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Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.
Assuntos
Cromatina , Entropia , Células-Tronco Pluripotentes Induzidas , Cromatina/metabolismo , Cromatina/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Protoplastos/metabolismo , Reprogramação Celular/genética , Histonas/metabolismo , Histonas/genética , Células Vegetais/metabolismo , Epigênese GenéticaRESUMO
The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.
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Cromatina , Histonas , RNA Longo não Codificante , Cromatina/metabolismo , Cromatina/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Histonas/metabolismo , Histonas/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células HeLa , Transcrição Gênica , RNA/metabolismo , RNA/genética , Animais , Regulação da Expressão GênicaRESUMO
The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear shape and gene expression. To understand how the Arabidopsis (Arabidopsis thaliana) nucleoskeleton physically connects to the nuclear periphery in plants, we investigated the Arabidopsis nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. We identified 3 conserved peptide motifs within the N-terminal region of KAKU4, which are required for intermolecular interactions of KAKU4 with itself, interaction with the nucleoskeleton protein CROWDED NUCLEI (CRWN), localization at the nuclear periphery, and nuclear elongation in differentiated tissues. Unexpectedly, we find these motifs to be present also in NUP82 and NUP136, 2 plant-specific nucleoporins from the nuclear pore basket. We further show that NUP82, NUP136, and KAKU4 have a common evolutionary history predating nonvascular land plants with KAKU4 mainly localizing outside the nuclear pore suggesting its divergence from an ancient nucleoporin into a new nucleoskeleton component. Finally, we demonstrate that both NUP82 and NUP136, through their shared N-terminal motifs, interact with CRWN and KAKU4 proteins revealing the existence of a physical continuum between the nuclear pore and the nucleoskeleton in plants.
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Proteínas de Arabidopsis , Arabidopsis , Poro Nuclear/genética , Poro Nuclear/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Motivos de Aminoácidos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Matriz Nuclear/metabolismoRESUMO
While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.
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Proteínas de Arabidopsis , Arabidopsis , Animais , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Telômero/metabolismo , Mamíferos/metabolismoRESUMO
Introduction: Differentiation of spore mother cells marks the somatic-to-reproductive transition in higher plants. Spore mother cells are critical for fitness because they differentiate into gametes, leading to fertilization and seed formation. The female spore mother cell is called the megaspore mother cell (MMC) and is specified in the ovule primordium. The number of MMCs varies by species and genetic background, but in most cases, only a single mature MMC enters meiosis to form the embryo sac. Multiple candidate MMC precursor cells have been identified in both rice and Arabidopsis, so variability in MMC number is likely due to conserved early morphogenetic events. In Arabidopsis, the restriction of a single MMC per ovule, or MMC singleness, is determined by ovule geometry. To look for potential conservation of MMC ontogeny and specification mechanisms, we undertook a morphogenetic description of ovule primordium growth at cellular resolution in the model crop maize. Methods: We generated a collection of 48 three-dimensional (3D) ovule primordium images for five developmental stages, annotated for 11 cell types. Quantitative analysis of ovule and cell morphological descriptors allowed the reconstruction of a plausible developmental trajectory of the MMC and its neighbors. Results: The MMC is specified within a niche of enlarged, homogenous L2 cells, forming a pool of candidate archesporial (MMC progenitor) cells. A prevalent periclinal division of the uppermost central archesporial cell formed the apical MMC and the underlying cell, a presumptive stack cell. The MMC stopped dividing and expanded, acquiring an anisotropic, trapezoidal shape. By contrast, periclinal divisions continued in L2 neighbor cells, resulting in a single central MMC. Discussion: We propose a model where anisotropic ovule growth in maize drives L2 divisions and MMC elongation, coupling ovule geometry with MMC fate.
RESUMO
Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.
Assuntos
Hordeum , Hordeum/genética , Endosperma/genética , Núcleo Celular/genética , Histonas/genética , Centrômero/genéticaRESUMO
Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.
