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Galactic outflows are believed to play a critical role in the evolution of galaxies by regulating their mass build-up and star formation1. Theoretical models assume bipolar shapes for the outflows that extend well into the circumgalactic medium (CGM), up to tens of kiloparsecs (kpc) perpendicular to the galaxies. They have been directly observed in the local Universe in several individual galaxies, for example, around the Milky Way and M82 (refs. 2,3). At higher redshifts, cosmological simulations of galaxy formation predict an increase in the frequency and efficiency of galactic outflows owing to the increasing star-formation activity4. Galactic outflows are usually of low gas density and low surface brightness and therefore difficult to observe in emission towards high redshifts. Here we present an ultra-deep Multi-Unit Spectroscopic Explorer (MUSE) image of the mean Mg II emission surrounding a sample of galaxies at z ≈ 1 that strongly suggests the presence of outflowing gas on physical scales of more than 10 kpc. We find a strong dependence of the detected signal on the inclination of the central galaxy, with edge-on galaxies clearly showing enhanced Mg II emission along the minor axis, whereas face-on galaxies show much weaker and more isotropic emission. We interpret these findings as supporting the idea that outflows typically have a bipolar cone geometry perpendicular to the galactic disk. We demonstrate that this CGM-scale outflow is prevalent among galaxies with stellar mass M* â³ 109.5Mâ.
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In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.
Assuntos
Epigenoma , Histonas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Histonas/metabolismo , Histonas/genética , Epigênese Genética , Genoma de Planta , Cromatina/metabolismo , Cromatina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Heterocromatina/metabolismo , Heterocromatina/genética , Código das Histonas/genéticaRESUMO
The 3D chromatin organization plays a major role in the control of gene expression. However, our comprehension of the governing principles behind nuclear organization remains incomplete. Particularly, the spatial segregation of loci with similar repressive transcriptional states in plants poses a significant yet poorly understood puzzle. In this study, employing a combination of genetics and advanced 3D genomics approaches, we demonstrated that a redistribution of facultative heterochromatin marks in regions usually occupied by constitutive heterochromatin marks disrupts the 3D genome compartmentalisation. This disturbance, in turn, triggers novel chromatin interactions between genic and transposable element (TE) regions. Interestingly, our results imply that epigenetic features, constrained by genetic factors, intricately mold the landscape of 3D genome organisation. This study sheds light on the profound genetic-epigenetic interplay that underlies the regulation of gene expression within the intricate framework of the 3D genome. Our findings highlight the complexity of the relationships between genetic determinants and epigenetic features in shaping the dynamic configuration of the 3D genome.
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BACKGROUND: Epigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1. PURPOSE: Here, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system. RESULTS: Methylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification. CONCLUSION: Collectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles.
Assuntos
Metilação de DNA , Epigênese Genética , Epigenoma , Genoma de Planta , Solanum lycopersicum , Solanum lycopersicum/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genéticaRESUMO
Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromossomos de Plantas/genética , Metilação de DNA , Meiose/genética , Proteínas de Arabidopsis/genética , Pareamento Cromossômico , Segregação de Cromossomos , Troca Genética , Quebras de DNA de Cadeia Dupla , Elementos de DNA Transponíveis/genética , Técnicas de Inativação de Genes , Plantas Geneticamente ModificadasRESUMO
In plants, cytosine methylation, an epigenetic mark critical for transposon silencing, is maintained over generations by key enzymes that directly methylate DNA and is facilitated by chromatin remodelers, like DECREASE IN DNA METHYLATION1 (DDM1). Short-interfering RNAs (siRNAs) also mediate transposon DNA methylation through a process called RNA-directed DNA methylation (RdDM). In tomato (Solanum lycopersicum), siRNAs are primarily mapped to gene-rich chromosome arms, and not to pericentromeric regions as in Arabidopsis thaliana Tomato encodes two DDM1 genes. To better understand their functions and interaction with the RdDM pathway, we targeted the corresponding genes via the CRISPR/Cas9 technology, resulting in the isolation of Slddm1a and Slddm1b knockout mutants. Unlike the single mutants, Slddm1a Slddm1b double mutant plants display pleiotropic vegetative and reproductive phenotypes, associated with severe hypomethylation of the heterochromatic transposons in both the CG and CHG methylation contexts. The methylation in the CHH context increased for some heterochromatic transposons and conversely decreased for others localized in euchromatin. We found that the number of heterochromatin-associated siRNAs, including RdDM-specific small RNAs, increased significantly, likely limiting the transcriptional reactivation of transposons in Slddm1a Slddm1b Taken together, we propose that the global production of siRNAs and the CHH methylation mediated by the RdDM pathway are restricted to chromosome arms in tomato. Our data suggest that both pathways are greatly enhanced in heterochromatin when DDM1 functions are lost, at the expense of silencing mechanisms normally occurring in euchromatin.
