RESUMEN
Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity.
Asunto(s)
Arabidopsis/inmunología , Diacetil/farmacología , Fosfatos/metabolismo , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/inmunología , Raíces de Plantas/inmunología , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Bacterias/inmunología , Bacterias/metabolismo , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Rizosfera , Simbiosis , Compuestos Orgánicos Volátiles/farmacologíaRESUMEN
Phosphorus nutrition has been known for a long time to influence floral transition in plants, but the underlying mechanism is unclear. Arabidopsis phosphate transporter PHOSPHATE1 (PHO1) plays a critical role in phosphate translocation from roots to shoots, but whether and how it regulates floral transition is unknown. Here, we show that knockout mutation of PHO1 delays flowering under both long- and short-day conditions. The late flowering of pho1 mutants can be partially rescued by Pi supplementation in rosettes or shoot apices. Grafting assay indicates that the late flowering of pho1 mutants is a result of impaired phosphate translocation from roots to shoots. Knockout mutation of SPX1 and SPX2, two negative regulators of the phosphate starvation response, partially rescues the late flowering of pho1 mutants. PHO1 is epistatic to PHO2, a negative regulator of PHO1, in flowering time regulation. Loss of PHO1 represses the expression of some floral activators, including FT encoding florigen, and induces the expression of some floral repressors in shoots. Genetic analyses indicate that at least jasmonic acid signaling is partially responsible for the late flowering of pho1 mutants. In addition, we find that rice PHO1;2, the homolog of PHO1, plays a similar role in floral transition. These results suggest that PHO1 integrates phosphorus nutrition and flowering time, and could be used as a potential target in modulating phosphorus nutrition-mediated flowering time in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Flores , Proteínas de Transporte de Fosfato , Fosfatos , Raíces de Plantas , Brotes de la Planta , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fosfatos/metabolismo , Fosfatos/deficiencia , Flores/crecimiento & desarrollo , Flores/genética , Flores/fisiología , Flores/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , Raíces de Plantas/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Transporte de Fosfato/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Transporte BiológicoRESUMEN
DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-binding domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases, and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , Proteínas de Arabidopsis/genética , Sitios de Unión , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
DNA methylation patterns are dynamically controlled by DNA methylation and active DNA demethylation, but the mechanisms of regulation of active DNA demethylation are not well understood. Through forward genetic screens for Arabidopsis mutants showing DNA hypermethylation at specific loci and increased silencing of reporter genes, we identified IDM2 (increased DNA methylation 2) as a regulator of DNA demethylation and gene silencing. IDM2 dysfunction causes DNA hypermethylation and silencing of reporter genes and some endogenous genes. These effects of idm2 mutations are similar to those of mutations in IDM1, a regulator of active DNA demethylation. IDM2 encodes an α-crystallin domain protein in the nucleus. IDM2 and IDM1 interact physically and partially colocalize at discrete subnuclear foci. IDM2 is required for the full activity of H3K18 acetylation but not H3K23 acetylation of IDM1 in planta. Our results suggest that IDM2 functions in active DNA demethylation and in antisilencing by regulating IDM1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Acetilación , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Metilación de ADN , ADN de Plantas , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histonas/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