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Núcleo Celular , Cromatina , Humanos , Fluxo de Trabalho , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Proteínas de Fluorescência VerdeRESUMO
BACKGROUND: Elucidating the genetic and molecular control of plant reproduction often requires the deployment of functional approaches based on reverse or forward genetic screens. The loss-of-function of essential genes, however, may lead to plant lethality prior to reproductive development or to the formation of sterile structures before the organ-of-interest can be analyzed. In these cases, inducible approaches that enable a spatial and temporal control of the genetic perturbation are extremely valuable. Genetic induction in reproductive organs, such as the ovule, deeply embedded in the flower, is a delicate procedure that requires both optimization and validation. RESULTS: Here we report on a streamlined procedure enabling reliable induction of gene expression in Arabidopsis ovule and anther tissues using the popular pOP/LhGR Dex-inducible system. We demonstrate its efficiency and reliability using fluorescent reporter proteins and histochemical detection of the GUS reporter gene. CONCLUSION: The pOP/LhGR system allows for a rapid, efficient, and reliable induction of transgenes in developing ovules without compromising developmental progression. This approach opens new possibilities for the functional analysis of candidate regulators in sporogenesis and gametogenesis, which is otherwise affected by early lethality in conventional, stable mutants.
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This Community Resource paper introduces the range of materials developed by the INDEPTH (Impact of Nuclear Domains on Gene Expression and Plant Traits) COST Action made available through the INDEPTH Academy. Recent rapid growth in understanding of the significance of epigenetic controls in plant and crop science has led to a need for shared, high-quality resources, standardization of protocols, and repositories for open access data. The INDEPTH Academy provides a range of masterclass tutorials, standardized protocols, and teaching webinars, together with a rapidly developing repository to support imaging and spatial analysis of the nucleus and deep learning for automated analysis. These resources were developed partly as a response to the COVID-19 pandemic, but also driven by needs and opportunities identified by the INDEPTH community of ~200 researchers in 80 laboratories from 32 countries. This community report outlines the resources produced and how they will be extended beyond the INDEPTH project, but also aims to encourage the wider community to engage with epigenetics and nuclear structure by accessing these resources.
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COVID-19 , Recursos Comunitários , Expressão Gênica , Humanos , Pandemias , Plantas/genéticaRESUMO
In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the female germline precursor differentiates as a single spore mother cell (SMC) as the ovule primordium forms. Here, we explored how organ growth contributes to SMC differentiation. We generated 92 annotated 3D images at cellular resolution in Arabidopsis. We identified the spatio-temporal pattern of cell division that acts in a domain-specific manner as the primordium forms. Tissue growth models uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis revealed that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in the fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry channels reproductive cell fate in the Arabidopsis ovule primordium.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Divisão Celular , Óvulo Vegetal/fisiologia , Arabidopsis/crescimento & desenvolvimento , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Mutação , Óvulo Vegetal/genéticaRESUMO
The evolution of the nucleus is an evolutionary milestone. By enabling genome compartmentalization, it contributes to the fine-tuning of genome functions. The genome is partitioned into functional domains differing in spatial positioning and topological folding at different scales. The rise of '3D Genomics' embracing experimental, theoretical, and modeling approaches allowed the proposal of a multiscale model of the eukaryotic genome, capturing its organizing principles and functionalities. In these efforts, resolving causality remains an important objective. Are positioning and folding the cause or consequence of functional states? This minireview presents emerging answers to this question, borrowing examples from recent studies of the three-dimensional genome in both plants and animals.
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Cromatina , Epigênese Genética , Animais , Núcleo Celular , Epigênese Genética/genética , Eucariotos/genética , Genoma/genéticaRESUMO
Transposable elements (TEs) constitute a large fraction of plant genomes and are mostly present in a transcriptionally silent state through repressive epigenetic modifications, such as DNA methylation. TE silencing is believed to influence the regulation of adjacent genes, possibly as DNA methylation spreads away from the TE. Whether this is a general principle or a context-dependent phenomenon is still under debate, pressing for studying the relationship between TEs, DNA methylation, and nearby gene expression in additional plant species. Here, we used the grass Brachypodium distachyon as a model and produced DNA methylation and transcriptome profiles for 11 natural accessions. In contrast to what is observed in Arabidopsis thaliana, we found that TEs have a limited impact on methylation spreading and that only few TE families are associated with a low expression of their adjacent genes. Interestingly, we found that a subset of TE insertion polymorphisms is associated with differential gene expression across accessions. Thus, although not having a global impact on gene expression, distinct TE insertions may contribute to specific gene expression patterns in B. distachyon.
