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Rachel M. Dent Marina N. Sharifi Alizée Malnoë Cat Haglund Robert H. Calderon Setsuko Wakao Krishna K. Niyogi 《The Plant journal : for cell and molecular biology》2015,82(2):337-351
Chlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large‐scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over‐represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date. 相似文献
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Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress 
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下载免费PDF全文 Nicolas Stankovic‐Valentin Katarzyna Drzewicka Cornelia König Elmar Schiebel Frauke Melchior 《The EMBO journal》2016,35(12):1312-1329
To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress. 相似文献
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The explanation for the continued existence of sex, despite its many costs, remains one of the major challenges of evolutionary biology. Previous experimental studies have demonstrated that sex increases the rate of adaptation in novel environments relative to asexual reproduction. Whereas these studies have investigated the impact of sex on adaptation to stressful abiotic environments, the potential for biotic interactions to influence this advantage of sex has been largely ignored. Species rarely exist in isolation in natural conditions, so the impact of sex on adaptation to a stressful abiotic environment may be altered by the interactions between coexisting species. To investigate the interplay of sex and competition on adaptation to deteriorating conditions, we allowed populations of the unicellular alga (Chlamydomonas reinhardtii) to evolve in an environment to which they were initially poorly adapted. We manipulated both their mode of reproduction and the presence of a competitor, and monitored population size and proportion of evolutionary rescue events for each mode of reproduction. The results indicate that sex may be the beneficial strategy in the presence of the competitor. Sexual populations had highest probability of evolutionary rescue irrespective of the presence of the competitor. The overall advantage of sex was also manifested through higher level of adaptedness of survived sexual populations relative to asexual populations. Since competitive interactions are commonplace in nature, one of the explanations for the maintenance of sex by natural selection may be the increased rate of adaptation of sexual populations both in the presence and absence of competitors. 相似文献
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Barbara Emonds‐Alt Nadine Coosemans Thomas Gerards Claire Remacle Pierre Cardol 《The Plant journal : for cell and molecular biology》2017,89(1):141-154
Phylloquinone (PhQ), or vitamin K1, is an essential electron carrier (A1) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A1 site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone‐deficient mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome. 相似文献
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Peeyush Ranjan Rudra Shankar Kashyap Manisha Goel Sindhu Kandoth Veetil Suneel Kateriya 《Journal of phycology》2014,50(6):1137-1145
GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADP – ribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch‐off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross‐linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20‐kDa monomeric and 40‐kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role. 相似文献
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Environments rarely remain the same over time, and populations are therefore frequently at risk of going extinct when changes are significant enough to reduce fitness. Although many studies have investigated what attributes of the new environments and of the populations experiencing these changes will affect their probability of going extinct, limited work has been directed towards determining the role of population history on the probability of going extinct during severe environmental change. Here, we compare the extinction risk of populations with a history of selection in a benign environment, to populations with a history of selection in one or two stressful environments. We exposed spores and lines of the green alga Chlamydomonas reinhardtii from these three different histories to a range of severe environmental changes. We found that the extinction risk was higher for populations with a history of selection in stressful environments compared to populations with a history of selection in a benign environment. This effect was not due to differences in initial population sizes. Finally, the rates of extinction were highly repeatable within histories, indicating strong historical contingency of extinction risk. Hence, information on the selection history of a population can be used to predict their probability of going extinct during environmental change. 相似文献
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Jaruswan Warakanont Chia‐Hong Tsai Elena J. S. Michel George R. Murphy III Peter Y. Hsueh Rebecca L. Roston Barbara B. Sears Christoph Benning 《The Plant journal : for cell and molecular biology》2015,84(5):1005-1020
In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant. 相似文献
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Valéria Nagy André Vidal‐Meireles Anna Podmaniczki Klára Szentmihályi Gábor Rákhely Laura Zsigmond László Kovács Szilvia Z. Tóth 《The Plant journal : for cell and molecular biology》2018,94(3):548-561
Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur‐limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur‐free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP‐L‐galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen‐evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation. 相似文献
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Laura Stoffels Henry N. Taunt Bambos Charalambous Saul Purton 《Plant biotechnology journal》2017,15(9):1130-1140
There is a pressing need to develop novel antibacterial agents given the widespread antibiotic resistance among pathogenic bacteria and the low specificity of the drugs available. Endolysins are antibacterial proteins that are produced by bacteriophage‐infected cells to digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These highly efficient enzymes show a considerable degree of specificity for the target bacterium of the phage. Furthermore, the emergence of resistance against endolysins appears to be rare as the enzymes have evolved to target molecules in the cell wall that are essential for bacterial viability. Taken together, these factors make recombinant endolysins promising novel antibacterial agents. The chloroplast of the green unicellular alga Chlamydomonas reinhardtii represents an attractive platform for production of therapeutic proteins in general, not least due to the availability of established techniques for foreign gene expression, a lack of endotoxins or potentially infectious agents in the algal host, and low cost of cultivation. The chloroplast is particularly well suited to the production of endolysins as it mimics the native bacterial expression environment of these proteins while being devoid of their cell wall target. In this study, the endolysins Cpl‐1 and Pal, specific to the major human pathogen Streptococcus pneumoniae, were produced in the C. reinhardtii chloroplast. The antibacterial activity of cell lysates and the isolated endolysins was demonstrated against different serotypes of S. pneumoniae, including clinical isolates and total recombinant protein yield was quantified at ~1.3 mg/g algal dry weight. 相似文献
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There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae. 相似文献
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Chia‐Hong Tsai Krzysztof Zienkiewicz Cynthia L. Amstutz Benedikt G. Brink Jaruswan Warakanont Rebecca Roston Christoph Benning 《The Plant journal : for cell and molecular biology》2015,83(4):650-660
In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed. 相似文献
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When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter. 相似文献
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Bo Xie Dan Stessman Jason H. Hart Haili Dong Yingjun Wang David A. Wright Basil J. Nikolau Martin H. Spalding Larry J. Halverson 《Plant biotechnology journal》2014,12(7):872-882
The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low‐lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence‐activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high‐lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid‐induction periods. 相似文献