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Protein Disulfide Isomerase 2 of chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

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Molecular Plant 2013年第5期6卷 1503-1517页

作者： Anna Filonova Paul Haemsch Christin Gebauer Wolfram Weisheit Volker Wagner Institute of General Botany and Plant Physiology Friedrich-Schiller-Universitat Jena 07743 Jena Germany

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.

关键词： chlamydomonas reinhardtii protein disulfide isomerase 2 circadian clock.

Characterization of arsenate transformation and identification of arsenate reductase in a green alga chlamydomonas reinhardtii

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Journal of Environmental Sciences 2011年第7期23卷 1186-1193页

作者： Xixiang Yin Lihong Wang Guilan Duan Guoxin Sun Key Laboratory of Urban Environment and Health Institute of Urban Environment Chinese Academy of Sciences Xiamen 361021 China State Key Laboratory of Urban and Regional Ecology Research Center for Eco-environmental Sciences Chinese Academy of Sciences Beijing 100085 China Shandong Analysis and Test Center Shandong Academy of Sciences Shandong 250014 China

Arsenic （As） is a pervasive and ubiquitous environmental toxin that has created catastrophic human health problems world-wide. chlamydomonas reinhardtii is a unicellular green alga, which exists ubiquitously in freshwater aquatic systems. Arsenic metabolism processes of this alga through arsenate reduction and sequent store and efflux were investigated. When supplied with 10 μmol/L arsenate, arsenic speciation analysis showed that arsenite concentration increased from 5.7 to 15.7 mg/kg dry weight during a 7-day period, accounting for 18%–24% of the total As in alga. When treated with different levels of arsenate （10, 20, 30, 40, 50 μmol/L） for 7 days, the arsenite concentration increased with increasing external arsenate concentrations, the proportion of arsenite was up to 23%–28% of the total As in alga. In efflux experiments, both arsenate and arsenite could be found in the efflux solutions. Additionally, the efflux of arsenate was more than that of arsenite. Furthermore, two arsenate reductase genes of C. reinhardtii （CrACR2s） were cloned and expressed in Escherichia coli strain WC3110 （？arsC） for the first time. The abilities of both CrACR2s genes to complement the arsenate- sensitive strain were examined. CrACR2.1 restored arsenate resistance at 0.8 mmol/L. However, CrACR2.2 showed much less ability to complement. The gene products were demonstrated to reduce arsenate to arsenite in vivo. In agreement with the complementation results, CrACR2.1 showed higher reduction ability than CrACR2.2, when treated with 0.4 mmol/L arsenate for 16 hr incubation.

关键词： arsenate accumulation reduction efflux arsenate reductase chlamydomonas reinhardtii

Response mechanism of chlamydomonas reinhardtii to nanoscale bismuth oxyiodide(nano-BiOI):Integrating analysis of mineral nutrient metabolism and metabolomics

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Journal of Environmental Sciences 2022年第11期34卷 13-24页

作者： Yan Huang Jinlong Lai Yang Huang Xuegang Luo Xu Yang Zewei Liu Yue Duan Chen Li School of Life Sciences Southwest University of Science and TechnologyMianyang 621010China College of Environment and Resources Southwest University of Science and TechnologyMianyang 621010China Laboratory of Solid Waste Treatment and Resource Recycle Ministry of EducationMianyang 621010China College of Chemical and Environment Science Shaanxi University of TechnologyHanzhong 723000China

Nanoscale bismuth oxyiodide(nano-BiOI) is widely studied and applied in environmental applications and biomedical fields, with the consequence that it may be deposited into aquatic environments. However, the impact of nano-Bi OI on aquatic ecosystems, especially freshwater microalga, remains limited. Herein, the nano-Bi OI was synthesized and its response mechanism towards microalga chlamydomonas reinhardtii was evaluated. Results showed that a low concentration of nano-Bi OI(5 mg/L) could stimulate algal growth at the early stage of stress. With the increase in concentration, the growth rate of algal cells was inhibited and showed a dose effect. Intracellular reactive oxygen species(ROS) were significantly induced and accompanied by enhanced lipid peroxidation, decreased nonspecific esterase activity, and significantly upregulated glutathione S-transferase activity(GST) activity. Mineral nutrient metabolism analysis showed that nano-Bi OI significantly interfered with the mineral nutrients of the algae. Non-targeted metabolomics identified 35 different metabolites(DEMs, 22 upregulated, and 13 downregulated) under 100 mg/L Bi OI *** pathway analysis demonstrated that a high concentration of nano-Bi OI significantly induced metabolic pathways related to amino acid biosynthesis, lipid biosynthesis,and glutathione biosynthesis, and significantly inhibited the sterol biosynthesis *** finding will contribute to understanding the toxicological mechanisms of nano-Bi OI on C. reinhardtii.

