Effects of Nano-TiO2 on Chlamydomonas reinhardtii Cell Surface under UV,Natural Light Conditions

Cell surface of aquatic organisms constitutes a primary site for the interaction and a barrier for the nano-TiO2 biological effects.In the present study,the biological effects of nano-TiO2 on a unicellular green algae Chlamydomonas reinhardtii were studied by observing the changes of the cell surface morphology and functional groups under UV or natural light.By SEM,the cell surface morphology of C.reinhardtii was changed under UV light,nano-TiO2 with UV light or natural light,which indicated that photocatalysis damaged cell surface.It was also observed that cell surface was surrounded by TiO2 nanoparticles.The ATR-FTIR spectra showed that the peaks of functional groups such as C-N,-C=O,-C-O-C and P=O,which were the important components of cell wall and membrane,were all depressed by the photocatalysis of nano-TiO2 under UV light or natural light.The photocatalysis of nano-TiO2 promoted peroxidation of functional groups on the surface of C.reinhardtii cells,which led to the damages of cell wall and membrane.

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

The unicellular green alga,Chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid metabolism.To find some novel genes regulating the lipid metabolism under various stress conditions,the paromomycin resistance gene aphVIII was transferred into the genome of C.reinhardtii to establish a mutant library.Two 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 deprivation.The 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,respectively.Since 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.

Effect of Putrescine on Low-Temperature Acclimation in Chlamydomonas reinhardtii

Putrescine is reported to be necessary for cold acclimation under low-temperature stress.In this study,the effect of low-temperature on some physiological and biochemical parameters has been investigated using the green algae Chlamydomonas reinhardtii.The lipid peroxidation rate,amount of Rubisco protein,activities of antioxidant enzymes and gene expression of polyamine biosynthesis(odc2,and spd1),heat shock proteins(hsp70c,hsp90a,and hsp90c),and PSII repair mechanisms(psba,rep27,and tba1)were determined to understand the low-temperature response.Exogenous putrescine application significantly increased Rubisco protein concentration and catalase enzyme activities under low-temperature stress.Moreover,real-time RT-PCR results and gene expression analysis showed that polyamine metabolism induced gene expression at low-temperatures in the first 24 h.In the same way,the gene expression of heat shock proteins(hsp70c,hsp90a,and hsp90c)decreased under low-temperature treatment for 72 h;however,application of putrescine enhanced the gene expression in the first 24 h.The results obtained indicated that molecular response in the first 24 h could be important for cold acclimation.The psba and tba1 expressions were reduced under low-temperatures depending on the exposure time.In contrast,the exogenous putrescine enhanced the expression level of the psba response to low-temperature at 24 and 72 h.The results obtained in this study indicate that putrescine could play a role in the PS II repair mechanisms under low-temperature stress.

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.

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

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 plants.However,the pattern and function of m^(6)A modification during the sexual reproduction of Chlamydomonas remain unknown.Here,we performed transcriptome and methylated RNA immunoprecipitation sequencing(MeRIP-seq)analyses on six samples from different stages during sexual reproduction of the Chlamydomonas life cycle.The results show that m^(6)A modification frequently occurs at the main motif of DRAC(D=G/A/U,R=A/G)in Chlamydomonas mRNAs.Moreover,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 stage.In 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 movement.In 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 stress.Metabolic 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 pathway.This finding will contribute to understanding the toxicological mechanisms of nano-Bi OI on C. reinhardtii.

The Deep Thioredoxome in Chlamydomonas reinhardtii： New Insights into Redox Regulation

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathio- nylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular pro- cesses. This study broadens not only the redox regulation to new enzymes involved in well-known thiore- doxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce （aromatic amino acid biosynthesis, nuclear transport, etc）. Moreover, we char- acterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.