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Brachypodium/genética , Metilação de DNA , Elementos de DNA Transponíveis , Expressão Gênica , Variação Genética , Genoma de PlantaRESUMO
BACKGROUND: Chromatin provides a tunable platform for gene expression control. Besides the well-studied core nucleosome, H1 linker histones are abundant chromatin components with intrinsic potential to influence chromatin function. Well studied in animals, little is known about the evolution of H1 function in other eukaryotic lineages for instance plants. Notably, in the model plant Arabidopsis, while H1 is known to influence heterochromatin and DNA methylation, its contribution to transcription, molecular, and cytological chromatin organization remains elusive. RESULTS: We provide a multi-scale functional study of Arabidopsis linker histones. We show that H1-deficient plants are viable yet show phenotypes in seed dormancy, flowering time, lateral root, and stomata formation-complemented by either or both of the major variants. H1 depletion also impairs pluripotent callus formation. Fine-scale chromatin analyses combined with transcriptome and nucleosome profiling reveal distinct roles of H1 on hetero- and euchromatin: H1 is necessary to form heterochromatic domains yet dispensable for silencing of most transposable elements; H1 depletion affects nucleosome density distribution and mobility in euchromatin, spatial arrangement of nanodomains, histone acetylation, and methylation. These drastic changes affect moderately the transcription but reveal a subset of H1-sensitive genes. CONCLUSIONS: H1 variants have a profound impact on the molecular and spatial (nuclear) chromatin organization in Arabidopsis with distinct roles in euchromatin and heterochromatin and a dual causality on gene expression. Phenotypical analyses further suggest the novel possibility that H1-mediated chromatin organization may contribute to the epigenetic control of developmental and cellular transitions.
Assuntos
Arabidopsis/genética , Cromatina/química , Histonas/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Epigênese Genética , Eucromatina/química , Regulação da Expressão Gênica de Plantas , Heterocromatina/química , Histonas/genética , Histonas/metabolismo , Mutação , NucleossomosRESUMO
The eukaryotic cell nucleus is a central organelle whose architecture determines genome function at multiple levels. Deciphering nuclear organizing principles influencing cellular responses and identity is a timely challenge. Despite many similarities between plant and animal nuclei, plant nuclei present intriguing specificities. Complementary to molecular and biochemical approaches, 3D microscopy is indispensable for resolving nuclear architecture. However, novel solutions are required for capturing cell-specific, sub-nuclear and dynamic processes. We provide a pointer for utilising high-to-super-resolution microscopy and image processing to probe plant nuclear architecture in 3D at the best possible spatial and temporal resolution and at quantitative and cell-specific levels. High-end imaging and image-processing solutions allow the community now to transcend conventional practices and benefit from continuously improving approaches. These promise to deliver a comprehensive, 3D view of plant nuclear architecture and to capture spatial dynamics of the nuclear compartment in relation to cellular states and responses. Abbreviations: 3D and 4D: Three and Four dimensional; AI: Artificial Intelligence; ant: antipodal nuclei (ant); CLSM: Confocal Laser Scanning Microscopy; CTs: Chromosome Territories; DL: Deep Learning; DLIm: Dynamic Live Imaging; ecn: egg nucleus; FACS: Fluorescence-Activated Cell Sorting; FISH: Fluorescent In Situ Hybridization; FP: Fluorescent Proteins (GFP, RFP, CFP, YFP, mCherry); FRAP: Fluorescence Recovery After Photobleaching; GPU: Graphics Processing Unit; KEEs: KNOT Engaged Elements; INTACT: Isolation of Nuclei TAgged in specific Cell Types; LADs: Lamin-Associated Domains; ML: Machine Learning; NA: Numerical Aperture; NADs: Nucleolar Associated Domains; PALM: Photo-Activated Localization Microscopy; Pixel: Picture element; pn: polar nuclei; PSF: Point Spread Function; RHF: Relative Heterochromatin Fraction; SIM: Structured Illumination Microscopy; SLIm: Static Live Imaging; SMC: Spore Mother Cell; SNR: Signal to Noise Ratio; SRM: Super-Resolution Microscopy; STED: STimulated Emission Depletion; STORM: STochastic Optical Reconstruction Microscopy; syn: synergid nuclei; TADs: Topologically Associating Domains; Voxel: Volumetric pixel.
