Publishing type：Research paper (scientific journal)  

DOI： 10.1093/pcp/pcad109

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Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig , together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.

DOI： 10.1073/pnas.2214765120

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

DOI： 10.1038/s41477-023-01377-1

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Other Link： https://www.nature.com/articles/s41477-023-01377-1

Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.2023.08.002

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Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga Cyanidioschyzon merolae after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted via a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1–4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching C. merolae strains in which phycocyanin was not degraded under N-depletion, via autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.

DOI： 10.3389/fpls.2022.1036839

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DOI： 10.1016/j.jbc.2022.102650

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DOI： 10.3390/antiox11122359

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DOI： 10.7554/eLife.82411

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Chloroplast protein import is mediated by translocons named TOC and TIC on the outer and inner envelope membranes, respectively. Translocon constituents are conserved among green lineages, including plants and green algae. However, it remains unclear whether Rhodophyta (red algae) share common chloroplast protein import mechanisms with the green lineages. We show that in the rhodophyte Cyanidioschyzon merolae , plastome-encoded Tic20 _pt localized to the chloroplast envelope and was transiently associated with preproteins during import, suggesting its conserved function as a TIC constituent. Besides plastome-encoded FtsH _pt and several chaperones, a class of GTP (guanosine 5′-triphosphate)-binding proteins distinct from the Toc34/159 GTPase family associated transiently with preproteins. This class of proteins resides mainly in the cytosol and shows sequence similarities with Sey1/RHD3, required for endoplasmic reticulum membrane fusion, and with the periplastid-localized import factor PPP1, previously identified in the Apicomplexa and diatoms. These GTP-binding proteins, named plastid targeting factor for protein import 1 (PTF1) to PTF3, may act as plastid targeting factors in Rhodophyta.

DOI： 10.1073/pnas.2208277119

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

DOI： 10.3389/FPLS.2022.821947

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

Abstract

The highly conserved Hik2–Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signaling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the cyclic GMP, adenylyl cyclase, FhlA (GAF) domain. Consistent with this, Rre1 phosphorylation occurred after the addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone but not after the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the culture medium, which corresponded to PQ-reducing or -oxidizing conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat-inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.

DOI： 10.1093/pcp/pcab158

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Other Link： https://academic.oup.com/pcp/article-pdf/63/2/176/42961720/pcab158.pdf

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

Acetate overflow refers to the metabolism by which a large part of carbon incorporated as glucose into Escherichia coli cells is catabolized and excreted as acetate into the medium. We previously found that mutants for the acetate overflow pathway enzymes phosphoacetyltransferase (Pta) and acetate kinase (AckA) showed significant diauxic growth after glucose depletion in E. coli. Here, we analyzed the underlying mechanism in the pta mutant. Proteomic and other analyses revealed an increase in pyruvate dehydrogenase complex subunits and a decrease in glyoxylate shunt enzymes, which resulted from pyruvate accumulation. Since restoration of these enzyme levels by overexpressing PdhR (pyruvate-sensing transcription factor) or deleting iclR (gene encoding a pyruvate- and glyoxylate-sensing transcription factor) alleviated the growth lag of the pta mutant after glucose depletion, these changes were considered as the reason for the phenotype. Given the evidence for decreased coenzyme A (HS-CoA) levels in the pta mutant, the growth inhibition after glucose depletion was partly explained by limited availability of HS-CoA in the cell. The findings provide insights into the role of acetate overflow in metabolic regulation, which may be useful for biotechnological applications.

