RESUMO
RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.
Assuntos
Arabidopsis/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Adenina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Biológicos , Sítio de Iniciação de TranscriçãoRESUMO
Understanding how plants adapt to ambient temperatures has become a major challenge prompted by global climate change. This has led to the identification of several genes regulating the thermal plasticity of plant growth and flowering time. However, the mechanisms accounting for the natural variation and evolution of such developmental plasticity remain mostly unknown. In this study, we determined that natural variation at ICARUS2 (ICA2), which interacts genetically with its homolog ICA1, alters growth and flowering time plasticity in relation to temperature in Arabidopsis (Arabidopsis thaliana). Transgenic analyses demonstrated multiple functional effects for ICA2 and supported the notion that structural polymorphisms in ICA2 likely underlie its natural variation. Two major ICA2 haplogroups carrying distinct functionally active alleles showed high frequency, strong geographic structure, and significant associations with climatic variables related to annual and daily fluctuations in temperature. Genome analyses across the plant phylogeny indicated that the prevalent plant ICA genes encoding two tRNAHis guanylyl transferase 1 units evolved â¼120 million years ago during the early divergence of mono- and dicotyledonous clades. In addition, ICA1/ICA2 duplication occurred specifically in the Camelineae tribe (Brassicaceae). Thus, ICA2 appears to be ubiquitous across plant evolution and likely contributes to climate adaptation through modifications of thermal developmental plasticity in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nucleotidiltransferases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Nucleotidiltransferases/genética , TemperaturaRESUMO
Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.
Assuntos
Arabidopsis/genética , Metilação de DNA , Epigenômica , Regulação da Expressão Gênica de Plantas , Análise por Conglomerados , Biologia Computacional , Ilhas de CpG , Epigênese Genética , Biblioteca Gênica , Genoma de Planta , Heterocromatina/química , Sequenciamento de Nucleotídeos em Larga Escala , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Análise de Sequência de RNA , SoftwareRESUMO
DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.
Assuntos
Metilação de DNA , DNA de Plantas/genética , DNA de Plantas/metabolismo , Picea/genética , Picea/metabolismo , Sequência de Bases , Células Cultivadas , Sequência Conservada , Cycadopsida/genética , Cycadopsida/metabolismo , Genoma de Planta , Filogenia , Picea/embriologia , Folhas de Planta/genética , Folhas de Planta/metabolismoRESUMO
Plants are highly sensitive to environmental changes and even small variations in ambient temperature have severe consequences on their growth and development. Temperature affects multiple aspects of plant development, but the processes and mechanisms underlying thermo-sensitive growth responses are mostly unknown. Here we exploit natural variation in Arabidopsis thaliana to identify and characterize novel components and processes mediating thermo-sensitive growth responses in plants. Phenotypic screening of wild accessions identified several strains displaying pleiotropic growth defects, at cellular and organism levels, specifically at high ambient temperatures. Positional cloning and characterization of the underlying gene revealed that ICARUS1 (ICA1), which encodes a protein of the tRNAHis guanylyl transferase (Thg1) superfamily, is required for plant growth at high temperatures. Transcriptome and gene marker analyses together with DNA content measurements show that ICA1 loss-of-function results in down regulation of cell cycle associated genes at high temperatures, which is linked with a block in G2/M transition and endoreduplication. In addition, plants with mutations in ICA1 show enhanced sensitivity to DNA damage. Characterization of additional strains that carry lesions in ICA1, but display normal growth, shows that alternative splicing is likely to alleviate the deleterious effects of some natural mutations. Furthermore, analyses of worldwide and regional collections of natural accessions indicate that ICA1 loss-of-function has arisen several times independently, and that these occur at high frequency in some local populations. Overall our results suggest that ICA1-mediated-modulation of fundamental processes such as tRNAHis maturation, modify plant growth responses to temperature changes in a quantitative and reversible manner, in natural populations.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proliferação de Células , Regulação da Expressão Gênica de Plantas , Variação Genética , Nucleotidiltransferases/genética , Alelos , Processamento Alternativo , Proteínas de Arabidopsis/metabolismo , Biologia Computacional , Dano ao DNA , Duplicação Gênica , Marcadores Genéticos , Temperatura Alta , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/metabolismo , Folhas de Planta/genética , TranscriptomaRESUMO
DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.
Assuntos
Metilação de DNA , Duplicação Gênica , Manihot/genéticaRESUMO
DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase domains rearranged methylase 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.