KEY MESSAGE: An efficient Agrobacterium-mediated transient expression method was developed, which contributed to the functional characterization of the transcription factor CqPHR1, and demonstrates the potential application of gene editing in quinoa. Chenopodium quinoa is a crop expected to ensure global food security in future due to its high resistance to multiple abiotic stresses and nutritional value. We cloned one of the paralogous genes of the Arabidopsis homolog PHR1 (PHOSPHATE STARVATION RESPONSE 1) in quinoa-inbred lines by reverse genetic approach. Overexpression of CqPHR1 driven by the constitutive CaMV 35S promoter in Arabidopsis phr1 mutant can complement its phenotypes, including the induction of phosphate starvation-induced (PSI) genes and anthocyanin accumulation in leaves. By Agrobacterium-mediated gene transient expression, we found that CqPHR1 localized in the nucleus of quinoa cells, and overexpression of CqPHR1 in quinoa cells promoted PSI genes expression, which further revealed the function of CqPHR1 as a transcription factor. We have also shown that the transient expression system can be used to express Cas9 protein in various quinoa-inbred lines and perform effective gene editing in quinoa tissue. The method developed in this study will be useful for verifying the effectiveness of gene-editing systems in quinoa cells and has potential application in the generation of gene-edited quinoa with heritable traits.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Chenopodium quinoa , Agrobacterium/genética , Agrobacterium/metabolismo , Antocianinas/genética , Antocianinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteína 9 Asociada a CRISPR/genética , Chenopodium quinoa/genética , Chenopodium quinoa/metabolismo , Edición Génica , Fosfatos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Desmetilación del ADN , Histonas/metabolismo , Complejos Multiproteicos/metabolismo , Acetilación , Cromatina/metabolismo , Silenciador del Gen , Modelos Biológicos , Mutación/genética , Unión Proteica , Subunidades de Proteína/metabolismoRESUMEN
Plasticity in root system architecture (RSA) allows plants to adapt to changing nutritional status in the soil. Phosphorus availability is a major determinant of crop yield, and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition. Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants, whether and how epigenetic regulatory mechanisms contribute is poorly understood. Here, we report that the Switch defective/sucrose non-fermentable (SWI/SNF) ATPase BRAHMA (BRM) is involved in the local response to phosphate (Pi) starvation. The loss of BRM function induces iron (Fe) accumulation through increased LOW PHOSPHATE ROOT1 (LPR1) and LPR2 expression, reducing primary root length under Pi deficiency. We also demonstrate that BRM recruits the histone deacetylase (HDA) complex HDA6-HDC1 to facilitate histone H3 deacetylation at LPR loci, thereby negatively regulating local Pi deficiency responses. BRM is degraded under Pi deficiency conditions through the 26 S proteasome pathway, leading to increased histone H3 acetylation at the LPR loci. Collectively, our data suggest that the chromatin remodeler BRM, in concert with HDA6, negatively regulates Fe-dependent local Pi starvation responses by transcriptionally repressing the RSA-related genes LPR1 and LPR2 in Arabidopsis thaliana.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Histonas/metabolismo , Cromatina/metabolismo , Fosfatos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fósforo/metabolismo , Regulación de la Expresión Génica de las Plantas , Histona Desacetilasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismoRESUMEN
DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Metilación de ADN/genética , Proteínas Portadoras/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas de Arabidopsis/metabolismo , Silenciador del Gen , ADN/metabolismoRESUMEN