DOI： 10.1002/1873-3468.14151

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Microalgae are considered one of the best resources for the production of biofuels and industrially important compounds. Various models have been developed to understand the fundamental mechanism underlying the accumulation of triacylglycerols (TAGs)/starch and to enhance its content in cells. Among various algae, the red alga Cyanidioschyzonmerolae has been considered an excellent model system to understand the fundamental mechanisms behind the accumulation of TAG/starch in the microalga, as it has a smaller genome size and various biotechnological methods are available for it. Furthermore, C. merolae can grow and survive under high temperature (40 °C) and low pH (2–3) conditions, where most other organisms would die, thus making it a choice alga for large-scale production. Investigations using this alga has revealed that the target of rapamycin (TOR) kinase is involved in the accumulation of carbon-reserved molecules, TAGs, and starch. Furthermore, detailed molecular mechanisms of the role of TOR in controlling the accumulation of TAGs and starch were uncovered via omics analyses. Based on these findings, genetic engineering of the key gene and proteins resulted in a drastic increment of the amount of TAGs and starch. In addition to these studies, other trials that attempted to achieve the TAG increment in C. merolae have been summarized in this article.

DOI： 10.3390/plants10061218

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga Cyanidioschyzon merolae were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in C. merolae.

DOI： 10.3390/plants10050971

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

DOI： 10.1002/1873-3468.14075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

<jats:title>Abstract</jats:title>
<jats:p>Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possesses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here, we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae—either alone or in comparative and combinatory studies with other photosynthetic organisms—can provide significant insights into the biology of photosynthetic eukaryotes.</jats:p>

DOI： 10.1093/pcp/pcab052

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.2020.02.003

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

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae. Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

DOI： 10.1098/rstb.2019.0488

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DOI： 10.1093/PCP/PCAA023

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DOI： 10.1038/S41589-019-0461-9

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DOI： 10.1002/1873-3468.13605

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.2019.05.002

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

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than in animals, the roles of ESCRT in cytokinesis are poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. merolae cells divide by furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I-IV, the four subcomplexes of ESCRT, are partially conserved in C. merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4-6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated-a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

DOI： 10.3389/fcell.2020.00169

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

DOI： 10.1073/pnas.1911251116

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The ε subunit of Fo F1 -ATPase/synthase (Fo F1 ) plays a crucial role in regulating Fo F1 activity. To understand the physiological significance of the ε subunit-mediated regulation of Fo F1 in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε∆C ). Analyses using inverted membrane vesicles revealed that the ε∆C mutation decreased ATPase activity and the ATP-dependent H+ -pumping activity of Fo F1 . To enhance the effects of ε∆C mutation, this mutation was introduced into a ∆rrn8 strain harboring only two of the 10 rrn (rRNA) operons (∆rrn8 ε∆C mutant strain). Interestingly, growth of the ∆rrn8 ε∆C mutant stalled at late-exponential phase. During the stalled growth phase, the membrane potential of the ∆rrn8 ε∆C mutant cells was significantly reduced, which led to a decrease in the cellular level of 70S ribosomes. The growth stalling was suppressed by adding glucose into the culture medium. Our findings suggest that the C-terminal region of the ε subunit is important for alleviating the temporal reduction in the membrane potential, by enhancing the ATP-dependent H+ -pumping activity of Fo F1 .

DOI： 10.1002/mbo3.815

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DOI： 10.21769/BIOPROTOC.3204

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DOI： 10.1016/J.ALGAL.2018.101396

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DOI： 10.21769/BIOPROTOC.3172

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DOI： 10.2323/JGAM.2018.01.002

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Other Link： http://orcid.org/0000-0001-6200-964X

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DOI： 10.1080/15592324.2019.1596718

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DOI： 10.1111/tpj.14136

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Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn-1/sn-3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.

DOI： 10.1038/s41598-018-30809-8

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DOI： 10.1093/pcp/pcy156

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DOI： 10.1111/tpj.13859

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DOI： 10.1007/978-981-10-6101-1_13

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DOI： 10.1002/1873-3468.12763

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DOI： 10.1371/journal.pone.0179181

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DOI： 10.3390/catal7050163

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DOI： 10.1038/s41598-017-00979-y

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DOI： 10.1111/mmi.13624

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DOI： 10.2323/jgam.2017.02.008

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DOI： 10.2323/jgam.2017.02.002

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DOI： 10.1007/978-981-10-6101-1_18

