Biology subjects

Shimakawa, G.

Publications and source records attributed to Shimakawa, G..

6 recordsLinked to original sources

bioRxiv2024

Different functions of Lhcx isoforms in photoprotective mechanism in the marine diatom Thalassiosira pseudonana

Photosynthesis needs light energy, but that exceeding the maximal capacity of photosynthesis enhances formation of reactive oxygen species, which potentially causes photodamages. Therefore, light-harvesting complexes (Lhc) in phototrophs harbor various proteins and pigments to function in both light capture and energy dissipation. Diatom Lhcx proteins are reported to be a critical component for thermal dissipation of excess light energy, but the molecular mechanism of photoprotection is still not fully understood and the functions of each Lhcx isoform are not yet differentiated. Here, we focused on two types of Lhcx isoforms in Thalassiosira pseudonana: TpLhcx1/2, putative major components for energy-dependent fluorescence quenching (qE); and TpLhcx6_1, functionally unknown isoform uniquely conserved in Thalassiosirales. TpLhcx1/2 proteins accumulated more under high light than under low light, while the TpLhcx6_1 protein level was constitutive irrespective of light intensities and CO2 concentrations. High-light induced photodamage of photosystem II was increased in the genome-editing transformants of these Lhcx isoforms relative to the wild-type. Transformants lacking TpLhcx1/2 showed significantly lowered qE capacities, strongly suggesting that these proteins are important for the fast thermal energy dissipation. While in contrast, genome-editing transformants lacking the TpLhcx6_1 protein rather increased the qE capacity. TpLhcx6_1 transformants were further evaluated by the low-temperature time-resolved chlorophyll fluorescence measurement, showing the longer fluorescence lifetime in transformants than that in the wild type cells even at the dark-acclimated state of these cells. These results suggest that TpLhcx6_1 functions in photoprotection through non-photochemical energy dissipation in the different way from qE. One sentence summaryThe marine diatom Thalassiosira pseudonana dissipates excess light energy for photoprotection via two types of mechanisms supported by different Lhc isofoms.

plant biology↗

bioRxiv2023

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation

Pyrenoids are subcompartments of algal chloroplasts that concentrate Rubisco enzymes and their CO2 substrate, thereby increasing the efficiency of carbon fixation. Diatoms perform up to 20% of global CO2 fixation, but their pyrenoids remain poorly characterized at a molecular level. Here, we used in vivo photo-crosslinking to catalogue components of diatom pyrenoids and identified a pyrenoid shell (PyShell) protein, which we localized to the pyrenoid periphery of both the pennate diatom, Pheaodactylum tricornutum, and the centric diatom, Thalassiosira pseudonana. In situ cryo-electron tomography (cryo-ET) revealed that the pyrenoids of both diatom species are encased in a lattice-like protein sheath. Disruption of PyShell expression in T. pseudonana resulted in the absence of this protein sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with impaired growth and reduced carbon fixation efficiency under standard atmospheric conditions. Pyrenoids in mutant cells were fragmented and lacked the thylakoid membranes that normally traverse the Rubisco matrix, demonstrating how the PyShell plays a guiding role in establishing pyrenoid architecture. Recombinant PyShell proteins self-assembled into helical tubes, enabling us to determine a 3.0 [A]-resolution PyShell structure. We then fit this in vitro structure into an in situ subtomogram average of the pyrenoids protein sheath, yielding a putative atomic model of the PyShell within diatom cells. The structure and function of the diatom PyShell provides a new molecular view of how CO2 is assimilated in the ocean, a crucial biome that is on the front lines of climate change.

