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1.
Nat Plants ; 7(9): 1314-1322, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34462576

RESUMO

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.


Assuntos
Adaptação Ocular/fisiologia , Proliferação de Células/fisiologia , Chlorella/metabolismo , Chlorella/ultraestrutura , Ritmo Circadiano/fisiologia , Temperatura Alta , Complexo de Proteína do Fotossistema I/metabolismo
2.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
3.
FEMS Microbiol Rev ; 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34165541

RESUMO

Biological soil crusts (BSCs) are found in drylands, cover ∼12% of the Earth's surface in arid and semi-arid lands and their destruction is considered an important promoter of desertification. These crusts are formed by the adhesion of soil particles to polysaccharides excreted mostly by filamentous cyanobacteria, which are the pioneers and main primary producers in BSCs. Desert BSCs survive in one of the harshest environments on Earth, and are exposed to daily fluctuations of extreme conditions. The cyanobacteria inhabiting these habitats must precisely read the changing conditions and predict, for example, the forthcoming desiccation. Moreover, they evolved a comprehensive regulation of multiple adaptation strategies to enhance their stress tolerance. Here we focus on what distinguishes cyanobacteria able to revive after dehydration from those that cannot. While important progress has been made in our understanding of physiological, biochemical and omics aspects, clarification of the sensing, signal transduction and responses enabling desiccation tolerance are just emerging. We plot the trajectory of current research and open questions ranging from general strategies and regulatory adaptations in the hydration/desiccation cycle, to recent advances in our understanding of photosynthetic adaptation. The acquired knowledge provides new insights to mitigate desertification and improve plant productivity under drought conditions.

4.
New Phytol ; 229(6): 3208-3220, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33533496

RESUMO

In multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit tri-methylation of lysine 27 of histone H3 (H3K27me3) to establish and maintain gene silencing, critical for developmentally regulated processes. The PRC2 complex is absent in both widely studied model yeasts, which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes. Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di- and tri-methylation of lysine 27 of histone H3. H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome-wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity. These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species, and highlight their ancestral function in a broader evolutionary context than currently is appreciated.


Assuntos
Histonas , Complexo Repressor Polycomb 2 , Diferenciação Celular/genética , Histonas/metabolismo , Metilação , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb
5.
Life (Basel) ; 10(9)2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32899164

RESUMO

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.

6.
Nat Plants ; 6(8): 1031-1043, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32719473

RESUMO

The unparalleled performance of Chlorella ohadii under irradiances of twice full sunlight underlines the gaps in our understanding of how the photosynthetic machinery operates, and what sets its upper functional limit. Rather than succumbing to photodamage under extreme irradiance, unique features of photosystem II function allow C. ohadii to maintain high rates of photosynthesis and growth, accompanied by major changes in composition and cellular structure. This remarkable resilience allowed us to investigate the systems response of photosynthesis and growth to extreme illumination in a metabolically active cell. Using redox proteomics, transcriptomics, metabolomics and lipidomics, we explored the cellular mechanisms that promote dissipation of excess redox energy, protein S-glutathionylation, inorganic carbon concentration, lipid and starch accumulation, and thylakoid stacking. C. ohadii possesses a readily available capacity to utilize a sudden excess of reducing power and carbon for growth and reserve formation, and post-translational redox regulation plays a pivotal role in this rapid response. Frequently the response in C. ohadii deviated from that of model species, reflecting its life history in desert sand crusts. Comparative global and case-specific analyses provided insights into the potential evolutionary role of effective reductant utilization in this extreme resistance of C. ohadii to extreme irradiation.


Assuntos
Chlorella/metabolismo , Proteínas de Algas/metabolismo , Proteínas de Algas/fisiologia , Chlorella/fisiologia , Chlorella/efeitos da radiação , Clima Desértico , Perfilação da Expressão Gênica , Lipidômica , Metabolômica , Oxirredução/efeitos da radiação , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/fisiologia , Proteômica
7.
Plant J ; 101(6): 1269-1286, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31657869

RESUMO

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N6 -methylation of adenosine ribonucleotides (m6 A-RNA) in plant mitochondria. m6 A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m6 A mediates structural switches that affect RNA stability and/or activity. Using m6 A-pulldown/RNA-seq (m6 A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m6 A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m6 A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m6 A modifications, N6 -methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.