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DOI： 10.3389/fpls.2016.01300

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DOI： 10.1073/pnas.1525538113

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DOI： 10.1080/15592324.2016.1149285

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DOI： 10.1093/pcp/pcv120

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DOI： 10.1111/tpj.12310

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DOI： 10.1093/pcp/pct119

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DOI： 10.1091/mbc.E13-04-0208

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DOI： 10.1126/science.1230397

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DOI： 10.3389/fpls.2012.00301

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DOI： 10.3390/ijms131012182

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DOI： 10.1074/jbc.M111.338251

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DOI： 10.1508/cytologia.77.73

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DOI： 10.4161/psb.7.1.18451

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Other Link： http://orcid.org/0000-0001-7560-7884

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DOI： 10.1271/nogeikagaku1924.71.1170

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DOI： 10.1073/pnas.90.8.3511

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Publisher：シーエムシー出版  

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Other Link： http://orcid.org/0000-0002-3040-4757

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Publisher：The Japanese Society of Plant Physiologists  

C. merolae is a photoautotrophic unicellular red algae living in acid hot springs, and has the simplest cell architecture among eukaryotes. In addition to the minimally redundant gene content, many nuclear and chloroplast genes are regulated by light conditions. These features provide a good model for studying light signal transduction in plant cells. <br>In higher plants, DET1 is known as a negative regulator of light-dependent transcription, and widely influences cellular functions as photomorphogenesis. DET1 binds nuclear chromatin and associates with CUL4-DDB1. While these lines of evidence suggest that DET1 might affect transcriptional regulation via chromatin modification, the underlying mechanism remains unclear. <br>In this study, we constructed a null mutant strain lacking the DET1 homolog gene in C. merolae. This mutant showed growth inhibition under light-dark cycles. As observed in higher plants, several light-regulated nuclear and chloroplast genes were highly expressed even under dark. Currently, we are analyzing the relationship between DET1 and histone H3 lysine 9 di-methylation as a representative chromatin mark for transcriptional silencing.

DOI： 10.14841/jspp.2011.0.0749.0

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Publisher：The Japanese Society of Plant Physiologists  

In higher plants, plastids can differentiate depending on tissues and developmental stages. Using the suspension culture of Arabidopsis T87 cell line, we developed simple systems for differentiation of several types of plastids. In detail, proplastids in dark-grown T87 cells could be easily differentiated into amyloplasts or chloroplasts by change of light or phytohormone conditions. In the tobacco BY-2 system reported by Miyazawa et al. (1999), differentiation of amyloplasts was accompanied with the induction of nuclear-encoded genes related to starch synthesis. We have recently demonstrated in BY-2 cells that inhibition of plastid gene expression by specific inhibitors resulted in inhibition of amyloplast differentiation as well as repression of gene expression for nuclear starch-synthesis genes. This observation was partly reproduced in the T87 system established here. To assess the possibility that plastid differentiation could be regulated by plastid-derived signals, we started to analyze functions of some regulatory factors using transgenic approaches.

DOI： 10.14841/jspp.2011.0.0076.0

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Publisher：The Japanese Society of Plant Physiologists  

Amyloplast is a subtype of plastids found in non-photoshynthetic tissues characterized by starch synthesis and storage. Tobacco BY-2 cultured cell is normally grown in an auxin-medium, and exchanging hormone from auxin to cytokinin induces differentiation of proplastid to amyloplast. In this system, expression of nuclear genes such as ADP-glucose pyrophosphatase (AGPS), which are required for starch biosynthesis, are induced. However, involvement of plastid gene expression in amyloplast differentiation remains unclear.<br>We here examined plastid gene expression by microarray analysis, and found that no major change during amyloplast differentiation was observed. Nevertheless, starch biosynthesis followed by amyloplast development was inhibited in the presence of plastid translation/transcription inhibitors. Interestingly, we found that expression of nuclear-encoded starch biosynthesis genes such as AGPS was repressed by addition of these inhibitors. These results suggest that plastid transcription/translation is required for amyloplast development via unknown signals regulating nuclear gene expression. Possible roles of plastid-derived signals will be also discussed with some data.