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Núcleo Celular , Imageamento Tridimensional , Células Vegetais , Animais , Inteligência Artificial , Núcleo Celular/química , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Subunidades Proteicas/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Óvulo Vegetal/genética , Pólen/genética , Ligação Proteica , Sementes/genética , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
"Seeds nourish, seeds unite, seeds endure, seeds defend, seeds travel," explains the science writer Thor Hanson in his book The Triumph of Seeds (2015). The seed is an ultimate product of land plant evolution. The nursing and protective organization of the seed enable a unique parental care of the progeny that has fueled seed plant radiation. Seeds promote dispersal and optimize offspring production and thus reproductive fitness through biological adaptations that integrate environmental and developmental cues. The composite structure of seeds, uniting tissues that originate from three distinct organisms, enables the partitioning of tasks during development, maturation, and storage, while a sophisticated interplay between the compartments allows the fine-tuning of embryonic growth, as well as seed maturation, dormancy, and germination. In this review, we will highlight peculiarities in the development and evolution of the different seed compartments and focus on the molecular mechanisms underlying the interactions between them.
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Evolução Biológica , Germinação , Magnoliopsida/crescimento & desenvolvimento , Proteínas de Plantas/genética , Reprodução , Sementes/crescimento & desenvolvimento , Biologia do Desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Magnoliopsida/genética , Sementes/genéticaRESUMO
Genomic imprinting confers parent-of-origin-specific gene expression, thus non-equivalent and complementary function of parental genomes. As a consequence, genomic imprinting poses an epigenetic barrier to parthenogenesis in sexual organisms. We report aberrant imprinting in Boechera, a genus in which apomicts evolved from sexuals multiple times. Maternal activation of a MADS-box gene, a homolog of which is imprinted and paternally expressed in the sexual relative Arabidopsis, is accompanied by locus-specific DNA methylation changes in apomicts where parental imprinting seems to be relaxed.
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Brassicaceae/genética , Impressão Genômica , Proteínas de Domínio MADS/genética , Partenogênese , Proteínas de Plantas/genética , Evolução Biológica , Metilação de DNA , Epigenômica , Regulação da Expressão Gênica de PlantasRESUMO
The precise location of chromatin domains within the cell nucleus has seen growing recognition in the past decade as an additional mechanism of controlling gene expression in both plants and animals (Dekker et al., 2017). Consequently, international efforts are devoted to understanding the organising principle of this organelle in plants, and notably the nature and the role of functional compartments on gene expression (Graumann et al., 2013; Sotelo-Silveira et al., 2018). The European cooperation 'Impact of Nuclear Domains on Gene Expression and Plant Traits' (INDEPTH) brings together molecular cell biologists, plant physiologists, bioinformaticians, image analysts and computer scientists. They aim to address the question of how nuclear architecture, chromatin organisation and gene expression are connected in plants, particularly in relation to traits of interest such as biomass, reproduction and resistance to pathogens (https://www.brookes.ac.uk/indepth/). The kick-off meeting of the INDEPTH consortium took place in Clermont-Ferrand, France, on 12-14th March 2018, where more than 80 researchers set the agenda for the coming four years of research and collaboration.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , HumanosRESUMO
Chromatin organization in eukaryotes is highly dynamic, playing fundamental roles in regulating diverse nuclear processes including DNA replication, transcription, and repair. Thus, the analysis of chromatin organization is of great importance for the elucidation of chromatin-mediated biological processes. Immunostaining coupled with imaging is one of the most powerful tools for chromatin analysis at the cellular level. However, in plants, it is sometimes technically challenging to apply this method due to the inaccessibility of certain cell types and/or poor penetration of the reagents into plant tissues and cells. To circumvent these limitations, we developed a highly efficient protocol enabling the analysis of chromatin modifications and nuclear organization at the single-cell level with high resolution in whole-mount plant tissues. The main procedure consists of five steps: (1) tissue fixation; (2) dissection and embedding; (3) tissue processing; (4) antibody incubation; and (5) imaging. This protocol has been simplified for the processing of multiple samples without the need for laborious tissue sectioning. Additionally, it preserves cellular morphology and chromatin organization, allowing comparative analyses of chromatin organization between different cell types or developmental stages. This protocol was successfully used for various tissues of different plant species, including Arabidopsis thaliana, Oryza sativa (rice), and Zea mays (maize). Importantly, this method is very useful to analyze poorly accessible tissues, such as female meiocytes, gametophytes, and embryos.
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Arabidopsis/metabolismo , Cromatina/metabolismo , Imunofluorescência/métodos , Oryza/metabolismo , Zea mays/metabolismo , Arabidopsis/citologia , Núcleo Celular , Cromatina/imunologia , Regulação da Expressão Gênica de Plantas , Especificidade de Órgãos , Oryza/citologia , Fixação de Tecidos , Zea mays/citologiaRESUMO
In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.