The past decade has witnessed unprecedented development in genome engineering, a process that enables targeted modification of genomes. The identification of sequence-specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system, in particular, has led to precise and efficient introduction of genetic variations into genomes of various organisms. Since the CRISPR/Cas system is highly versatile, cost-effective and much superior to ZFNs and TALENs, its widespread adoption by the research community has been inevitable. In plants, a number of studies have shown that CRISPR/Cas could be a potential tool in basic research where insertion, deletion and/or substitution in the genetic sequence could help answer fundamental questions about plant processes, and in applied research these technologies could help build or reverse-engineer plant systems to make them more useful. In this review article, we summarize technologies for precise editing of genomes with a special focus on the CRISPR/Cas system, highlight the latest developments in the CRISPR/Cas system and discuss the challenges and prospects in using the system for plant biology research.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Genoma de Planta/genéticaRESUMEN
KEY MESSAGE: TPST is involved in fructose signaling to regulate the root development and expression of genes in biological processes including auxin biosynthesis and accumulation in Arabidopsis. Sulfonation of proteins by tyrosine protein sulfotransferases (TPST) has been implicated in many important biological processes in eukaryotic organisms. Arabidopsis possesses a single TPST gene and its role in auxin homeostasis and root development has been reported. Here we show that the Arabidopsis tpst mutants are hypersensitive to fructose. In contrast to sucrose and glucose, fructose represses primary root growth of various ecotypes of Arabidopsis at low concentrations. RNA-seq analysis identified 636 differentially expressed genes (DEGs) in Col-0 seedlings in response to fructose verses glucose. GO and KEGG analyses of the DEGs revealed that fructose down-regulates genes involved in photosynthesis, glucosinolate biosynthesis and IAA biosynthesis, but up-regulates genes involved in the degradation of branched amino acids, sucrose starvation response, and dark response. The fructose responsive DEGs in the tpst mutant largely overlapped with that in Col-0, and most DEGs in tpst displayed larger changes than in Col-0. Interestingly, the fructose up-regulated DEGs includes genes encoding two AtTPST substrate proteins, Phytosulfokine 2 (PSK2) and Root Meristem Growth Factor 7 (RGF7). Synthesized peptides of PSK-α and RGF7 could restore the fructose hypersensitivity of tpst mutant plants. Furthermore, auxin distribution and accumulation at the root tip were affected by fructose and the tpst mutation. Our findings suggest that fructose serves as a signal to regulate the expression of genes involved in various biological processes including auxin biosynthesis and accumulation, and that modulation of auxin accumulation and distribution in roots by fructose might be partly mediated by the TPST substrate genes PSK-α and RGF7.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fructosa/metabolismo , Raíces de Plantas/metabolismo , Sulfotransferasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Glucosa/metabolismo , Ácidos Indolacéticos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meristema/metabolismo , Hormonas Peptídicas/genética , Hormonas Peptídicas/metabolismo , Proteínas de Plantas , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Plantones/crecimiento & desarrollo , Transducción de Señal , Sulfotransferasas/genética , TranscriptomaRESUMEN
In vascular (Arabidopsis thaliana) and non-vascular (Physcomitrella patens) plants, PHOSPHATE 1 (PHO1) homologs play important roles in the acquisition and transfer of phosphate. The tomato genome contains six genes (SlPHO1;1-SlPHO1;6) homologous to AtPHO1. The six proteins have typical characteristics of the plant PHO1 family, such as the three Syg1/Pho81/XPRI (SPX) subdomains in the N-terminal portion and one ERD1/XPR1/SYG1 (EXS) domain in the C-terminal portion. Phylogenetic analysis revealed that the SlPHO1 family is subdivided into three clusters. A pairwise comparison indicated that SlPHO1;1 showed the highest level of sequence identity/similarity (67.39/76.21%) to AtPHO1. SlPHO1;1 deletion mutants induced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 displayed typical phenotypes of Pi starvation, such as decreased shoot fresh weight and increased root fresh weight, therefore having a greater root-to-shoot ratio. Mutants also accumulated more anthocyanin and had more soluble Pi content in the root and less in the shoot. These results indicate that SlPHO1;1 plays an important role in Pi transport in the tomato at seedling stage.
