, the entire set of FCPs has not been characterized. Here, we identified 46 FCPs in the newly assembled genome and transcriptome of C. gracilis. Phylogenetic analyses suggested that these FCPs could be classified into five subfamilies: Lhcr, Lhcf, Lhcx, Lhcz, and novel Lhcq, in addition to a distinct type of Lhcr, CgLhcr9. The FCPs in Lhcr, including CgLhcr9 and some Lhcqs, had orthologous proteins in other diatoms, particularly those found in the PSI-FCPI structure. By contrast, the Lhcf subfamily, some of which were found in the PSII-FCPII complex, seemed to be diversified in each diatom species, and the number of Lhcqs differed among species, indicating that their diversification may contribute to species-specific adaptations to light. Further phylogenetic analyses of FCPs/light-harvesting complex (LHC) proteins using genome data and assembled transcriptomes of other diatoms and microalgae in public databases suggest that our proposed classification of FCPs was common among various red-lineage algae derived from secondary endosymbiosis of red algae, including Haptophyta. These results provided insights into the loss and gain of FCP/LHC subfamilies during the evolutionary history of the red algal lineage. One sentence summaryPhylogenetic analysis of fucoxanthin-chlorophyll a/c proteins in C. gracilis revealed five major subfamilies and one minor subfamily, providing insights into the diversification of light-harvesting systems in red algae.

plant biology↗

bioRxiv2020

Circumstances and factors of sleep-related sudden infant deaths in Japan

Background Sudden unexpected death in infancy (SUDI) comprises both natural and unnatural causes of death. However, a few epidemiological surveys have investigated SUDI in Japan.Objective This retrospective study was conducted to investigate the recent trends of circumstances and risk factors of sleep-related SUDI cases.Methods Forensic pathology sections from eight universities participated in the selection of subjects from 2013 to 2018. Data obtained from the checklist form were analyzed based on information at postmortem.Results There were 259 SUDI cases consisting of 145 male infants and 114 female infants with a mean birth weight of 2888 ± 553 and 2750 ± 370 g, respectively. Deaths most frequently occurred among infants at 1 month of age (18%). According to population-based analyses, the odds ratio (95% confidence interval) of mother’s age ≤19 years was 11.1 (6.9–17.7) compared with ages 30–39. The odds ratio for the fourth- and later born infants was 5.2 (3.4–7.9) compared with the frequency of first-born infants. The most frequent time of day for discovery was between 7 and 8 o’clock. Co-sleeping was recorded for 61%, and the prone position was found for 40% of cases at discovery. Mother’s smoking habit exhibited an adds ratio of 4.5 (2.9–5.8).Conclusion This study confirmed the trends that have been observed for sudden infant death syndrome; particularly, very high odds ratios were evident for teenage mothers and later birth order in comparison with those in other developed countries. The child of a young mother tended to die within 2 months of age. To our knowledge, this is the first report of an extensive survey of sleep-related SUDI in Japan.View Full Text

pathology↗

bioRxiv2020

Open microfluidic coculture reveals paracrine signaling from human kidney epithelial cells promotes kidney specificity of Endothelial Cells

Endothelial cells (ECs) from different human organs possess organ-specific characteristics that support specific tissue regeneration and organ development. EC specificity are identified by both intrinsic and extrinsic cues, among which, parenchyma and organ-specific microenvironment are critical contributors. These extrinsic cues are, however, largely lost during ex vivo cultures. Outstanding challenges remain to understand and re-establish EC organ-specificity for in vitro studies to recapitulate human organ-specific physiology. Here, we designed an open microfluidic platform to study the role of human kidney tubular epithelial cells in supporting EC specificity. The platform consists of two independent cell culture regions segregated with a half wall; culture media is added to connect the two culture regions at a desired timepoint, and signaling molecules can travel across the half wall (paracrine signaling). Specifically, we report that in the microscale coculture device, primary human kidney proximal tubular epithelial cells (HPTECs) rescued primary human kidney peritubular microvascular EC (HKMEC) monolayer integrity and fenestra formation, and HPTECs upregulated key HKMEC kidney-specific genes (HNF1B, AJAP1, KCNJ16) and endothelial activation genes (VCAM1, MMP7, MMP10) in coculture. Co-culturing with HPTECs also promoted kidney-specific genotype expression in human umbilical vein ECs (HUVECs), and human pluripotent stem cell-derived ECs (hPSC-ECs). In comparison to the culture in HPTEC conditioned media, co-culture of ECs with HPTECs showed increased upregulation of kidney specific genes, suggesting potential bidirectional paracrine signaling. Importantly, our device is compatible with standard pipettes, incubators, and imaging readouts, and could also be easily adapted to study cell signaling between other rare or sensitive cells.

bioengineering↗