RESUMEN
In plants, epitranscriptomic mark N6-adenine methylation (m6A) is dynamically regulated in response to environmental cues. However, little is known about m6A dynamics under biotic stresses and their role in environmental adaptation. Additionally, current methodologies limit the investigation of m6A dynamics at single-nucleotide resolution on specific RNA molecules. Using Oxford Nanopore Technology direct RNA sequencing (ONT-DRS) and a neural network model, we show transcript-specific dynamics of m6A modification at single-nucleotide resolution during Hyaloperonospera arabidopsidis (Hpa) infection in Arabidopsis (Arabidopsis thaliana). In wild-type seedlings, pathogen infection causes a significant reduction of global m6A ratios, which corresponds with the activation of m6A-modified transcripts. Defect of m6A deposition in the m6A mutant hakai-1 mimics m6A reduction from Hpa infection at â¼70% of sites, resulting in constitutive overexpression of basal defense genes and enhanced resistance against the pathogen. Our results demonstrate that m6A dynamics impact defense response against Hpa, providing a promising target for future crop improvement strategies.
RESUMEN
Transposable elements (TEs) contribute to plant evolution, development, and adaptation to environmental changes, but the regulatory mechanisms are largely unknown. RNA-directed DNA methylation (RdDM) is 1 TE regulatory mechanism in plants. Here, we identified that novel ARGONAUTE 1 (AGO1)-binding Tudor domain proteins Precocious dissociation of sisters C/E (PDS5C/E) are involved in 24-nt siRNA production to establish RdDM on TEs in Arabidopsis thaliana. PDS5 family proteins are subunits of the eukaryote-conserved cohesin complex. However, the double mutant lacking angiosperm-specific subfamily PDS5C and PDS5E (pds5c/e) exhibited different developmental phenotypes and transcriptome compared with those of the double mutant lacking eukaryote-conserved subfamily PDS5A and PDS5B (pds5a/b), suggesting that the angiosperm-specific PDS5C/E subfamily has a unique function in angiosperm plants. Proteome and imaging analyses revealed that PDS5C/E interact with AGO1. The pds5c/e double mutant had defects in 24-nt siRNA accumulation and CHH DNA methylation on TEs. In addition, some lncRNAs that accumulated in the pds5c/e mutant were targeted by AGO1-loading 21-nt miRNAs and 21-nt siRNAs. These results indicate that PDS5C/E and AGO1 participate in 24-nt siRNA production for RdDM in the cytoplasm. These findings indicate that angiosperm plants evolved a new regulator, the PDS5C/E subfamily, to control the increase in TEs during angiosperm evolution.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Metilación de ADN , ARN Interferente Pequeño , Metilación de ADN/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Regulación de la Expresión Génica de las Plantas , Dominio Tudor/genética , Elementos Transponibles de ADN/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Mutación/genéticaRESUMEN
Transposable elements (TEs) are accumulated in both intergenic and intragenic regions in plant genomes. Intragenic TEs often act as regulatory elements of associated genes and are also co-transcribed with genes, generating chimeric TE-gene transcripts. Despite the potential impact on mRNA regulation and gene function, the prevalence and transcriptional regulation of TE-gene transcripts are poorly understood. By long-read direct RNA sequencing and a dedicated bioinformatics pipeline, ParasiTE, we investigated the transcription and RNA processing of TE-gene transcripts in Arabidopsis thaliana. We identified a global production of TE-gene transcripts in thousands of A. thaliana gene loci, with TE sequences often being associated with alternative transcription start sites or transcription termination sites. The epigenetic state of intragenic TEs affects RNAPII elongation and usage of alternative poly(A) signals within TE sequences, regulating alternative TE-gene isoform production. Co-transcription and inclusion of TE-derived sequences into gene transcripts impact regulation of RNA stability and environmental responses of some loci. Our study provides insights into TE-gene interactions that contributes to mRNA regulation, transcriptome diversity, and environmental responses in plants.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Elementos Transponibles de ADN/genética , ARN Interferente Pequeño/genética , ARN Mensajero/genética , Análisis de Secuencia de ARNRESUMEN
Transposable elements are present in a wide variety of organisms; however, our understanding of the diversity of mechanisms involved in their activation is incomplete. In this study, we analyzed the transcriptional activation of the ONSEN retrotransposon, which is activated by high-temperature stress in Arabidopsis thaliana. We found that its transcription is significantly higher in the Japanese ecotype Kyoto. Considering that transposons are epigenetically regulated, DNA methylation levels were analyzed, revealing that CHH methylation was reduced in Kyoto compared to the standard ecotype, Col-0. A mutation was also detected in the Kyoto CMT2 gene, encoding a CHH methyltransferase, suggesting that it may be responsible for increased expression of ONSEN. CHH methylation is controlled by histone modifications through a self-reinforcing loop between DNA methyltransferase and histone methyltransferase. Analysis of these modifications revealed that the level of H3K9me2, a repressive histone marker for gene expression, was lower in Kyoto than in Col-0. The level of another repressive histone marker, H3K27me1, was decreased in Kyoto; however, it was not impacted in a Col-0 cmt2 mutant. Therefore, in addition to the CMT2 mutation, other factors may reduce repressive histone modifications in Kyoto.
