Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Taylor and Francis Inc.  

Chloroplasts have evolved from a cyanobacterial endosymbiont and multiply by dividing. Chloroplast division is performed by constriction of the ring-like protein complex (the PD machinery), which forms at the division site. The PD machinery is composed of cyanobacteriadescended components such as FtsZ and eukaryote-derived proteins such as the dynamin-related protein, DRP5B. In the red alga Cyanidioschyzon merolae, FtsZ ring formation on the stromal side precedes PDR1 and DRP5B ring formation on the cytosolic side. In this study, we impaired FtsZ ring formation in C. merolae by overexpressing FtsZ just before FtsZ ring formation. As a result, PDR1 and DRP5B failed to localize at the chloroplast division site, suggesting that FtsZ ring formation is required for the PDR1 and DRP5B rings. We further found, by expressing a dominant negative form of DRP5B, that DRP5B ring formation begins on the nuclear side of the chloroplast division site. These findings provide insight into how the PD machinery forms in red algae.

DOI： 10.1080/19420889.2017.1294298

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase-specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplastdivision machinery by the overexpression of Filamenting temperaturesensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

DOI： 10.1073/pnas.1612872113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation.

DOI： 10.1093/pcp/pcv120

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.

DOI： 10.3389/fpls.2015.00657

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50 degrees C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

DOI： 10.1371/journal.pone.0111261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain unknown. Here, using the unicellular red alga Cyanidioschyzon merolae, we show that the G1/S regulator RBR-E2F-DP complex links the G1/S transition to circadian rhythms. Time-dependent E2F phosphorylation promotes the G1/S transition during subjective night and this phosphorylation event occurs independently of cell cycle progression, even under continuous dark or when cytosolic translation is inhibited. Constitutive expression of a phospho-mimic of E2F or depletion of RBR unlinks cell cycle progression from circadian rhythms. These transgenic lines are exposed to higher oxidative stress than the wild type. Circadian inhibition of cell cycle progression during the daytime by RBR-E2F-DP pathway likely protects cells from photosynthetic oxidative stress by temporally compartmentalizing photosynthesis and cell cycle progression.

DOI： 10.1038/ncomms4807

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Language：English   Publishing type：Research paper (scientific journal)  

Septins are a group of GTP-binding proteins that are multi-functional, with a well-known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog-possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae. © 2013 John Wiley &amp
Sons Ltd.

DOI： 10.1111/tpj.12147

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient-enriched conditions have multiple rings of FtsZ, a prokaryote-derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient-enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti-BrdU antibody. BrdU-labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient-enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod-like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient-rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.

DOI： 10.1111/j.1529-8817.2012.01204.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV TOKYO CYTOLOGIA  

We have developed a convenient method to visualize triacylglycerol-filled lipid droplets (LDs) in some species of bacteria, algae and fungi by staining with borondipyrromethene difluoride (BODIPY). When BODIPY was excited by blue light, LDs emitted green fluorescence, which was distinguished easily from the red autofluorescence of chloroplasts. This makes BODIPY staining suitable for the identification of small amounts of LDs, especially in plants. We first ensured that in Chlamydomonas reinhardtii cells growing in nitrogen-replete (+N) and -deficient (-N) media, the spots of BODIPY-stained LDs coincided with those of Nile Red-stained LDs. In addition, it was shown that the LD content per cell in N-starved cells was 200-fold higher than those of the control (+N) using a video-intensified microscope photoncounting system (VIMPCS). BODIPY staining was applied to visualize LD in bacteria, algae and fungi, and included those algae regarded as non-oleaginous. We identified LD spots in unicellular and multicellular bacteria and eukaryotes, namely Cyanidioschyzon merolae, Cyanidium caldarium delta, Chlamydomonas reinhardtii, Klebsormidium nitens and Penicillium sp., but not in Anabaena flos-aquae. We also examined the relationship between the contents of LDs and the genome size in the algae and fungi using VIMPCS but were unable to find a strong relationship between genome size and production of LDs. Finally, the location of LDs was considered in relation to organelles including the endoplasmic reticulum and chloroplasts, which are related to the formation of LDs.

