RESUMO
Chromatin is a dynamic platform within which gene expression is controlled by epigenetic modifications, notably targeting amino acid residues of histone H3. Among them is lysine 27 of H3 (H3K27), the trimethylation of which by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatiotemporal patterns of key developmental genes. H3K27 is also subjected to acetylation and is found at sites of active transcription. Most information on the function of histone residues and their associated modifications in plants was obtained from studies of loss-of-function mutants for the complexes that modify them. To decrypt the genuine function of H3K27, we expressed a non-modifiable variant of H3 at residue K27 (H3.3K27A ) in Arabidopsis, and developed a multi-scale approach combining in-depth phenotypical and cytological analyses, with transcriptomics and metabolomics. We uncovered that the H3.3K27A variant causes severe developmental defects, part of them are reminiscent of PRC2 mutants, part of them are new. They include early flowering, increased callus formation and short stems with thicker xylem cell layer. This latest phenotype correlates with mis-regulation of phenylpropanoid biosynthesis. Overall, our results reveal novel roles of H3K27 in plant cell fates and metabolic pathways, and highlight an epigenetic control point for elongation and lignin composition of the stem.
Assuntos
Arabidopsis , Histonas , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Lisina/metabolismo , Lignina/metabolismo , Metilação , Epigênese Genética , Genes Controladores do DesenvolvimentoRESUMO
An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of "second generation", chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark.
Assuntos
Biotecnologia/métodos , Cromatina/genética , Epigênese Genética , Epigenômica/métodos , Edição de Genes/métodos , Plantas/genética , Animais , Cromatina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , HumanosRESUMO
The ATP-dependent Switch/Sucrose non-fermenting (SWI/SNF) chromatin remodeling complex (CRC) regulates the transcription of many genes by destabilizing interactions between DNA and histones. In plants, BRAHMA (BRM), one of the two catalytic ATPase subunits of the complex, is the closest homolog of the yeast and animal SWI2/SNF2 ATPases. We summarize here the advances describing the roles of BRM in plant development as well as its recently reported chromatin-independent role in pri-miRNA processing in vitro and in vivo. We also enlighten the roles of plant-specific partners that physically interact with BRM. Three main types of partners can be distinguished: (i) DNA-binding proteins such as transcription factors which mostly cooperate with BRM in developmental processes, (ii) enzymes such as kinases or proteasome-related proteins that use BRM as substrate and are often involved in response to abiotic stress, and (iii) an RNA-binding protein which is involved with BRM in chromatin-independent pri-miRNA processing. This overview contributes to the understanding of the central position occupied by BRM within regulatory networks controlling fundamental biological processes in plants.
Assuntos
Adenosina Trifosfatases , Proteínas de Plantas , Plantas , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Plantas/genética , Proteólise , Precursores de RNA/biossíntese , Precursores de RNA/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase Ð hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Elementos Facilitadores Genéticos , Flores/fisiologia , Imunoprecipitação da Cromatina , Genes de Plantas , Estudo de Associação Genômica Ampla , Histonas/química , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNARESUMO
Transcription factors (TFs) are key cellular components that control gene expression. They recognize specific DNA sequences, the TF binding sites (TFBSs), and thus are targeted to specific regions of the genome where they can recruit transcriptional co-factors and/or chromatin regulators to fine-tune spatiotemporal gene regulation. Therefore, the identification of TFBSs in genomic sequences and their subsequent quantitative modeling is of crucial importance for understanding and predicting gene expression. Here, we review how TFBSs can be determined experimentally, how the TFBS models can be constructed in silico, and how they can be optimized by taking into account features such as position interdependence within TFBSs, DNA shape, and/or by introducing state-of-the-art computational algorithms such as deep learning methods. In addition, we discuss the integration of context variables into the TFBS modeling, including nucleosome positioning, chromatin states, methylation patterns, 3D genome architectures, and TF cooperative binding, in order to better predict TF binding under cellular contexts. Finally, we explore the possibilities of combining the optimized TFBS model with technological advances, such as targeted TFBS perturbation by CRISPR, to better understand gene regulation, evolution, and plant diversity.