RESUMEN
Mangroves are adapted to harsh environments, such as high ultraviolet (UV) light, low nutrition, and fluctuating salinity in coastal zones. However, little is known about the transcriptomic and epigenomic basis of the resilience of mangroves due to limited available genome resources. We performed a de novo genome assembly and in natura epigenome analyses of the mangrove Bruguiera gymnorhiza, one of the dominant mangrove species. We also performed the first genome-guided transcriptome assembly for mangrove species. The 309 Mb of the genome is predicted to encode 34 403 genes and has a repeat content of 48%. Depending on its growing environment, the natural B. gymnorhiza population showed drastic morphological changes associated with expression changes in thousands of genes. Moreover, high-salinity environments induced genome-wide DNA hypermethylation of transposable elements (TEs) in the B. gymnorhiza. DNA hypermethylation was concurrent with the transcriptional regulation of chromatin modifier genes, suggesting robust epigenome regulation of TEs in the B. gymnorhiza genome under high-salinity environments. The genome and epigenome data in this study provide novel insights into the epigenome regulation of mangroves and a better understanding of the adaptation of plants to fluctuating, harsh natural environments.
Asunto(s)
Rhizophoraceae , Metilación de ADN/genética , Epigenómica , Perfilación de la Expresión Génica , Rhizophoraceae/genética , Salinidad , Árboles/genéticaRESUMEN
Mangroves are salt-tolerant plant species that grow in coastal saline water and are adapted to harsh environmental conditions. In this study, we de novo assembled and functionally annotated the transcriptome of Rhizophora stylosa, the widely distributed mangrove from the largest mangrove family (Rhizophoraceae). The final transcriptome consists of 200,491 unigenes with an average length, and N50 of 912.7 and 1334 base pair, respectively. We then compared the genome-wide expression profiles between the two morphologically distinct natural populations of this species growing under different levels of salinity depending on their distance from the ocean. Among the 200,491 unigenes, 40,253 were identified as differentially expressed between the two populations, while 15,741 and 24,512 were up- and down-regulated, respectively. Functional annotation assigned thousands of upregulated genes in saline environment to the categories related to abiotic stresses such as response to salt-, osmotic-, and oxidative-stress. Validation of those genes may contribute to a better understanding of adaptation in mangroves species. This study reported, for the first time, the transcriptome of R. stylosa, and the dynamic of it in response to salt stress and provided a valuable resource for elucidation of the molecular mechanism underlying the salt stress response in mangroves and other plants that live under stress.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Rhizophoraceae/metabolismo , Tolerancia a la Sal , Estrés Fisiológico , Transcriptoma , Árboles/metabolismo , Adaptación Fisiológica , Perfilación de la Expresión Génica , Proteínas de Plantas/genética , Rhizophoraceae/genética , Rhizophoraceae/crecimiento & desarrollo , Árboles/genética , Árboles/crecimiento & desarrolloRESUMEN
Their high adaptability to difficult coastal conditions makes mangrove trees a valuable resource and an interesting model system for understanding the molecular mechanisms underlying stress tolerance and adaptation of plants to the stressful environmental conditions. In this study, we used RNA sequencing (RNA-Seq) for de novo assembling and characterizing the Bruguiera gymnorhiza (L.) Lamk leaf transcriptome. B. gymnorhiza is one of the most widely distributed mangrove species from the biggest family of mangroves; Rhizophoraceae. The de novo assembly was followed by functional annotations and identification of individual transcripts and gene families that are involved in abiotic stress response. We then compared the genome-wide expression profiles between two populations of B. gymnorhiza, growing under different levels of stress, in their natural habitats. One population living in high salinity environment, in the shore of the Pacific Ocean- Japan, and the other population living about one kilometre farther from the ocean, and next to the estuary of a river; in less saline and more brackish condition. Many genes involved in response to salt and osmotic stress, showed elevated expression levels in trees growing next to the ocean in high salinity condition. Validation of these genes may contribute to future salt-resistance research in mangroves and other woody plants. Furthermore, the sequences and transcriptome data provided in this study are valuable scientific resources for future comparative transcriptome research in plants growing under stressful conditions.
