Biology subjects

Wakao, S.

Publications and source records attributed to Wakao, S..

7 recordsLinked to original sources

bioRxiv2026

A Natural qE- and qZ-Deficient Alga Retains Functional Plant-Like and Chlorophycean Violaxanthin De-Epoxidases

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

plant biology↗

bioRxiv2026

Simple Electroporation of Chlamydomonas reinhardtii Strains with an Intact Cell Wall

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI

molecular biology↗

bioRxiv2026

Ovothiol A mediates singlet oxygen resistance and acclimation in Chlamydomonas

Light is essential for photosynthetic organisms, but excess light can generate toxic levels of reactive oxygen species (ROS). To neutralize these ROS, plants and algae produce a variety of antioxidants like carotenoids, tocopherols, and glutathione. However, the role of alternative ROS scavengers, such as ovothiols, has not been studied in the context of oxidative stress in photosynthetic organisms. Here, we report that many algal groups have the potential for the biosynthesis of ovothiols, a group of thiohistidines. We discovered that the model green microalga Chlamydomonas reinhardtii produces millimolar concentrations of ovothiol A, whose biosynthesis is mediated by the ovothiol synthase OVOA1. Using CRISPR-generated ovoa1 knockout mutants, we found that ovothiol production is essential for resistance and acclimation to singlet oxygen, a prominent ROS in photosynthetic organisms. Finally, we demonstrated that OVOA1 expression is activated by singlet oxygen and light signaling pathways in which we identified the major regulatory factors. Overall, our results show that ovothiol A is a major, previously overlooked antioxidant in Chlamydomonas. This work broadens our understanding of cellular mechanisms that combat the damaging effects of oxidative stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/702910v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cddb9corg.highwire.dtl.DTLVardef@10d0a43org.highwire.dtl.DTLVardef@11cc087org.highwire.dtl.DTLVardef@a40cc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2023

Tumor suppressor let-7 acts as a key regulator for pluripotency gene expression in Muse cells

In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the expression of an RNA-binding pluripotency-relevant protein, LIN28, and the absence of its antagonist, the tumor-suppressor microRNA (miRNA) let-7, play a key role in maintaining pluripotency. Muse cells are non-tumorigenic pluripotent-like stem cells residing in the bone marrow, peripheral blood, and organ connective tissues as pluripotent surface marker SSEA-3(+). They express pluripotency genes, differentiate into triploblastic-lineage cells, and self-renew at the single cell level. Muse cells do not express LIN28 but do express let-7 at higher levels than in iPSCs. In Muse cells, we demonstrated that let-7 inhibited the PI3K-AKT pathway, leading to sustainable expression of the key pluripotency regulator KLF4 as well as its downstream genes, POU5F1, SOX2, and NANOG. Let-7 also suppressed proliferation and glycolysis by inhibiting the PI3K-AKT pathway, suggesting its involvement in non-tumorigenicity. Furthermore, the MEK/ERK pathway is not controlled by let-7 and may have a pivotal role in maintaining self-renewal and suppression of senescence. The system found in Muse cells, in which the tumor suppressor let-7, but not LIN28, tunes the expression of pluripotency genes, might be a rational cell system conferring both pluripotency-like properties and a low risk for tumorigenicity.

cell biology↗

bioRxiv2023

Mathematical model of nuclear speckle morphology

Nuclear speckles are nuclear bodies consisting of populations of small and irregularly shaped droplet-like molecular condensates that contain various splicing factors. Recent experiments have shown the following morphological features of nuclear speckles: (I) Each molecular condensate contains SON and SRRM2 proteins, and MALAT1 non-coding RNA surrounds these condensates; (II) In the normal interphase of the cell cycle, these condensates are broadly distributed throughout the nucleus in multicellular organisms. In contrast, the fusion of condensates leads to the formation of strongly condensed spherical droplets when cell transcription is suppressed; (III) SON is dispersed spatially in MALAT1 knocked-down cells, whereas MALAT1 is dispersed in SON knocked-down cells by the collapse of nuclear speckles. However, the detailed interactions among molecules that reveal the mechanisms of this rich variety of morphologies remain unknown. In this study, a coarse-grained molecular dynamics model of the nuclear speckle was developed considering the dynamics of SON, SRRM2 or SRSF2, MALAT1, and pre-mRNA as representative components of condensates. The simulations reproduced the abovementioned morphological changes, by which the interaction strength among the representative components of the condensates was predicted.

cell biology↗

bioRxiv2021

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and 1405 genes in total were affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 273 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 55 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 68 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria, 15 genes encode proteins previously shown to have functions unrelated to photosynthesis, and 190 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, represents a rich resource for gene discovery and protein functional analysis in photosynthesis.

plant biology↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