Assuntos
Fatores de Transcrição/metabolismo , Algoritmos , Sítios de Ligação , Biologia Computacional/métodos , Flores/metabolismo , Regulação da Expressão Gênica/fisiologia , Ligação ProteicaRESUMO
Targeted changes in chromatin state at thousands of genes are central to eukaryotic development. RELATIVE OF EARLY FLOWERING 6 (REF6) is a Jumonji-type histone demethylase that counteracts Polycomb repressive complex 2 (PRC2)-mediated gene silencing in plants and was reported to select its binding sites in a direct, sequence-specific manner1-3. Here we show that REF6 and its two close paralogues determine spatial 'boundaries' of the repressive histone H3K27me3 mark in the genome and control the tissue-specific release from PRC2-mediated gene repression. Targeted mutagenesis revealed that these histone demethylases display pleiotropic, redundant functions in plant development, several of which depend on trans factor-mediated recruitment. Thus, Jumonji-type histone demethylases restrict repressive chromatin domains and contribute to tissue-specific gene activation via complementary targeting mechanisms.
Assuntos
Arabidopsis/metabolismo , Histona Desmetilases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Histonas/metabolismo , Filogenia , Complexo Repressor Polycomb 2 , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Chromatin modifiers and remodelers are involved in generating dynamic changes at the chromatin, which allow differential and specific readouts of the genome. While genetic evidence indicates that several chromatin factors play a key role in controlling basic developmental programs for inflorescence and flower morphogenesis, it remained unknown until recently how they exert their specificity toward gene expression, both temporally and spatially. An emerging topic is the recruitment or eviction of chromatin factors through the activity of sequence-specific DNA-binding domains, present in the chromatin factors themselves or in partnering transcription factors. Here we summarize recent progress that has been made in this regard in the model plant Arabidopsis thaliana. We further outline the different possible modes through which chromatin complexes specifically target genes involved in flower development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromatina/metabolismo , Flores/crescimento & desenvolvimento , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Fatores de Transcrição/metabolismoRESUMO
The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.
Assuntos
Proteína AGAMOUS de Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Flores/fisiologia , Proteínas de Homeodomínio/metabolismo , Meristema/fisiologia , Fatores de Transcrição/metabolismo , Proteína AGAMOUS de Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Mutação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Multimerização Proteica , Fatores de Transcrição/genéticaRESUMO
Covalent histone modifications and their effects on chromatin state and accessibility play a key role in the regulation of gene expression in eukaryotes. To gain insights into their functions during plant growth and development, the distribution of histone modifications can be analyzed at a genome-wide scale through chromatin immunoprecipitation assays followed by sequencing of the isolated genomic DNA. Here, we present a protocol for systematic analysis of the distribution and dynamic changes of selected histone modifications, during flower development in the model plant Arabidopsis thaliana. This protocol utilizes a previously established floral induction system to synchronize flower development, which allows the collection of sufficient plant material for analysis by genomic technologies. In this chapter, we describe how to use this system to study, from the same set of samples, chromatin and transcriptome dynamics during early stages of flower formation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Imunoprecipitação da Cromatina/métodos , Flores/crescimento & desenvolvimento , Histonas/metabolismo , Análise de Sequência de DNA/métodos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Flores/genética , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Código das HistonasRESUMO
The shoot apical meristem (SAM) of angiosperm plants is a small, highly organized structure that gives rise to all above-ground organs. The SAM is divided into three functional domains: the central zone (CZ) at the SAM tip harbors the self-renewing pluripotent stem cells and the organizing center, providing daughter cells that are continuously displaced into the interior rib zone (RZ) or the surrounding peripheral zone (PZ), from which organ primordia are initiated. Despite the constant flow of cells from the CZ into the RZ or PZ, and cell recruitment for primordium formation, a stable balance is maintained between the distinct cell populations in the SAM. Here we combined an in-depth phenotypic analysis with a comparative RNA-Seq approach to characterize meristems from selected combinations of clavata3 (clv3), jabba-1D (jba-1D) and erecta (er) mutants of Arabidopsis thaliana We demonstrate that CLV3 restricts meristem expansion along the apical-basal axis, whereas class III HD-ZIP and ER pathways restrict meristem expansion laterally, but in distinct and possibly perpendicular orientations. Our k-means analysis reveals that clv3, jba-1D/+ and er lead to meristem enlargement by affecting different aspects of meristem function; for example, clv3 displays an increase in the stem cell population, whereas jba-1D/+ er exhibits an increase in mitotic activity and in the meristematic cell population. Our analyses demonstrate that a combined genetic and mRNA-Seq comparative approach provides a precise and sensitive method to identify cell type-specific transcriptomes in a small structure, such as the SAM.