Asunto(s)
Presión Osmótica/efectos de los fármacos , Rhizophoraceae/genética , Estrés Fisiológico/genética , Transcriptoma/genética , Adaptación Fisiológica/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Anotación de Secuencia Molecular , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Rhizophoraceae/crecimiento & desarrollo , Salinidad , Tolerancia a la Sal/genética , Cloruro de Sodio/efectos adversos , ÁrbolesRESUMEN
In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.
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Arabidopsis/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Transcripción Genética , Secuencia de Bases , Secuencia de Consenso/genética , Metilación de ADN/genética , ADN Polimerasa beta/metabolismo , Elementos Transponibles de ADN/genética , Genes de Plantas , Mutación/genética , ARN Polimerasa II/metabolismo , Sitio de Iniciación de la Transcripción , Transcriptoma/genéticaRESUMEN
Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.
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Flores/crecimiento & desarrollo , MicroARNs/metabolismo , Oryza/crecimiento & desarrollo , ARN de Planta/metabolismo , ARN Interferente Pequeño/biosíntesis , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Mutación , Organogénesis de las Plantas/genética , Oryza/genética , Epidermis de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Intronic regions of eukaryotic genomes accumulate many Transposable Elements (TEs). Intronic TEs often trigger the formation of transcriptionally repressive heterochromatin, even within transcription-permissive chromatin environments. Although TE-bearing introns are widely observed in eukaryotic genomes, their epigenetic states, impacts on gene regulation and function, and their contributions to genetic diversity and evolution, remain poorly understood. In this study, we investigated the genome-wide distribution of intronic TEs and their epigenetic states in the Oryza sativa genome, where TEs comprise 35% of the genome. We found that over 10% of rice genes contain intronic heterochromatin, most of which are associated with TEs and repetitive sequences. These heterochromatic introns are longer and highly enriched in promoter-proximal positions. On the other hand, introns also accumulate hypomethylated short TEs. Genes with heterochromatic introns are implicated in various biological functions. Transcription of genes bearing intronic heterochromatin is regulated by an epigenetic mechanism involving the conserved factor OsIBM2, mutation of which results in severe developmental and reproductive defects. Furthermore, we found that heterochromatic introns evolve rapidly compared to non-heterochromatic introns. Our study demonstrates that heterochromatin is a common epigenetic feature associated with actively transcribed genes in the rice genome.
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Genoma de Planta/genética , Heterocromatina/genética , Intrones/genética , Oryza/genética , Transcripción Genética/genética , Cromatina/genética , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Histone proteins are crucial in the study of chromatin dynamics owing to their wide-ranging implications in the regulation of gene expression. Modifications of histones are integral to these regulatory processes in concert with associated proteins, such as transcription factors and coactivators. One of the biochemical techniques available to enhance analysis of histone proteins is chemical derivatization using propionic anhydride. In this protocol, we describe the use of propionylation to efficiently derivatize acid-extracted histones from rice. We also synthesize H3 and H4 tryptic peptides, thus mimicking the nature of derivatized extracted peptides to aid in identification and quantification using targeted-mass spectrometry. Here we make available the masses of the precursor ions and the retention times (RT) of each synthesized peptide. These provide useful information to facilitate histone data analysis. Lastly, we note that we will distribute these synthetic peptides in nanomolar (nM) concentrations to those who wish to utilize them for assays and further experimental studies.
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Histonas/genética , Oryza/genética , Péptidos/genética , Acetilación , Anhídridos/metabolismo , Cromatina/genética , Expresión Génica/genética , Propionatos/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Genomes of animals and plants contain a large number of transposable elements (TEs). TEs often transpose into genic regions, affecting expression of surrounding genes. Intragenic TEs mostly reside in introns, and in much the same way as intergenic TEs, they are targeted by repressive epigenetic marks for transcriptional silencing. Silenced intragenic TEs generally co-repress expression of associated genes, while in some cases they significantly enhance splicing and transcript elongation. Genomes have evolved molecular mechanisms that allow the presence of silenced TEs within transcriptionally permissive chromatin environments. Epigenetic modulation of intragenic TEs often contributes to gene regulation, phenotypic expression, and genome evolution.