Assuntos
Arabidopsis/enzimologia , Metilação de DNA/fisiologia , RNA Polimerases Dirigidas por DNA/genética , Metiltransferases/fisiologia , RNA Mensageiro/genética , Arabidopsis/genética , Genes de Plantas , Metiltransferases/genética , RNA Mensageiro/metabolismoRESUMO
DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA methyltransferase1 (MET1), chromomethylase3 (CMT3), domains rearranged methyltransferase2 (DRM2), and chromomethylase2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases dicer-like 2, 3, and 4, and involved in de novo2 (IDN2) interactors IDN2-like 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, as the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we used coexpression data to identify closely related genes that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-ring-helicase-like1 and -2 (FRG1 and -2) that are putative chromatin modifiers belonging to the SNF2 family of helicase-like proteins. Analysis of genome-wide bisulfite sequencing shows that simultaneous mutations of FRG1 and -2 cause defects in methylation at specific RdDM targeted loci. We also show that FRG1 physically associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as previously unidentified components of the RdDM machinery.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , RNA de Plantas/química , Arabidopsis/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Mutagênese , Mutação , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
In many flowering plants, the transition to flowering is primarily affected by seasonal changes in day length (photoperiod). An inductive photoperiod promotes flowering via synthesis of a floral stimulus, called florigen. In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is an essential component of florigen, which is synthesized in leaf phloem companion cells and is transported through phloem tissue to the shoot apical meristem where floral morphogenesis is initiated. However, the molecular mechanism involved in the long-distance transport of FT protein remains elusive. In this study, we characterized the classic Arabidopsis mutant fe, which is involved in the photoperiodic induction of flowering, and showed that FE encodes a phloem-specific Myb-related protein that was previously reported as ALTERED PHLOEM DEVELOPMENT. Phenotypic analyses of the fe mutant showed that FT expression is reduced in leaf phloem companion cells. In addition, the transport of FT protein from leaves to the shoot apex is impaired in the fe mutant. Expression analyses further demonstrated that FE is also required for transcriptional activation of FLOWERING LOCUS T INTERACTING PROTEIN 1 (FTIP1), an essential regulator for selective trafficking of the FT protein from companion cells to sieve elements. These findings indicate that FE plays a dual role in the photoperiodic induction of flowering: as a transcriptional activator of FT on the one hand, and its transport machinery component, FTIP1, on the other hand. Thus, FE is likely to play a role in regulating FT by coordinating FT synthesis and FT transport in phloem companion cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Flores/metabolismo , Proteínas de Membrana/genética , Mutação , Floema/genética , Floema/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , Fatores de Transcrição/genéticaRESUMO
The timing of flowering initiation depends strongly on the environment, a property termed as the plasticity of flowering. Such plasticity determines the adaptive potential of plants because it provides phenotypic buffer against environmental changes, and its natural variation contributes to evolutionary adaptation. We addressed the genetic mechanisms of the natural variation for this plasticity in Arabidopsis thaliana by analysing a population of recombinant inbred lines derived from Don-0 and Ler accessions collected from distinct climates. Quantitative trait locus (QTL) mapping in four environmental conditions differing in photoperiod, vernalization treatment and ambient temperature detected the folllowing: (i) FLOWERING LOCUS C (FLC) as a large effect QTL affecting flowering time differentially in all environments; (ii) numerous QTL displaying smaller effects specifically in some conditions; and (iii) significant genetic interactions between FLC and other loci. Hence, the variation for the plasticity of flowering is determined by a combination of environmentally sensitive and specific QTL, and epistasis. Analysis of FLC from Don identified a new and more active allele likely caused by a cis-regulatory deletion covering the non-coding RNA COLDAIR. Further characterization of four FLC natural alleles showed different environmental and genetic interactions. Thus, FLC appears as a major modulator of the natural variation for the plasticity of flowering to multiple environmental factors.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Meio Ambiente , Flores/genética , Flores/fisiologia , Variação Genética , Proteínas de Domínio MADS/genética , Alelos , Arabidopsis/crescimento & desenvolvimento , Mapeamento Cromossômico , Ecótipo , Flores/crescimento & desenvolvimento , Genótipo , Endogamia , Locos de Características Quantitativas/genética , Reprodutibilidade dos TestesRESUMO
At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metilação de DNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Northern Blotting , Western Blotting , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , RNA de Plantas/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Transcriptoma/genéticaRESUMO
Roots are essential to terrestrial plants, as their growth and morphology are crucial for plant development. The growth of the roots is affected and regulated by several internal and external environmental signals and metabolic pathways. Among them, chromatin modification plays an important regulatory role. In this study, we explore the potential roles of the histone deacetylase AtHD2D in root development and lay the foundation for further research on the biological processes and molecular mechanisms of AtHD2D in the future. Our study indicates that AtHD2D affects the root tip microenvironment homeostasis by affecting the gene transcription levels required to maintain the root tip microenvironment. In addition, we confirmed that AtHD2D is involved in regulating Arabidopsis lateral root development and further explained the possible role of AtHD2D in auxin-mediated lateral root development. AtHD2D can effectively enhance the resistance of Arabidopsis thaliana to abiotic stress. We believe that AtHD2D is involved in coping with abiotic stress by promoting the development of lateral roots. Overexpression of AtHD2D promotes the accumulation of reactive oxygen species (ROS) in roots, indicating that AtHD2D is also involved in developing lateral roots mediated by ROS. Previous studies have shown that the overexpression of AtHD2D can effectively enhance the resistance of Arabidopsis thaliana to abiotic stress. Based on our data, we believe that AtHD2D participates in the response to abiotic stress by promoting the development of lateral roots. AtHD2D-mediated lateral root development provides new ideas for studying the mechanism of HDAC protein in regulating root development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Histona Desacetilases , Raízes de Plantas , Estresse Fisiológico , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The initiation of flowering in plants is controlled by environmental and endogenous signals. Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Topos Floridos/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Temperatura Baixa , Topos Floridos/genética , Topos Floridos/metabolismo , Dados de Sequência Molecular , Proteína do Retinoblastoma/genética , Fatores de TempoRESUMO
Histone demethylases-both lysine-specific demethylase 1 (LSD1) and Jumonji-C (JmjC) domain-containing proteins-are broadly implicated in the regulation of chromatin-dependent processes. In Arabidopsis thaliana, histone marks directly affect DNA methylation, and mutations in LSD1 homologues show reduced DNA methylation at some loci. We screened transfer DNA mutations in genes encoding JmjC domains for defects in DNA methylation. Mutations in jmj14 result in reduced DNA methylation in non-CG contexts at targets of DRM2 (domains rearranged methyltransferase 2)-mediated RNA-directed DNA methylation (RdDM), which is associated with an increase in H3K4m3. Unlike other components of RdDM, JMJ14 is not required for de novo methylation of a transgene, suggesting that JMJ14 is specifically involved in the maintenance phase of DRM2-mediated RdDM.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Metilação de DNA/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metiltransferases/metabolismo , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Teste de Complementação Genética , Loci Gênicos/genética , Histonas/metabolismo , Lisina/metabolismo , Modelos Biológicos , Mutação/genética , RNA de Plantas/metabolismoRESUMO
Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.
Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Epigênese Genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteômica , Proteínas de ArabidopsisRESUMO
In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Histonas/genética , Chaperonas Moleculares/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Sequenciamento Completo do Genoma/métodosRESUMO
Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C has not been thoroughly characterized. Here, we aim to identify interacting partners of a conserved Arabidopsis SWR1 subunit ACTIN-RELATED PROTEIN 6 (ARP6). We isolate nine predicted components and identify additional interactors implicated in histone acetylation and chromatin biology. One of the interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacts with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 is required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 is preferentially bound to nucleosome-depleted regions at the 5' ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica de Plantas , Histona Acetiltransferases/genética , Histonas/genética , Proteínas dos Microfilamentos/genética , Ligação ProteicaRESUMO
The timing of flower initiation is a highly plastic developmental process. To achieve reproductive success, plants must select the most favourable season to initiate reproductive development; this in turn requires continuous monitoring of environmental factors and a properly response. Environmental factors which change in a predictable fashion along the year, such as light and temperature, are the most relevant in terms of selection of the flowering season. In Arabidopsis and more recently in a few other species, molecular genetic analyses are providing a way to identify the genes involved in the regulation of flowering time. From gene sequences it is possible to develop hypotheses regarding molecular function and to infer some of the molecular mechanisms involved in the environmental regulation of flowering time. In this paper, we summarize recent discoveries concerning the mechanisms which plants use to perceive and respond to major environmental factors (light and temperature) and their different components. We focus mainly on annual plants and especially on Arabidopsis because most of the available molecular and functional data come from this species. However, additional information arising from other plant systems is also considered.
Assuntos
Flores/crescimento & desenvolvimento , Desenvolvimento Vegetal , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Meio Ambiente , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Modelos Biológicos , Fotobiologia , Fotoperíodo , Complexo de Proteínas do Centro de Reação Fotossintética/fisiologia , Plantas/genética , Plantas/efeitos da radiação , TemperaturaRESUMO
Cytosine DNA methylation is an epigenetic mark frequently associated with silencing of genes and transposons. In Arabidopsis, the establishment of cytosine DNA methylation is performed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). DRM2 is guided to target sequences by small interfering RNAs (siRNAs) in a pathway termed RNA-directed DNA methylation (RdDM). We performed a screen for mutants that affect the establishment of DNA methylation by investigating genes that contain predicted RNA-interacting domains. After transforming FWA into 429 T-DNA insertion lines, we assayed for mutants that exhibited a late-flowering phenotype due to hypomethylated, thus ectopically expressed, copies of FWA. A T-DNA insertion line within the coding region of the spliceosome gene SR45 (sr45-1) flowered late after FWA transformation. Additionally, sr45-1 mutants display defects in the maintenance of DNA methylation. DNA methylation establishment and maintenance defects present in sr45-1 mutants are enhanced in dcl3-1 mutant background, suggesting a synergistic cooperation between SR45 and DICER-LIKE3 (DCL3) in the RdDM pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Metilação de DNA , Splicing de RNA , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , MutaçãoRESUMO
De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.