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Meristema/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Arabidopsis/genética , Diferenciação Celular , Proliferação de Células , Meristema/citologia , MicroRNAs/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , Transdução de Sinais/fisiologiaRESUMO
Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Meristema/crescimento & desenvolvimento , Morfogênese/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Ensaio de Desvio de Mobilidade Eletroforética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Folhas de Planta/crescimento & desenvolvimento , Caules de Planta/crescimento & desenvolvimento , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Células-Tronco/citologia , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Redes Reguladoras de Genes , Genes de Plantas , Arabidopsis/genética , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , RNA de Plantas/genéticaRESUMO
BACKGROUND AND AIMS: The morphological variability of the flower in angiosperms, combined with its relatively simple structure, makes it an excellent model to study cell specification and the establishment of morphogenetic patterns. Flowers are the products of floral meristems, which are determinate structures that generate four different types of floral organs before terminating. The precise organization of the flower in whorls, each defined by the identity and number of organs it contains, is controlled by a multi-layered network involving numerous transcriptional regulators. In particular, the AGAMOUS (AG) MADS domain-containing transcription factor plays a major role in controlling floral determinacy in Arabidopsis thaliana in addition to specifying reproductive organ identity. This study aims to characterize the genetic interactions between the ULTRAPETALA1 (ULT1) and LEAFY (LFY) transcriptional regulators during flower morphogenesis, with a focus on AG regulation. METHODS: Genetic and molecular approaches were used to address the question of redundancy and reciprocal interdependency for the establishment of flower meristem initiation, identity and termination. In particular, the effects of loss of both ULT1 and LFY function were determined by analysing flower developmental phenotypes of double-mutant plants. The dependency of each factor on the other for activating developmental genes was also investigated in gain-of-function experiments. KEY RESULTS: The ULT1 and LFY pathways, while both activating AG expression in the centre of the flower meristem, functioned independently in floral meristem determinacy. Ectopic transcriptional activation by ULT1 of AG and AP3, another gene encoding a MADS domain-containing flower architect, did not depend on LFY function. Similarly, LFY did not require ULT1 function to ectopically determine floral fate. CONCLUSIONS: The results indicate that the ULT1 and LFY pathways act separately in regulating identity and determinacy at the floral meristem. In particular, they independently induce AG expression in the centre of the flower to terminate meristem activity. A model is proposed whereby these independent contributions bring about a switch at the AG locus from an inactive to an active transcriptional state at the correct time and place during flower development.
Assuntos
Proteína AGAMOUS de Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Alelos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Flores/fisiologia , Genes Reporter , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Modelos Biológicos , Mutação , Organogênese Vegetal , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Fatores de Transcrição/metabolismoRESUMO
In plants, environment-adaptable organogenesis extends throughout the lifespan, and iterative development requires repetitive rounds of activation and repression of several sets of genes. Eukaryotic genome compaction into chromatin forms a physical barrier for transcription; therefore, induction of gene expression requires alteration in chromatin structure. One of the present great challenges in molecular and developmental biology is to understand how chromatin is brought from a repressive to permissive state on specific loci and in a very specific cluster of cells, as well as how this state is further maintained and propagated through time and cell division in a cell lineage. In this review, we report recent discoveries implementing our knowledge on chromatin dynamics that modulate developmental gene expression. We also discuss how new data sets highlight plant specificities, likely reflecting requirement for a highly dynamic chromatin.