关键词： chlamydomonas reinhardtii Nano-BiOI Mineral nutrient Metabolomics

Characteristics of N^(6)-methyladenosine Modification During Sexual Reproduction of chlamydomonas reinhardtii

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Genomics, Proteomics & Bioinformatics 2023年第4期21卷 756-768页

作者： Ying Lv Fei Han Mengxia Liu Ting Zhang Guanshen Cui Jiaojiao Wang Ying Yang Yun-Gui Yang Wenqiang Yang Photosynthesis Research Center Key Laboratory of PhotobiologyInstitute of BotanyChinese Academy of SciencesBeijing 100093China China National Botanical Garden Beijing 100093China College of Advanced Agricultural Sciences University of Chinese Academy of SciencesBeijing 100049China CAS Key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of Sciences and China National Center for BioinformationBeijing 100101China Sino-Danish College University of Chinese Academy of SciencesBeijing 100049China Institute of Stem Cell and Regeneration Chinese Academy of SciencesBeijing 100101China Innovative Academy of Seed Design Chinese Academy of SciencesBeijing 100093China

The unicellular green alga chlamydomonas reinhardtii(hereafter chlamydomonas)possesses both plant and animal attributes,and it is an ideal model organism for studying fundamental processes such as photosynthesis,sexual reproduction,and life cycle.N^(6)-methyladenosine(m^(6)A)is the most prevalent mRNA modification,and it plays important roles during sexual reproduction in animals and ***,the pattern and function of m^(6)A modification during the sexual reproduction of chlamydomonas remain ***,we performed transcriptome and methylated RNA immunoprecipitation sequencing(MeRIP-seq)analyses on six samples from different stages during sexual reproduction of the chlamydomonas life *** results show that m^(6)A modification frequently occurs at the main motif of DRAC(D=G/A/U,R=A/G)in chlamydomonas ***,m^(6)A peaks in chlamydomonas mRNAs are mainly enriched in the 30 untranslated regions(30 UTRs)and negatively correlated with the abundance of transcripts at each *** particular,there is a significant negative correlation between the expression levels and the m^(6)A levels of genes involved in the microtubule-associated pathway,indicating that m^(6)A modification influences the sexual reproduction and the life cycle of chlamydomonas by regulating microtubule-based *** summary,our findings are the first to demonstrate the distribution and the functions of m^(6)A modification in chlamydomonas mRNAs and provide new evolutionary insights into m^(6)A modification in the process of sexual reproduction in other plant organisms.

关键词： N6-methyladenosine m^(6)A sequencing Sexual reproduction Microtubule-associated pathway Photosynthesis chlamydomonas reinhardtii

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in chlamydomonas reinhardtii

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Journal of Integrative Plant Biology 2007年第7期49卷 1054-1061页

作者： Jing-Jing Ma Liang-Bi Li Yu-Xiang Jing Ting-Yun Kuang Key Laboratory of Photosynthesis and Environmental Molecular Physiology Institute of Botany Chinese Academy of Sciences Beijing 100093 China Graduate University of Chinese Academy of Sciences Beijing 100049 China

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.

关键词： chlamydomonas reinhardtii chloroplast transformation cytochrome b559 mutation photosystem Ⅱ.

Cyclic adenosine monophosphate signal pathway is involved in regulation of triacylglycerol biosynthesis following nitrogen deprivation in chlamydomonas reinhardtii

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Journal of Oceanology and Limnology 2020年第2期38卷 517-528页

作者： MIAO Rongli HUANG Kaiyao Key Laboratory of Algal Biology Institute of HydrobiologyChinese Academy of SciencesWuhan 430072China University of Chinese Academy of Sciences Beijing 100049China

The unicellular green alga,chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid *** find some novel genes regulating the lipid metabolism under various stress conditions,the paromomycin resistance gene aphVIII was transferred into the genome of *** to establish a mutant *** genes mutated in two of the TAG-reduced mutants(Cre06.g278111 in M2 mutant,Cre06.g278110 in M6 mutants)were neighboring in the genome,and their expression levels were down-regulated in their corresponding mutants in parallel with their reduced TAG levels following N *** proteins encoded by these two genes(KCN11 by Cre06.g278111,ACYC3 by Cre06.g278110)contained a conversed cyclic mononucleotide phosphate(cNMP)binding protein and an adenylate domain,*** cNMP binding protein and adenylate domain have been known as important components of cyclic adenosine monophosphate(cAMP)signaling pathway,suggesting that these two genes might af fect cellular TAG biosynthesis through cAMP signal pathway.