cell biology↗

bioRxiv2023

A complex and dynamic redox network regulating oxygen reduction at photosystem I

Thiol-dependent redox regulations of enzyme activities play a central role in regulating photosynthesis. Beside the regulation of metabolic pathways, alternative electron transport has been shown to be subjected to thiol-dependent regulation. We investigated the regulation of O2 reduction at photosystem I. The level of O2 reduction in leaves and isolated thylakoid membranes depends on the photoperiod in which plants are grown. We used a set of Arabidopsis mutant plants affected in the stromal, membrane and lumenal thiol network to study the redox protein partners involved in regulating O2 reduction. Light-dependent O2 reduction was determined in leaves and in thylakoids of plants grown in short day and long day conditions using a spin-trapping EPR assay. In wild type samples from short day, ROS generation was twice the amount of that in samples from long day, while this difference was abolished in several redoxin mutants. An in vitro reconstitution assays showed that thioredoxin m, NADPH-dependent reductase C (NTRC) and NADPH are required for high O2 reduction levels in long day thylakoids. Using isolated photosystem I, we also show that reduction of a PSI protein is responsible for the increase in O2 reduction. Furthermore, differences in the membrane localization of thioredoxins m and 2-Cys peroxiredoxin were demonstrated between thylakoids of short day and long day plants. Finally, we propose a model of redox regulation of O2 reduction according to the reduction power of the stroma and the capabilities of the different thiol-containing proteins to form a network of redox interactions.

biochemistry↗

bioRxiv2023

Low-CO2 inducible bestrophins in diatom thylakoid membranes sustain high photosynthetic efficacy at distant locations from the pyrenoid

Anion transporters are important to sustain a variety of physiological states in cells. Bestrophins are a family of Cl- and/or HCO3- transporters conserved in bacteria, animals, algae, and plants. Recently, bestrophin paralogs were found in the green alga Chlamydomonas reinhardtii as up- regulated components in low CO2 conditions that play an essential role in the CO2- concentrating mechanism (CCM). Bestrophin orthologs are also conserved in diatoms, a group of secondary endosymbiotic algae harboring red-type plastids, but their physiological functions are not known yet. Here, we characterized the subcellular localization and expression profile of bestrophins in the marine diatoms Phaeodactylum tricornutum (PtBST1-4) and Thalassiosira pseudonana (TpBST1 and 2). PtBST1 and PtBST2 were localized at the stromal thylakoid membrane outside of the pyrenoid, and PtBST3 was localized in the pyrenoid. Contrarily, TpBST1 and TpBST2 were both localized in the pyrenoid. These bestrophin proteins were accumulated in cells grown in atmospheric CO2 but not in 1% CO2-grown cells. To assess the physiological functions, we generated knock-out mutants for PtBST1 by genome editing. The lack of PtBST1 decreased affinity of photosynthesis for dissolved inorganic carbon closer to that of the cells grown in 1% CO2. Additionally, non-photochemical quenching was 1.5-2.0 times higher in the mutants than that of the wild type cells. These data suggests that HCO3- transport at the stroma thylakoid membranes by PtBST1 is a critical part of the CO2 evolving system of the pyrenoid in the fully induced CCM, and simultaneously that PtBST1 modulates photoprotection in response to CO2 availability in P. tricornutum. Significant statementMarine diatoms are responsible for nearly half of oceanic primary production, owing to the high-affinity photosynthesis for dissolved inorganic carbon which is supported by CO2- concentrating mechanism (CCM). This study uncovered that a bestrophin family protein at the stoma thylakoid membrane operates to import HCO3- to the thylakoid lumen and mobilizes it towards the CO2 evolving system at the pyrenoid-penetrating thylakoid in the diatom Phaeodactylum tricornutum. This HCO3- collecting system not only enhances the CCM but also down regulates the photoprotection capacity of photosystem II, presumably by affecting the thylakoid lumen acidification. This study experimentally demonstrates the molecular mechanism how diatoms optimize the use of CO2 and light energy, giving an insight into the reason of ecological successfulness of marine diatoms.

plant biology↗

bioRxiv2021

Coral symbionts exhibit a polycistronic flavodiiron gene leading to functional proteins in photosynthesis

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

plant biology↗

bioRxiv2020

A quantitative demonstration of NADP+/NADPH redox homeostasis in cyanobacterial cells

In photosynthetic organisms, it is recognized that the intracellular NADP+/NADPH ratio is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADP+/NADPH ratio quantitatively estimated to date. In the present study, the light-response of the NADP+/NADPH ratio was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light-response of NADP(H) observed using PCI extraction was qualitatively consistent with the NADPH fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light-response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition, indicating that some NADP(H) is not light-responsive. Specifically, 64% of total NADP(H) was observed as non-light-responsive in the wild-type cells. The variation of the intracellular NADP+/NADPH ratio is limited to a narrow range due to the presence of non-light-responsive NADP(H).

plant biology↗