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Proteínas de Plantas/genética , RNA Interferente Pequeno/genética , Solanum lycopersicum/genética , Proteínas de Arabidopsis/genética , Metilação de DNA/genética , Eucromatina/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica/fisiologia , Heterocromatina/genéticaRESUMO
Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Inativação Gênica , RNA Polimerase Dependente de RNA/genética , Proteínas de Arabidopsis/metabolismo , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Genéticos , Plantas Geneticamente Modificadas/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Repressive epigenetic marks, such as DNA and histone methylation, are sometimes located within introns. In Arabidopsis (Arabidopsis thaliana), INCREASE IN BONSAI METHYLATION2 (IBM2), an RNA-binding protein containing a bromo-adjacent homology domain, is required to process functional transcript isoforms of genes carrying intronic heterochromatin. In a genetic screen for suppressors of the ibm2 mutation, we identified FPA, an RNA-binding protein that promotes use of proximal polyadenylation sites in genes targeted by IBM2, including IBM1 encoding an essential H3K9 histone demethylase and the disease resistance gene RECOGNITION OF PERONOSPORA PARASITICA7 Both IBM2 and FPA are involved in the processing of their common mRNA targets: Transcription of IBM2 target genes is restored when FPA is mutated in ibm2 and impaired in transgenic plants overexpressing FPA By contrast, transposons targeted by IBM2 and localized outside introns are not under this antagonistic control. The DNA methylation patterns of some genes and transposons are modified in fpa plants, including the large intron of IBM1, but these changes are rather limited and reversed when the mutant is complemented, indicating that FPA has a restricted role in mediating silencing. These data reveal a complex regulation by IBM2 and FPA pathways in processing mRNAs of genes bearing heterochromatic marks.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Heterocromatina/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Arabidopsis/genética , Metilação de DNA , Elementos de DNA Transponíveis , Genes Supressores , Íntrons , Histona Desmetilases com o Domínio Jumonji/genética , Mutação , Plantas Geneticamente Modificadas , Poliadenilação , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The extent of epigenetic variation is currently well documented, but the number of natural epialleles described so far remains very limited. Determining the relevance of epigenetic changes for natural variation is an important question of research that we investigate by isolating natural epialleles segregating in Arabidopsis recombinant populations. We previously described a genetic incompatibility among Arabidopsis strains based on the silencing of a gene involved in fitness. Here, we isolated a new epiallele resulting from the silencing of a transfer-RNA editing gene in an Arabidopsis accession from the Netherlands (Nok-1). Crosses with the reference accession Col-0 show a complete incompatibility between this epiallele and another locus localized on a different chromosome. We demonstrate that conversion of an unmethylated version of this allele occurs in hybrids, associated with modifications of small RNA populations. These epialleles can also spontaneously revert within the population. Furthermore, we bring evidence that neither METHYLTRANSFERASE 1, maintaining methylation at CGs, nor components of RNA-directed DNA methylation, are key factors for the transmission of the epiallele over generations. This depends only on the self-reinforcing loop between CHROMOMETHYLASE 3 and KRYPTONITE, involving DNA methylated in the CHG context and histone H3 lysine 9 methylation. Our findings reveal a predominant role of this loop in maintaining a natural epiallele.