关键词： chlamydomonas reinhardtii mutant lipid droplet(LD) triacylglycerol(TAG) cyclic adenosine monophosphate(cAMP)

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in chlamydomonas reinhardtii

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2013年第12期14卷 1121-1131页

作者： Xiao-dong DENG Jia-jia CAI Xiao-wen FEI Key Laboratory of Tropical Crop Biotechnology Ministry of Agriculture Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences School of Science Hainan Medical College

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

关键词： Phosphatidate phosphohydrolase 2 Triacylglycerol biosynthesis RNAi chlamydomonas reinhardtii Nitrogen deprivation Over-expression

Functional Accumulation of Antenna Proteins in Chlorophyll b-Less Mutants of chlamydomonas reinhardtii

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Molecular Plant 2017年第1期10卷 115-130页

作者： Sandrine Bujaldon Natsumi Kodama Fabrice Rappaport Rajagopal Subramanyam Catherine de Vitry Yuichiro Takahashi FrancisAndre Wollman Institut de Biologie Physico-Chimique UMR7141 CNRS-UPMC Paris 75005 France Research Institute for Interdisciplinary Science Okayama University Okayama 700-8530 Japan JST-CREST Okayama University Okayama 700-8530 Japan Department of Plant Sciences School of Life Sciences University of Hyderabad Hyderabad 500046 India These authors contributed equally to this article Deceased while manuscript was in progress

The green alga chlamydomonas reinhardtii contains several light-harvesting chlorophyll a/b complexes （LHC）- four major LHCIIs, two minor LHClIs, and nine LHCIs. We characterized three chlorophyll b-less mutants to assess the effect of chlorophyll b deficiency on the function, assembly, and stability of these chlorophyll a/b binding proteins. We identified point mutations in two mutants that inactivate the CAO gene responsible for chlorophyll a to chlorophyll b conversion. All LHClIs accumulated to wild-type levels in a CAO mutant but their light-harvesting function for photosystem II was impaired. In contrast, most LHCIs accumulated to wild-type levels in the mutant and their light-harvesting capability for photosystem I remained unaltered. Unexpectedly, LHCl accumulation and the photosystem I functional antenna size increased in the mutant compared with in the wild type when grown in dim light. When the CAO mutation was placed in a yellow-in-the-dark background （yid-BF3）, in which chlorophyll a synthesis remains limited in dim light, accumulation of the major LHClIs and of most LHCls was markedly reduced, indicating that sustained synthesis of chlorophyll a is required to preserve the proteolytic resistance of antenna pro- teins. Indeed, after crossing yid-BF3 with a mutant defective for the thylakoid FtsH protease activity, yid- BF3-ftshl restored wild-type levels of LHCI, which defines LHCI as a new substrate for the FtsH protease.

关键词： chlamydomonas reinhardtii chlorophyll b-less mutant antenna protein CAO gene

The High Light Response and Redox Control of Thylakoid FtsH Protease in chlamydomonas reinhardtii

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Molecular Plant 2017年第1期10卷 99-114页

作者： Fei Wang Yafei Qi Alizee Malnoe Yves Choquet Francis-Andre Wollman Institut de Biologie Physico-Chimique Unite Mixte de Recherche 7141 Centre National de la Recherche Scientifique/Universite Pierre et Marie Curie Paris 75005 France

In chlamydomonas reinhardtii, the major protease involved in the maintenance of photosynthetic machinery in thylakoid membranes, the FtsH protease, mostly forms large hetero-oligomers （-1 MDa） comprising FtsH1 and FtsH2 subunits, whatever the light intensity for growth. Upon high light exposure, the FtsH subunits display a shorter half-life, which is counterbalanced by an increase in FTSH1/2 mRNA levels, resulting in the modest upregulation of FtsH1/2 proteins. Furthermore, we found that high light increases the protease activity through a hitherto unnoticed redox-controlled reduction of intermolecular disulfide bridges. We iso- lated a chlamydomonas FTSH1 promoter-deficient mutant, ftsh1-3, resulting from the insertion of a TOC1 transposon, in which the high light-induced upregulation of FTSH1 gene expression is largely lost. In ftsh1- 3, the abundance of FtsH1 and FtsH2 proteins are loosely coupled （decreased by 70% and 30%, respectively） with no formation of large and stable homo-oligomers. Using strains exhibiting different accumulation levels of the FtsH1 subunit after complementation of ftsh1-3, we demonstrate that high light tolerance is tightly correlated with the abundance of the FtsH protease. Thus, the response of chlamydomonas to light stress involves higher levels of FtsH 1/2 subunits associated into large complexes with increased proteolytic activity.

关键词： chloroplast protease regulation of gene expression photoinhibition chlamydomonas reinhardtii