DOI： 10.14841/jspp.2011.0.0517.0

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Publisher：The Japanese Society of Plant Physiologists  

Nitrogen is an essential nutrient for plant cells. We are using a unicellular red alga, Cyanidioschyzon merolae, as the model system to analyze the nitrogen regulation in photosynthetic eukaryotes. In this study, we examined in detail the physiological changes of C. merolae in case of nitrogen depletion. In addition, we identified and characterized a transcription factor that is responsible for expression of nitrogen assimilation genes in C. merolae. DNA microarray and Northern blot analyses revealed that transcript of the gene encoding CmMYB1, an R2R3-type MYB transcription factor, increased 1 hour after nitrogen depletion. The CmMYB1 protein started to accumulate after 2 hours and reached a peak after 4 hours after the nitrogen depletion, correlating with the expression of key nitrogen assimilation genes. The nitrogen depletion-induced gene expression disappeared in a CmMYB1 null mutant. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assays using crude cell extract demonstrated that CmMYB1 specifically occupied these nitrogen-responsive promoter regions only under nitrogen-depleted conditions. <br>Ref) Imamura et al. (2009) PNAS 106, 12548-1255

DOI： 10.14841/jspp.2011.0.0700.0

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Publisher：The Japanese Society of Plant Physiologists  

Chloroplasts have their own DNA and gene expression systems. In order to study transcriptional regulation, genetic approaches have been historically used. However, this approach may include some indirect effects, which make it difficult to understand specific regulation by the transcription factors. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool that can obtain information for binding sites for transcription factors, and directly detect dynamic changes of their interaction patterns in vivo.<br>To further understand the roles of plastid sigma factors in Arabidopsis thaliana, we here developed ChIP-based method, and analyzed binding pattern of SIG5, a stress-induced chloroplast sigma factor. We found SIG5 specifically binds to novel target promoters as well as psbA or psbD BLRP that are already known, and this binding depends on several kinds of stress conditions. We further analyzed and identified target promoters for SIG1, an essential sigma factor by ChIP analysis. These results suggest that ChIP analysis is useful to understand transcriptional regulation of chloroplast genes, which can overcome several problems from traditional methods.

DOI： 10.14841/jspp.2011.0.0723.0

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Publisher：The Japanese Society of Plant Physiologists  

Plastids and mitochondria have their own genomes descended from their ancestors, and the organelle DNA replications (ODR) of plant cells are coordinated with the nuclear DNA replication (NDR) as ODR precedes NDR during a cell cycle progression. Recently, we identified that a tetrapyrrole compound, Mg-Protoporphyrin IX (Mg-ProtoIX), is a cell-cycle coordinator from organelle to NDR in plant cells. Mg-ProtoIX somehow activates CDKA to direct the G1/S transition¹. However, the molecular mechanism for CDKA activation remained elusive. Here we identified an F-box protein Fbx3, which inhibits CDKA by ubiquitinating the cyclin 1 and inducing the degradation. Mg-ProtoIX binds to Fbx3 and inhibits the cyclin 1 ubiquitination. We will provide a model of molecular mechanisms for coordination among organelles and nuclear DNA replication.<br>1 Kobayashi, Y. et al. Proc. Natl. Acad. Sci. USA 106, 803 (2009).

DOI： 10.14841/jspp.2011.0.0868.0

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Publisher：The Japanese Society of Plant Physiologists  

Chloroplasts have their own DNA and gene expression systems that are derived from endosymbiosis of ancestral cyanobacteria. However, during long history of evolution, chloroplasts lost their autonomy and most of regulatory system became under control of the nucleus. In this work, we used the primitive, unicellular red alga Cyanidioschyzon merolae, which shows ancestral characteristics on many aspects including chloroplast genome and transcription systems. Therefore, gene expression in C. merolae chloroplasts can be regulated more autonomously than that in higher plants.<br>We previously found that the chloroplast two-component system, which is composed of the unique histidine kinase (HIK) and one of chloroplast-encoded response regulators (Ycf27), is involved in this regulation. In this work, we further analyzed function of HIK. Expression level of HIK was constant in various light conditions, suggesting that activity of HIK could be regulated post-translationally. Including other data as well as structural characteristics of HIK, the system for light-responsive transcription regulation in C. merolae chloroplasts will be discussed.