DOI： 10.1508/cytologia.77.289

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV TOKYO CYTOLOGIA  

In plants, the prokaryote-derived chloroplast division protein FtsZ is divided into the FtsZ1 and FtsZ2 families. To clarify the relationship between FtsZ1 and FIsZ2 expression and chloroplast division in the unicellular green alga Nannochloris bacillaris, we compared FtsZ1 and FriZ2 expression in alga cultured in 2 nutritionally different media. Cells grown in a rich medium had 1 FtsZ ring throughout the cell division cycle, whereas cells in an inorganic medium showed only 1 FtsZ ring during the chloroplast division phase. Both FtsZ1 and FtsZ2 mRNA levels in the rich medium were higher than those in the inorganic medium. However, a much larger difference in the amount of transcripts between cells cultured in the rich and in the inorganic media was observed in FtsZ2 than that in FtsZ1, suggesting different gene regulatory mechanisms between the FtsZ genes. N. bacillaris cells cultured in inorganic medium showed cell division photoperiodicity, whereas cells cultured in organic media proliferated continuously under a light/dark cycle. Although FtsZ1 was expressed constantly throughout the chloroplast division cycle in the rich medium, the increase in FisZ1 mRNA level was simultaneous with chloroplast division under a light/dark cycle in the inorganic medium. FtsZ2 was expressed constantly in both media. The control of chloroplast division by regulating FtsZ ring formation is discussed.

DOI： 10.1508/cytologia.77.59

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate-limited (no beta-glycerophosphate) and phosphate-enriched (2 mM beta-glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote-derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate-enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate-enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate-enriched than in phosphate-limited culture and decreased in cells cultured in phosphate-enriched medium containing 5-fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate-enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.

DOI： 10.1111/j.1529-8817.2008.00589.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING INC  

Two FtsZ paralogues (NbFtsZ1 and NbFtsZ2) were isolated from the unicellular green alga Nannochloris bacillaris Naumann. These sequences encoded proteins of 435 and 439 amino acids with tubulin signature motifs (GGGTG[T/S]G), which are important for GTP binding activity. NbFtsZ1 and NbFtsZ2 had four and three introns, respectively, and two different putative core promoters; a TATA box (TATAAAA) and an initiator element (CCCAGG) were located 40 bp and 80 bp upstream of the coding regions of NbFtsZ1 and NbFtsZ2, respectively. Southern blot hybridization and contour-clamped homogeneous electric field electrophoresis showed that N. bacillaris contained at least one copy of each gene and that NbFtsZ1 was located on chromosome 5 and NbFtsZ2 on chromosome 3 or 4. Phylogenetically, NbFtsZ1 and NbFtsZ2 belong to the vascular plant protein families FtsZ1 and FtsZ2, respectively. The FtsZ1 proteins do not contain carboxy-terminal consensus sequences, whereas all FtsZ2 proteins possess the consensus sequence (I/V)PxFL(R/K)(K/R)(K/R). Our study has shown that NbFtsZ2 possesses a similar consensus sequence (VPDFLRRK), whereas NbFtsZ1 does not, further supporting their classification as FtsZ2 and FtsZ1. Escherichia coli ftsZ mutants transformed with cloned NbFtsZ1, and NbFtsZ2 cDNAs were restored for the capacity to divide by binary fission, suggesting that the proteins retained the ability to function in the bacterium. An anti-NbFtsZ2 antibody specifically recognized a single protein band of approximately 51 kDa on an immunoblot of N. bacillaris cellular proteins. Immunostaining of the algal cells with this antibody produced an intense fluorescent signal as a ring near the middle of the cell, which corresponded to the chloroplast division site.

DOI： 10.1111/j.1529-8817.2004.03105.x

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)  

Venue：札幌  

国立遺伝学研究所以外との共同発表あり

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Presentation type：Oral presentation (general)  

Venue：札幌  

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Presentation type：Poster presentation  

Venue：札幌  

国立遺伝学研究所以外との共同発表あり

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Presentation type：Poster presentation  

Venue：姫路  

国立遺伝学研究所以外との共同発表あり

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