The Heme-Binding Protein SOUL3 of Chlamydomonas reinhardtii Influences Size and Position of the Eyespot

The flagellated green alga Chlamydomonas reinhardtii has a primitive visual system, the eyespot. It is situated at the cells equator and allows the cell to phototax. In a previous proteomic analysis of the eyespot, the SOUL3 protein was identified among 202 proteins. Here, we investigate the properties and functions of SOUL3. Heterologously expressed SOUL3 is able to bind specifically to hemin. In C. reinhardtii, SOUL3 is expressed at a constant level over the diurnal cycle, but forms protein complexes that differ in size during day and night phases. SOUL3 is primarily localized in the eyespot and it is situated in the pigment globule layer thereof. This is in contrast to the channelrhodopsin photoreceptors, which are localized in the plasma membrane region of the eyespot. Knockdown lines with a significantly reduced SOUL3 level are characterized by mislocalized eyespots, a decreased eyespot size, and alterations in phototactic behavior. Mislocalizations were either anterior or posterior and did not affect association with acetylated microtubules of the daughter four-membered rootlet. Our data suggest that SOUL3 is involved in the organization and placement of the eyespot within the cell.

Effects of CO_2 Enrichment on Microstructure and Ultrastructure of Two Species of Freshwater Green Algae

order to investigate the morphological response of freshwater green algae to elevated CO2 concentration, Chlamydomonas reinhardtii Dang and Scenedesmus obliquus Kutz were cultured with enriched CO2, and their microstructure and ultrastructure were examined by microscopy and electron microscopy. The effect of CO2 enrichment to 186 mumol/L, was insignificant on the shape and size of C. reinhardtii, but significant in reducing the volume of S. obliquus. High-CO2 increased the amount of chloroplast. The pyrenoids occurred in low-CO2-grown cells but not in high-CO2-grown ones and more starch granules were observed in the former.

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

The chloroplasts genome （plastome） occurs at high copy numbers per cell. Several chloroplast genome copies are densely packed into nucleoprotein particles called nucleoids. How genome packaging occurs and which proteins organize chloroplast nucleoids are largely unknown. Here, we have analyzed the Chlamydornonas reinhardtii homolog of the bacterial architectural DNA-binding protein HU, the histone-like protein HLP. We show that the Chlarnydornonas HLP protein is targeted to chloroplasts and associates with nucleoids. Knockdown of HLP gene expression by RNA interference （RNAi） alters the structure of chloroplast nucleoids and appears to reduce the level of compaction of chloroplast DNA. Unexpectedly, also chloroplast genome copy numbers are significantly decreased in the RNAi strains, suggesting that, in addition to its architectural role in nucleoid formation, the HIP protein is also involved in chloroplast genome maintenance.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

We asked what peptide features govern targeting to the mitochondria versus the chloroplast,using antimicrobial peptides as a starting point.This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell,to which organelle progenitors became resistant.To explore the molecular changes required to convert antimicrobial into targeting peptides,we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii.Peptides were systematically modified to test distinctive features of mitochondrion-and chloroplast-targeting peptides,and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein.Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences.Targeting to both organelles is improved by replacing lysines with arginines.Chloroplast targeting is enabled by the presence of flanking unstructured sequences,additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria.If indeed targeting peptides evolved from antimicrobial peptides,then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.

Impacts of Elevated CO2 Concentration on Biochemical Composition, Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

: To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186 ümol/L and the biochemical composition, carbonic anhydrase (CA), and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate, and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186 ümol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 ümol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

Loss of algal Proton Gradient Regulation 5 increases reactive oxygen species scavenging and H2 evolution

Summary We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5 （PGRS） capable of producing hydrogen （H2） for 25 days with more than 3o-fold yield increase compared to wild type. Thus, hpm91 displays a higher capacity of H2 production than a previously characterized pgr5 mutant. Physiological and biochemical characterization of hpm91 reveal that the prolonged H2 production is due to enhanced stability of PSII, which correlates with increased reactive oxygen species （ROS） scavenging capacity during sulfur depriva- tion. This anti-ROS response appears to protect the photosynthetic electron transport chain from photo- oxidative damage and thereby ensures electron supply to the hydrogenase.