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Arabidopsis/genética , Metilação de DNA , Epigênese Genética , Retroalimentação Fisiológica , Inativação Gênica , Histonas/metabolismo , Alelos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , Histonas/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
The present analysis revisits the impact of extremely low-frequency magnetic fields (ELF-MF) on melatonin (MLT) levels in human and rat subjects using both a parametric and non-parametric approach. In this analysis, we use 62 studies from review articles. The parametric approach consists of a Bayesian logistic regression (LR) analysis and the non-parametric approach consists of a Support Vector analysis, both of which are robust against spurious/false results. Both approaches reveal a unique well-ordered pattern, and show that human and rat studies are consistent with each other once the MF strength is restricted to cover the same range (with B â² 50 µT). In addition, the data reveal that chronic exposure (longer than â¼22 days) to ELF-MF appears to decrease MLT levels only when the MF strength is below a threshold of ~30 µT ( log B thr [ µ T ] = 1 . 4 - 0 . 4 + 0 . 7 ), i.e., when the man-made ELF-MF intensity is below that of the static geomagnetic field. Studies reporting an association between ELF-MF and changes to MLT levels and the opposite (no association with ELF-MF) can be reconciled under a single framework. Bioelectromagnetics. 2019;40:539-552. © 2019 Bioelectromagnetics Society.
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Campos Magnéticos , Melatonina/metabolismo , Animais , Teorema de Bayes , Humanos , Modelos Logísticos , RatosRESUMO
Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS.
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Interferência de RNA , RNA Antissenso/metabolismo , Transgenes , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Mutação , Estabilidade de RNA , RNA Antissenso/biossíntese , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Plant microRNAs (miRNAs) typically form near-perfect duplexes with their targets and mediate mRNA cleavage. Here, we describe an unconventional miRNA target of miR398 in Arabidopsis, an mRNA encoding the blue copper-binding protein (BCBP). BCBP mRNA carries an miR398 complementary site in its 5'-untranslated region (UTR) with a bulge of six nucleotides opposite to the 5' region of the miRNA. Despite the disruption of a target site region thought to be especially critical for function, BCBP mRNAs are cleaved by ARGONAUTE1 between nucleotides 10th and 11th, opposite to the miRNA, like conventional plant target sites. Levels of BCBP mRNAs are inversely correlated to levels of miR398 in mutants lacking the miRNA, or transgenic plants overexpressing it. Introducing two mutations that disrupt the miRNA complementarity around the cleavage site renders the target cleavage-resistant. The BCBP site functions outside of the context of the BCBP mRNA and does not depend on 5'-UTR location. Reducing the bulge does not interfere with miR398-mediated regulation and completely removing it increases the efficiency of the slicing. Analysis of degradome data and target predictions revealed that the miR398-BCBP interaction seems to be rather unique. Nevertheless, our results imply that functional target sites with non-perfect pairings in the 5' region of an ancient conserved miRNA exist in plants.
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Regiões 5' não Traduzidas , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , RNA de Plantas/química , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pareamento de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , MicroRNAs/química , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5'-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/embriologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Quimera/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Meristema/citologia , Meristema/metabolismo , MicroRNAs/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Estrutura Terciária de Proteína , Plântula/metabolismo , Sementes/citologia , Sementes/metabolismo , Supressão Genética , TransgenesRESUMO
Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here, we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence, and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.
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Células Satélites de Músculo Esquelético , Células-Tronco , Envelhecimento , Animais , Diferenciação Celular , Encapsulamento de Células , Camundongos , Músculo Esquelético/metabolismo , Células-Tronco/metabolismo , TranscriptomaRESUMO
microRNA398 (miR398) is a conserved miRNA of plants that targets two of the three copper/zinc superoxide dismutases (SOD) of Arabidopsis (CSD1 and CSD2) by triggering cleavage or inhibiting translation of their mRNAs. We analysed the transcriptomes of mutants impaired in miR398 production, and found that the mRNAs encoding the copper chaperone for superoxide dismutase (CCS1), which delivers copper to CSD1 and CSD2 apoproteins in different cellular compartments, are undiscovered targets of miR398. We identified the cleavage site in CCS1 mRNAs by 5'-RACE PCR. We further show that both CCS1 protein and mRNA levels are tightly linked to the quantities of miR398, which are themselves dependent on the copper content in the medium. We generated transgenic plants carrying a CCS1 mRNA version resistant to cleavage by miR398, and demonstrated that both CCS1 mRNAs and proteins accumulate in these plants when miR398 is abundant and copper limiting. Moreover, we show that one of the ten ARGONAUTE proteins of Arabidopsis (AGO10) is involved in miR398-directed translational inhibition of CCS1 mRNAs, as CCS1 protein, but not CCS1 mRNAs accumulates in ago10 (zll) mutants. Thus, miR398 mediates the cleavage and translational inhibition of mRNAs encoding CCS1, the chaperone protein that is essential for generating the mature copper/zinc SODs of Arabidopsis. Our results also imply that new targets that have not been identified by computing analyses have yet to be discovered, even for an extensively studied miRNA such as miR398.