DOI： 10.14841/jspp.2011.0.0869.0

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Language：Japanese   Publisher：日本生物工学会  

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Publisher：日本植物生理学会  

Metabolic engineering of photosynthetic organisms is required for utilization of light energy and reducing carbon emission. We previously showed that a group 2 sigma factor SigE of &lt;I&gt;Synechocystis&lt;/I&gt; sp. PCC 6803 globally activates transcription of sugar catabolic genes. In this study, we generated the strain overexpressing SigE and microarray analysis revealed that genes for the oxidative pentose phosphate pathway and glycogen catabolism increased in this strain. Immunoblotting revealed that protein levels of sugar catabolic enzymes such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glycogen phosphorylase, and isoamylase increased and the level of glycogen decreased in the SigE-overexpressing strain under light growth conditions. CE/MS analysis unraveled that metabolites of the TCA cycle and acetyl-CoA are altered by SigE overexpression. We also found that SigE-overexpressing strain exhibits defective growth under mixotrophic or dark conditions. We thus demonstrate that SigE overexpression activates sugar catabolism at transcript to phenotype levels, opening a sigma factor-based engineering for the modification of carbon metabolism in photosynthetic organisms.

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Plastids and mitochondria have their own genomes descended from their bacterial ancestors. In C. merolae, dark incubation arrests cell cycle at the G1 phase, and the cell cycle synchronously initiates after light illumination. During the G1 to S phase transition, organelle DNA replication (ODR) precedes the nuclear DNA replication (NDR), and ODR is required for the NDR initiation. Thus, NDR is tightly coupled with ODR in C. merolae. As the molecular mechanism, we recently found that intracellular accumulation of Mg-Protoporphyrin IX, which is induced by ODR, activates CDKA and thus initiate NDR^{1, 2}. However, little information is available on the ODR control mechanism. We have recently made clear that CDK-type kinase other than CDKA is involved in the ODR initiation. In this meeting, we will present recent results on the characterization of this CDK enzyme.<br>¹ Kobayashi et al. Proc. Natl. Acad. Sci. U S A 106:803-807 (2009)<br>² Kanesaki et al. Plant Signal. Behav. in press (2009)

DOI： 10.14841/jspp.2010.0.0562.0

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Chloroplasts have their own DNA and gene expression systems. In order to study transcriptional regulation, biochemical and genetic approaches have been historically used. However, biochemical approaches does not usually demonstrate functions under physiological conditions, and genetic approaches may include some indirect effects, which make it difficult to understand specific regulation by the transcription factors. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool that can obtain information for binding sites for transcription factors, and directly detect dynamic changes of their interaction patterns in vivo.<br>To further understand transcriptional regulation in Arabidopsis thaliana chloroplasts, we here developed ChIP-based method, and analyzed binding pattern of SIG5, a stress-induced chloroplast sigma factor. We found SIG5 specifically binds to target promoters such as psbA or psbD BLRP, and this binding depends on several kinds of stress conditions. This result suggests that ChIP analysis is useful to understand transcriptional regulation of chloroplast genes, which can overcome several problems from traditional methods.