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Elementos Transponibles de ADN/genética , Epigénesis Genética/genética , Animales , Humanos , Plantas/genética , Plantas/metabolismoRESUMEN
Defense mechanisms of plant genomes can epigenetically inactivate repetitive sequences and exogenous transgenes. Loss of mutant phenotypes in intronic T-DNA insertion lines by interaction with another T-DNA locus, termed T-DNA suppression, has been observed in Arabidopsis thaliana, although the molecular basis of establishment and maintenance of T-DNA suppression is poorly understood. Here we show that maintenance of T-DNA suppression requires heterochromatinisation of T-DNA sequences and the nuclear proteins, INCREASED IN BONSAI METHYLATION 2 (IBM2) and ENHANCED DOWNY MILDEW 2 (EDM2), which prevent ectopic 3' end processing of mRNA in atypically long introns containing T-DNA sequences. Initiation of T-DNA suppression is mediated by the canonical RdDM pathway after hybridisation of two T-DNA strains, accompanied by DNA hypermethylation of T-DNA sequences in the F1 generation. Our results reveal the presence of a genome surveillance mechanism through genome hybridisation that masks repetitive DNAs intruding into transcription units.
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Arabidopsis/genética , Epigénesis Genética , Intrones , Transgenes , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Immune recognition of pathogen-associated molecular patterns or effectors leads to defense activation at the pathogen challenged sites. This is followed by systemic defense activation at distant non-challenged sites, termed systemic acquired resistance (SAR). These inducible defenses are accompanied by extensive transcriptional reprogramming of defense-related genes. SAR is associated with priming, in which a subset of these genes is kept at a poised state to facilitate subsequent transcriptional regulation. Transgenerational inheritance of defense-related priming in plants indicates the stability of such primed states. Recent studies have revealed the importance and dynamic engagement of epigenetic mechanisms, such as DNA methylation and histone modifications that are closely linked to chromatin reconfiguration, in plant adaptation to different biotic stresses. Herein we review current knowledge regarding the biological significance and underlying mechanisms of epigenetic control for immune responses in plants. We also argue for the importance of host transposable elements as critical regulators of interactions in the evolutionary "arms race" between plants and pathogens.
RESUMEN
Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively "turn on" TEs when stressed.
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Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Retroelementos , Adaptación Fisiológica , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilación de ADN , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Cloruro de Sodio , Estrés FisiológicoRESUMEN
DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation.
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Metilación de ADN/genética , ADN Intergénico/genética , ADN de Plantas/genética , Plantas/genéticaRESUMEN
Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3' regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.
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Proteínas de Arabidopsis/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Retroalimentación Fisiológica , Genoma de Planta , Factores de Transcripción/genética , Arabidopsis , Proteínas de Arabidopsis/biosíntesis , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Citosina , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/biosíntesis , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Mutación , Factores de Transcripción/biosíntesisRESUMEN
Genomes of higher eukaryotes, including plants, contain numerous transposable elements (TEs), that are often silenced by epigenetic mechanisms, such as histone modifications and DNA methylation. Although TE silencing adversely affects expression of nearby genes, recent studies reveal the presence of intragenic TEs marked by repressive heterochromatic epigenetic marks within transcribed genes. However, even for the well-studied plant model Arabidopsis thaliana, the abundance of intragenic TEs, how they are epigenetically regulated, and their potential impacts on host gene expression, remain unexplored. In this study, we comprehensively analyzed genome-wide distribution and epigenetic regulation of intragenic TEs in A. thaliana. Our analysis revealed that about 3% of TEs are located within gene bodies, dominantly at intronic regions. Most of them are shorter and less methylated than intergenic TEs, but they are still targeted by RNA-directed DNA methylation-dependent and independent pathways. Surprisingly, the heterochromatic epigenetic marks at TEs are maintained within actively transcribed genes. Moreover, the heterochromatic state of intronic TEs is critical for proper transcription of associated genes. Our study provides the first insight into how intragenic TEs affect the transcriptional landscape of the A. thaliana genome, and suggests the importance of epigenetic mechanisms for regulation of TEs within transcriptional gene units.