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Arabidopsis/genética , Cobre/metabolismo , MicroRNAs/metabolismo , Chaperonas Moleculares/metabolismo , Superóxido Dismutase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genéticaRESUMO
Auxin is a key plant hormone that regulates various aspects of plant development. However, the mechanisms integrating auxin growth effects with stress responses are not fully understood. In this study, we investigated the possible role of calmodulin-binding transcription activator 1 (CAMTA1), an Arabidopsis thaliana calcium/calmodulin-binding transcription activator, in auxin signaling and its responses to different stresses. Plants harboring the AtCAMTA1 promoter fused to the GUS reporter gene revealed cell-specific expression patterns reminiscent of auxin responses. The responsiveness of CAMTA1 to auxin was further assessed by chemical disturbances in polar auxin transport, and by RT-PCR analysis of gene expression of dissected leaf sections from plants exposed to the auxin transport inhibitor NPA. Furthermore, the intensity and cell-specific expression patterns of CAMTA1 changed significantly and differentially on exposure to increasing salt concentrations and heat. Transcriptome analysis of a camta1 T-DNA insertion mutant revealed 63 up-regulated genes, of which 17 are associated with auxin signaling. Finally, analysis of hypocotyl elongation in the presence and absence of auxin revealed that camta1 T-DNA insertion mutants and CAMTA1-repressor lines are hyper-responsive to auxin compared to wild-type seedlings. Thus, CAMTA1 participates in auxin signaling and responds to abiotic stresses.
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Arabidopsis/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Ácidos Indolacéticos/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico , Arabidopsis/metabolismo , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Primers do DNA , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Calmodulin CaM is the most prominent Ca2+ transducer in eukaryotic cells, regulating the activity of numerous proteins with diverse cellular functions. Many features of CaM and its downstream targets are similar in plants and other eukaryotes. However, plants possess a unique set of CaM-related proteins, and several unique CaM target proteins. This review discusses recent progress in identifying plant-specific CaM-binding proteins and their roles in response to biotic and abiotic stresses and development. The review also addresses aspects emerging from recent structural studies of CaM interactions with target proteins relevant to plants.
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Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Cálcio/metabolismo , Transdução de SinaisRESUMO
Gene expression is regulated by transcriptional and post-transcriptional pathways, which are crucial for optimizing gene output and for coordinating cellular programs. MicroRNAs (miRNAs) regulate gene expression networks necessary for proper development, cell viability and stress responses. In plants and animals, 20-24-nt miRNAs direct cleavage and translational repression of partially complementary mRNA target transcripts, through conserved ARGONAUTE proteins. In plants, certain miRNAs indirectly regulate developmental programs by instigating the production of small interfering RNAs (siRNAs). In addition, non-cleavable plant miRNA targets sequester miRNAs, thus regulating miRNA availability. This review summarizes the complexities and diversity of plant miRNA-directed gene regulatory mechanisms and highlights the use of miRNAs for the specific knockdown of gene expression in plants.
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Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , RNA de Plantas/genética , MicroRNAs/metabolismo , Modelos Biológicos , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
In the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T-DNA insertion lines. In addition to the GABI-Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI-Kat lines, we found that the 1'2' bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI-Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T-DNA constructs, transcript read-through from selectable markers is also possible, which again has the potential to be exploited in activation/suppression studies. Finally, we show that, by selecting and characterizing lines in which the T-DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.
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DNA Bacteriano/genética , DNA de Plantas/genética , Mutagênese Insercional/métodos , Elementos Antissenso (Genética) , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Vetores Genéticos , Genoma de Planta , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Transcrição GênicaRESUMO
trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.