Assuntos
Adenosina/metabolismo , Expressão Gênica , Mitocôndrias/metabolismo , Organelas/metabolismo , Plantas/metabolismo , Arabidopsis/metabolismo , Brassica/metabolismo , Regulação da Expressão Gênica de Plantas , Metilação
8.
Environ Microbiol Rep ; 11(5): 621-629, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31390482

RESUMO

Various approaches have been proposed to control/eliminate toxic Microcystis sp. blooms including H2 O2 treatments. Earlier studies showed that pre-exposure of various algae to oxidative stress induced massive cell death when cultures were exposed to an additional H2 O2 treatment. We examined the vulnerability of exponential and stationary-phase Microcystis sp. strain MGK cultures to single and double H2 O2 applications. Stationary cultures show a much higher ability to decompose H2 O2 than younger cultures. Nevertheless, they are more sensitive to an additional H2 O2 dose given 1-6 h after the first one. Transcript analyses following H2 O2 application showed a fast rise in glutathione peroxidase abundance (227-fold within an hour) followed by a steep decline thereafter. Other genes potentially engaged in oxidative stress were far less affected. Metabolic-related genes were downregulated after H2 O2 treatments. Among those examined, the transcript level of prk (encoding phosphoribulose kinase) was the slowest to recover in agreement with the decline in photosynthetic rate revealed by fluorescence measurements. Our findings shed light on the response of Microcystis MGK to oxidative stress suggesting that two consecutive H2 O2 applications of low concentrations are far more effective in controlling Microcystis sp. population than a single dose of a higher concentration.


Assuntos
Peróxido de Hidrogênio/farmacologia , Microcystis/efeitos dos fármacos , Estresse Oxidativo , Microcystis/crescimento & desenvolvimento , Fotossíntese
9.
Metabolites ; 9(6)2019 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-31181869

RESUMO

Aeromonas veronii strain A134 was isolated from Microcystis aeruginosa colonies collected from Lake Kinneret (Sea of Galilee), Israel. The Aeromonas culture media inhibited the growth of M. aeruginosa (strain MGK). The crude extract of a large-scale culture of A. veronii A134 was separated in a few chromatographic steps to yield three new secondary metabolites, 9-chlorolumichrome (1), veronimide (2) and veronipyrazine (3), along with a known lumichrome and several known diketopiperazines. The structures of the new compounds were established by analyses of the data from 1D and 2D NMR experiments and HRMS data of the compounds, as well as a single-crystal x-ray analysis of synthetic 1. The structure elucidation and proposed biogenesis of the new compounds are described below.

10.
Mol Ecol ; 28(9): 2305-2320, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31025457

RESUMO

Cyanobacteria inhabiting desert biological soil crusts must prepare towards dehydration, or their revival after rewetting is severely impaired. The mechanisms involved are unknown but signalling of forthcoming dehydration by dawn illumination was demonstrated. Accurate and reproducible simulation of desert conditions enabled examination of physiological activities and transcript profiles in a model organism, Leptolyngbya ohadii, in response to specific conditions. Exposure to far red light or lack of ground warming during dawn severely reduced revival after rewetting and altered the network of gene expression. The data implicated phytochromes in light and temperature sensing. Many genes were up- or down-regulated before water content decline, while others were strongly affected by the progression of dehydration and desiccation. Transcription continues during the desiccated phase but only barely during early rewetting, although photosynthetic activity was regained. Application of rifampicin with or without a preceding dehydration phase demonstrated that RNA is stabilized/protected during desiccation, possibly by intrinsically disordered proteins. We conclude that increasing light and temperature at dawn activates a network of genes that prepare the cells towards dehydration. Quick resumption of photosynthesis upon rewetting in contrast to the slow change in the transcript profile suggested that in addition to preparing towards dehydration the cells also prepare for forthcoming rewetting, during dehydration. Unravelling the presently unknown function of many responding genes will help to clarify the networks involved.