Asunto(s)
Sistemas CRISPR-Cas , Proteínas de Transporte de Fosfato/genética , Fosfatos/fisiología , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/fisiología , Filogenia , Plantas Modificadas Genéticamente , Plantones/genética , Plantones/fisiologíaRESUMEN
The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1/genética , Ácido Abscísico/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas y Péptidos de Choque por Frío/genética , Proteínas y Péptidos de Choque por Frío/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1/biosíntesis , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Transducción de SeñalRESUMEN
DNA methylation is typically regarded as a repressive epigenetic marker for gene expression. Genome-wide DNA methylation patterns in plants are dynamically regulated by the opposing activities of DNA methylation and demethylation reactions. In Arabidopsis, a DNA methylation monitoring sequence (MEMS) in the promoter of the DNA demethylase gene ROS1 functions as a methylstat that senses these opposing activities and regulates genome DNA methylation levels by adjusting ROS1 expression. How DNA methylation in the MEMS region promotes ROS1 expression is not known. Here, we show that several Su(var)3-9 homologs (SUVHs) can sense DNA methylation levels at the MEMS region and function redundantly to promote ROS1 expression. The SUVHs bind to the MEMS region, and the extent of binding is correlated with the methylation level of the MEMS. Mutations in the SUVHs lead to decreased ROS1 expression, causing DNA hypermethylation at more than 1,000 genomic regions. Thus, the SUVHs function to mediate the activation of gene transcription by DNA methylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
In eukaryotic cells, transport of macromolecules across the nuclear envelope is an essential process that ensures rapid exchange of cellular components, including protein and RNA molecules. Chromatin regulators involved in epigenetic control are among the molecules exported across the nuclear envelope, but the significance of this nucleo-cytoplasmic trafficking is not well understood. Here, we use a forward screen to isolate XPO1A (a nuclear export receptor in Arabidopsis) as an anti-silencing factor that protects transgenes from transcriptional silencing. Loss-of-function of XPO1A leads to locus-specific DNA hypermethylation at transgene promoters and some endogenous loci. We found that XPO1A directly interacts with histone deacetylase HDA6 in vivo and that the xpo1a mutation causes increased nuclear retention of HDA6 protein and results in reduced histone acetylation and enhanced transgene silencing. Our results reveal a new mechanism of epigenetic regulation through the modulation of XPO1A-dependent nucleo-cytoplasm partitioning of a chromatin regulator.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Silenciador del Gen , Histona Desacetilasas/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transgenes , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Secuencia de Bases , Metilación de ADN/genética , Sitios Genéticos , Genoma de Planta , Carioferinas/química , Modelos Biológicos , Mutación/genética , Regiones Promotoras Genéticas , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/químicaRESUMEN
De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5' UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.
Asunto(s)
Arabidopsis/genética , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/genética , Secuencia de Bases , Cruzamientos Genéticos , Silenciador del Gen , Proteínas de Transporte de Membrana/genética , Mutación/genética , Proteínas Nucleares/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN de Planta/metabolismo , TransgenesRESUMEN
DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Metilación de ADN/genética , Epigénesis Genética , Proteínas Hierro-Azufre/genética , Proteínas Nucleares/genética , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras GenéticasRESUMEN
DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Histona Demetilasas con Dominio de Jumonji/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/metabolismo , ADN de Plantas/genética , Metanosulfonato de Etilo/química , Silenciador del Gen , Genoma de Planta , Heterocromatina/metabolismo , Histonas/química , Modelos Genéticos , Datos de Secuencia Molecular , Péptidos/química , Poliadenilación , ARN Mensajero/metabolismo , Sulfitos/química , TransgenesRESUMEN
DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3' distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Metilación de ADN/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Heterocromatina/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Arabidopsis/genética , Secuencia de Bases , Northern Blotting , Inmunoprecipitación de Cromatina , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Intrones/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Datos de Secuencia Molecular , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes to cope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated an Arabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almost all aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused by overexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) in both its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-type plants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pi starvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants can be induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genes include several important Pi signaling components and those that are directly involved in Pi transport, mobilization, and distribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other both synergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compelling evidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate the signaling mechanism that controls plant responses to this particular nutritional stress.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Genómica , Fosfatos/deficiencia , Sacarosa/metabolismo , Fosfatasa Ácida/metabolismo , Antocianinas/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Homeostasis , Iones , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación/genética , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantones/metabolismo , Almidón/metabolismoRESUMEN
Phosphorus (P) is obtained by plants as phosphate (Pi) from the soil and low Pi levels affects plant growth and development. Adaptation to low Pi condition entails sensing internal and external Pi levels and translating those signals to molecular and morphophysiological changes in the plant. In this review, we present findings related to local and systemin Pi sensing with focus the molecular mechanisms behind root system architectural changes and the impact of hormones and epigenetic mechanisms affecting those changes. We also present some of the recent advances in the Pi sensing and signaling mechanisms focusing on inositol pyrophosphate InsP8 and its interaction with SPX domain proteins to regulate the activity of the central regulator of the Pi starvation response, PHR.