DOI： 10.14841/jspp.2010.0.0355.0

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DOI： 10.14841/jspp.2009.0.0728.0

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DOI： 10.1271/kagakutoseibutsu.47.740

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Publisher：The Japanese Society of Plant Physiologists  

TFIIB and its related protein, BRF, are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro. In vitro transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody, and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate with the RNA polymerase I subunit, CmRPA190, and to co-localize in the nucleolus.Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

DOI： 10.14841/jspp.2009.0.0272.0

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Publisher：The Japanese Society of Plant Physiologists  

Chloroplasts have their own genome and genetic systems derived from endosymbiosis of ancestral cyanobacteria. However, during long evolution, chloroplasts lost their autonomy and most regulatory systems became under nuclear control. We here used the primitive, unicellular red alga Cyanidioschyzon merolae, which shows ancestral characteristics on many aspects including chloroplast genome and transcription systems. Therefore, transcription in C.merolae can be regulated more autonomously than that in higher plants.<br>To understand light-dependent transcription regulation in C.merolae chloroplasts, we performed run-on transcription and ChIP analyses. We found that the chloroplast two-component system, which is composed of the unique histidine kinase(HIK) and one of chloroplast-encoded response regulators(Ycf27), is involved in this regulation. In addition, we performed these assays using photosynthetic electron transport inhibitors, and showed that redox level of plastoquinone did not affect this regulation. Including structural characteristics of HIK, the system for light-responsive transcription in C.merolae is discussed.

DOI： 10.14841/jspp.2009.0.0283.0

J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Publisher：The Japanese Society of Plant Physiologists  

Amyloplast is a subtype of plastids found in non-photoshynthetic tissues characterized by starch synthesis and storage. Tobacco BY-2 cultured cell is normally grown in an auxin-medium, and exchanging hormone from auxin to cytokinin induces differentiation of proplastid to amyloplast. In this system, expression of nuclear genes such as ADP-glucose pyrophosphatase(Agp), which are required for starch biosynthesis, are induced. However, involvement of plastid gene expression in amyloplast differentiation remains unclear.<br>We here examined plastid gene expression by northern analysis, and found that no major change during amyloplast differentiation was observed. Nevertheless, starch biosynthesis followed by amyloplast development was inhibited in the presence of plastid translation/transcription inhibitors, spectynomycin or rifampicin. Interestingly, we found that expression of nuclear-encoded starch biosynthesis genes such as Agp was repressed by addition of these inhibitors. These results suggest that plastid transcription/translation is required for amyloplast development via unknown signals regulating nuclear gene expression.

DOI： 10.14841/jspp.2008.0.0703.0

J-GLOBAL

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Publisher：The Japanese Society of Plant Physiologists  

Chloroplasts have their own genome and gene expression systems derived from endosymbiosis of ancestral cyanobacteria. In the course of subsequent evolution, chloroplasts lost their autonomy and most regulatory systems became under nuclear control. In this work, we used the unicellular red alga Cyanidioschyzon merolae. This alga shows ancestral characteristics in many aspects including chloroplast genome structure and transcription regulation. Therefore, some regulatory systems in C. merolae show more autonomous features than those in higher plants.<br>Here we performed chromatin immunoprecipitation (ChIP) assay to understand light-dependent transcription regulation. Previously, we found transcription of ycf27 and psbD is specifically activated under light condition. Ycf27, one of the chloroplast-encoded transcription factors, is a candidate in this regulation. We performed ChIP assay and found that Ycf27 actually binds to those promoters and this binding depends on light-condition. This suggests that transcription of particular genes is regulated by chloroplast-specific mechanisms independent of the nucleus.

DOI： 10.14841/jspp.2008.0.0704.0

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

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Language：English  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)  

DOI： 10.14841/jspp.2006.0.653.0

Web of Science

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

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Language：English  

DOI： 10.1073/pnas.072587199

Web of Science

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Language：English   Publisher：Japanese Society of Plant Physiologists  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184564

J-GLOBAL

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Language：English   Publisher：American Association for the Advancement of Science  

DOI： 10.1126/science.272.5270.1932

Scopus

PubMed

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Language：English  

DOI： 10.1073/pnas.90.8.3511

Scopus

PubMed

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Language：English  

DOI： 10.1126/science.3194753

Scopus

PubMed

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