Assuntos
Cianobactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/efeitos dos fármacos , Desidratação , Clima Desértico , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Luz , Fotossíntese/fisiologia , Rifampina/farmacologia , Microbiologia do Solo , Temperatura , Trealose , Água
11.
Environ Microbiol ; 21(3): 1140-1150, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30761715

RESUMO

Toxic Microcystis spp. blooms constitute a serious threat to water quality worldwide. Aeromonas veronii was isolated from Microcystis sp. colonies collected in Lake Kinneret. Spent Aeromonas media inhibits the growth of Microcystis aeruginosa MGK isolated from Lake Kinneret. The inhibition was much stronger when Aeromonas growth medium contained spent media from MGK suggesting that Aeromonas recognized its presence and produced secondary metabolites that inhibit Microcystis growth. Fractionations of the crude extract and analyses of the active fractions identified several secondary metabolites including lumichrome in Aeromonas media. Application of lumichrome at concentrations as low as 4 nM severely inhibited Microcystis growth. Inactivation of aviH in the lumichrome biosynthetic pathway altered the lumichrome level in Aeromonas and the extent of MGK growth inhibition. Conversely, the initial lag in Aeromonas growth was significantly longer when provided with Microcystis spent media but Aeromonas was able to resume normal growth. The longer was pre-exposure to Microcystis spent media the shorter was the lag phase in Aeromonas growth indicating the presence of, and acclimation to, secondary MGK metabolite(s) the nature of which was not revealed. Our study may help to control toxic Microcystis blooms taking advantage of chemical languages used in the interspecies communication.


Assuntos
Aeromonas veronii/fisiologia , Microcystis/fisiologia , Aeromonas/fisiologia , Antibiose/fisiologia , Meios de Cultura , Lagos/microbiologia , Microcystis/metabolismo
12.
New Phytol ; 221(3): 1303-1316, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30216452

RESUMO

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.


Assuntos
Organismos Aquáticos/enzimologia , Cloroplastos/metabolismo , Diatomáceas/enzimologia , Diatomáceas/fisiologia , Regulação para Baixo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Antioxidantes/metabolismo , Organismos Aquáticos/fisiologia , Glutationa/metabolismo , Metabolômica , Oxirredução , Fotossíntese , Transcriptoma/genética
13.
Curr Biol ; 27(19): R1056-R1057, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-29017037

RESUMO

Desert biological soil crusts (BSC), among the harshest environments on Earth, are formed by the adhesion of soil particles to polysaccharides excreted mainly by filamentous cyanobacteria (see [1] and references therein). These species are the main primary producers in this habitat where they cope with various stressors including frequent hydration-dehydration cycles. Water is mainly provided as early-morning dew, followed by dehydration with rising temperatures and declining relative humidity. Earlier studies focused on community structure and cyanobacterial activities in various BSCs [1,2]. They identified genes present in dehydration-tolerant, but not -sensitive cyanobacteria [3], and suggested that abiotic conditions during natural dehydration (Figure 1A) are critical for the recovery upon rewetting. Inability of Leptolyngbya ohadii, which is abundant in the BSC examined here, to recover after rapid desiccation (Figure 1B) [4] suggested that the cells must prepare themselves toward forthcoming dehydration, but the nature of the signal involved was unknown. We show here that the rising dawn illumination, perceived by photo-sensors, serves as the signal inciting BSC-inhabiting cyanobacteria to prepare for forthcoming dehydration. L. ohadii filaments were exposed to simulated natural conditions from the morning of October 14th 2009, using our environmental chamber that enables accurate reproduction of BSC environment [4] (Supplemental Figure S1A). Samples were withdrawn at specific time points (Figure 1A), followed by RNA extraction and global transcript profiling (accession PRJNA391854). Four hours of dehydration led to up-regulation of 567 genes and down-regulation of 1597 (by more than 2-fold). Since BSC-inhabiting organisms have not been used as genetic models, the functions of 3258 (43.5% of the 7487 L. ohadii genes [3]) are unknown. Nevertheless, a pronounced rise in transcript levels of genes involved in carbon metabolism, transport, osmolyte production, energy dissipation and other cellular activities was observed. On the other hand, a declining transcript abundance for genes involved in light harvesting, photosynthetic metabolism, protein biosynthesis, cell division and other pathways was detected. The analysis unraveled clear distinctions between early- and late-responding genes. Supplemental Table S1 lists the 40 strongest differentially expressed genes verified by RT-qPCR and used in further analyses.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/efeitos da radiação , Dessecação , Regulação da Expressão Gênica/efeitos da radiação , Luz , Proteínas de Bactérias/metabolismo , Ritmo Circadiano , Cianobactérias/genética , Cianobactérias/fisiologia , Clima Desértico
14.
Curr Biol ; 27(16): 2559-2567.e3, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28803869