Assuntos
Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal , Plantas/genética , Genes de Plantas , Histonas/genética , Histonas/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Ativação TranscricionalRESUMO
In plants, the shoot apical meristem (SAM) serves as a reservoir of pluripotent stem cells from which all above ground organs originate. To sustain proper growth, the SAM must maintain homeostasis between the self-renewal of pluripotent stem cells and cell recruitment for lateral organ formation. At the core of the network that regulates this homeostasis in Arabidopsis are the WUSCHEL (WUS) transcription factor specifying stem cell fate and the CLAVATA (CLV) ligand-receptor system limiting WUS expression. In this study, we identified the ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Homeodomínio/metabolismo , Meristema/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteína AGAMOUS de Arabidopsis/metabolismo , Biologia Computacional , Primers do DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Hibridização In Situ , Meristema/citologia , Microscopia Eletrônica de Varredura , Mutagênese , Brotos de Planta/citologia , Plantas Geneticamente Modificadas , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genéticaRESUMO
The epigenetic regulation of gene expression is critical for ensuring the proper deployment and stability of defined genome transcription programs at specific developmental stages. The cellular memory of stable gene expression states during animal and plant development is mediated by the opposing activities of Polycomb group (PcG) factors and trithorax group (trxG) factors. Yet, despite their importance, only a few trxG factors have been characterized in plants and their roles in regulating plant development are poorly defined. In this work, we report that the closely related Arabidopsis trxG genes ULTRAPETALA1 (ULT1) and ULT2 have overlapping functions in regulating shoot and floral stem cell accumulation, with ULT1 playing a major role but ULT2 also making a minor contribution. The two genes also have a novel, redundant activity in establishing the apicalbasal polarity axis of the gynoecium, indicating that they function in differentiating tissues. Like ULT1 proteins, ULT2 proteins have a dual nuclear and cytoplasmic localization, and the two proteins physically associate in planta. Finally, we demonstrate that ULT1 and ULT2 have very similar overexpression phenotypes and regulate a common set of key development target genes, including floral MADS-box genes and class I KNOX genes. Our results reveal that chromatin remodeling mediated by the ULT1 and ULT2 proteins is necessary to control the development of meristems and reproductive organs. They also suggest that, like their animal counterparts, plant trxG proteins may function in multi-protein complexes to up-regulate the expression of key stage- and tissue-specific developmental regulatory genes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Desenvolvimento Vegetal/genética , Fatores de Transcrição/genética , Arabidopsis/anatomia & histologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Polaridade Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Inflorescência/anatomia & histologia , Inflorescência/genética , Inflorescência/ultraestrutura , Meristema/anatomia & histologia , Meristema/genética , Meristema/ultraestrutura , Mutação/genética , Tamanho do Órgão/genética , Fenótipo , Plantas Geneticamente Modificadas , Ligação Proteica/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Flores , Proteínas de Domínio MADS/metabolismo , Arabidopsis/metabolismo , Cromatografia de Afinidade , Espectrometria de MassasRESUMO
In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/química , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Teste de Complementação Genética , Metilação , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estabilidade de RNA , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Alinhamento de SequênciaRESUMO
The shoot apical meristem of Arabidopsis thaliana contains a reservoir of pluripotent stem cells that functions as a continuous source of new cells for organ formation during development. The SAM forms during embryogenesis, when it becomes stratified into specific cell layers and zones that can be delineated based on morphological and molecular criteria. The primary SAM produces all the aerial structures of the adult plant, and alterations in SAM organization or function can have profound effects on vegetative and reproductive plant morphology. Such SAM-specific defects can be identified, evaluated, and quantified using specialized microscopic and histological techniques.