RESUMO

The factors rate-limiting growth of photosynthetic organisms under optimal conditions are controversial [1-8]. Adaptation to extreme environments is usually accompanied by reduced performance under optimal conditions [9, 10]. However, the green alga Chlorella ohadii, isolated from a harsh desert biological soil crust [11-17], does not obey this rule. In addition to resistance to photodamage [17, 18], it performs the fastest growth ever reported for photosynthetic eukaryotes. A multiphasic growth pattern (very fast growth [phase I], followed by growth retardation [phase II] and additional fast growth [phase III]) observed under constant illumination and temperature indicates synchronization of the algal population. Large physiological changes at transitions between growth phases suggest metabolic shifts. Indeed, metabolome analyses at points along the growth phases revealed large changes in the levels of many metabolites during growth with an overall rise during phase I and decline in phase II. Multivariate analysis of the metabolome data highlighted growth phase as the main factor contributing to observed metabolite variance. The analyses identified putrescine as the strongest predictive metabolite for growth phase and a putative growth regulator. Indeed, extracellular additions of polyamines strongly affected the growth rate in phase I and the growth arrest in phase II, with a marked effect on O2 exchange. Our data implicate polyamines as the signals harmonizing metabolic shifts and suggest that metabolic flexibility enables the immense growth capabilities of C. ohadii. The data provide a new dimension to current models focusing on growth-limiting processes in photosynthetic organisms where the anabolic and catabolic metabolisms must be strictly regulated.


Assuntos
Adaptação Biológica , Chlorella/fisiologia , Clima Desértico , Fotossíntese , Chlorella/crescimento & desenvolvimento , Metaboloma , Solo
15.
Environ Microbiol ; 19(2): 535-550, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27501380

RESUMO

Filamentous cyanobacteria are the main founders and primary producers in biological desert soil crusts (BSCs) and are likely equipped to cope with one of the harshest environmental conditions on earth including daily hydration/dehydration cycles, high irradiance and extreme temperatures. Here, we resolved and report on the genome sequence of Leptolyngbya ohadii, an important constituent of the BSC. Comparative genomics identified a set of genes present in desiccation-tolerant but not in dehydration-sensitive cyanobacteria. RT qPCR analyses showed that the transcript abundance of many of them is upregulated during desiccation in L. ohadii. In addition, we identified genes where the orthologs detected in desiccation-tolerant cyanobacteria differs substantially from that found in desiccation-sensitive cells. We present two examples, treS and fbpA (encoding trehalose synthase and fructose 1,6-bisphosphate aldolase respectively) where, in addition to the orthologs present in the desiccation-sensitive strains, the resistant cyanobacteria also possess genes with different predicted structures. We show that in both cases the two orthologs are transcribed during controlled dehydration of L. ohadii and discuss the genetic basis for the acclimation of cyanobacteria to the desiccation conditions in desert BSC.


Assuntos
Cianobactérias/genética , Cianobactérias/fisiologia , Genoma Bacteriano , Microbiologia do Solo , Solo/química , Água , Aclimatação , Desidratação , Clima Desértico , Regulação Bacteriana da Expressão Gênica , Fotossíntese
16.
BMC Bioinformatics ; 17(1): 261, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27363443

RESUMO

BACKGROUND: With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. RESULTS: The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. CONCLUSIONS: PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.


Assuntos
Sistemas CRISPR-Cas , Genoma Humano , Internet , Humanos
17.
New Phytol ; 210(4): 1229-43, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26853530

RESUMO

Excess illumination damages the photosynthetic apparatus with severe implications with regard to plant productivity. Unlike model organisms, the growth of Chlorella ohadii, isolated from desert soil crust, remains unchanged and photosynthetic O2 evolution increases, even when exposed to irradiation twice that of maximal sunlight. Spectroscopic, biochemical and molecular approaches were applied to uncover the mechanisms involved. D1 protein in photosystem II (PSII) is barely degraded, even when exposed to antibiotics that prevent its replenishment. Measurements of various PSII parameters indicate that this complex functions differently from that in model organisms and suggest that C. ohadii activates a nonradiative electron recombination route which minimizes singlet oxygen formation and the resulting photoinhibition. The light-harvesting antenna is very small and carotene composition is hardly affected by excess illumination. Instead of succumbing to photodamage, C. ohadii activates additional means to dissipate excess light energy. It undergoes major structural, compositional and physiological changes, leading to a large rise in photosynthetic rate, lipids and carbohydrate content and inorganic carbon cycling. The ability of C. ohadii to avoid photodamage relies on a modified function of PSII and the dissipation of excess reductants downstream of the photosynthetic reaction centers. The biotechnological potential as a gene source for crop plant improvement is self-evident.


Assuntos
Chlorella/fisiologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Chlorella/efeitos da radiação , Clima Desértico , Solo , Estresse Fisiológico , Luz Solar
18.
Nature ; 524(7565): 366-9, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26168400

RESUMO

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.


Assuntos
Organismos Aquáticos/metabolismo , Dióxido de Carbono/metabolismo , Diatomáceas/citologia , Diatomáceas/metabolismo , Mitocôndrias/metabolismo , Fotossíntese , Plastídeos/metabolismo , Força Próton-Motriz , Trifosfato de Adenosina/metabolismo , Organismos Aquáticos/citologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Ciclo do Carbono , Diatomáceas/enzimologia , Diatomáceas/genética , Ecossistema , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , NADP/metabolismo , Oceanos e Mares , Oxirredução , Oxirredutases/deficiência , Oxirredutases/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo
19.
Genome Biol ; 16: 102, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-25990474

RESUMO

BACKGROUND: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. RESULTS: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. CONCLUSIONS: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.


Assuntos
Diatomáceas/metabolismo , Código das Histonas , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Cromatina/metabolismo , Diatomáceas/genética , Expressão Gênica , Genômica , Espectrometria de Massas , Nitratos/metabolismo , Nucleossomos/metabolismo
20.
New Phytol ; 202(2): 471-484, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24345283

RESUMO

Chlamydomonas reinhardtii tolerates relatively high H2 O2 levels that induce an array of antioxidant activities. However, rather than rendering the cells more resistant to oxidative stress, the cells become far more sensitive to an additional H2 O2 dose. If H2 O2 is provided 1.5-9 h after an initial dose, it induces programmed cell death (PCD) in the wild-type, but not in the dum1 mutant impaired in the mitochondrial respiratory complex III. This mutant does not exhibit a secondary oxidative burst 4-5 h after the inducing H2 O2 , nor does it activate metacaspase-1 after the second H2 O2 treatment. The intracellular dehydroascorbate level, a product of ascorbate peroxidase, increases under conditions leading to PCD. The addition of dehydroascorbate induces PCD in the wild-type and dum1 cultures, but higher levels are required in dum1 cells, where it is metabolized faster. The application of dehydroascorbate induces the expression of metacaspase-2, which is much stronger than the expression of metacaspase-1. The presence or absence of oxidative stress, in addition to the rise in internal dehydroascorbate, may determine which metacaspase is activated during Chlamydomonas PCD. Cell death is strongly affected by the timing of H2 O2 or dehydroascorbate admission to synchronously grown cultures, suggesting that the cell cycle phase may distinguish cells that perish from those that do not.


Assuntos
Apoptose , Chlamydomonas reinhardtii/metabolismo , Ácido Desidroascórbico/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Ascorbato Peroxidases/metabolismo , Caspases/metabolismo , Ciclo Celular , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/fisiologia , Ácido Desidroascórbico/farmacologia , Peróxido de Hidrogênio/farmacologia , Mutação , Oxirredução
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