RESUMEN
In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.
Asunto(s)
Epigenoma , Histonas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Histonas/metabolismo , Histonas/genética , Epigénesis Genética , Genoma de Planta , Cromatina/metabolismo , Cromatina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Heterocromatina/metabolismo , Heterocromatina/genética , Código de Histonas/genéticaRESUMEN
The 3D chromatin organization plays a major role in the control of gene expression. However, our comprehension of the governing principles behind nuclear organization remains incomplete. Particularly, the spatial segregation of loci with similar repressive transcriptional states in plants poses a significant yet poorly understood puzzle. In this study, employing a combination of genetics and advanced 3D genomics approaches, we demonstrated that a redistribution of facultative heterochromatin marks in regions usually occupied by constitutive heterochromatin marks disrupts the 3D genome compartmentalisation. This disturbance, in turn, triggers novel chromatin interactions between genic and transposable element (TE) regions. Interestingly, our results imply that epigenetic features, constrained by genetic factors, intricately mold the landscape of 3D genome organisation. This study sheds light on the profound genetic-epigenetic interplay that underlies the regulation of gene expression within the intricate framework of the 3D genome. Our findings highlight the complexity of the relationships between genetic determinants and epigenetic features in shaping the dynamic configuration of the 3D genome.
RESUMEN
Embedded ß-barrel proteins in the outer envelope membrane mediate most cellular trafficking between the cytoplasm and plastids. Although the TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 75-V (TOC75-V)/OUTER ENVELOPE PROTEIN OF 80 KDA (OEP80) complex has been implicated in the insertion and assembly of ß-barrel proteins in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts, relatively little is known about this process. CRUMPLED LEAF (CRL) encodes a chloroplast outer envelope membrane-localized protein, and its loss-of-function mutation results in pleiotropic defects, including altered plant morphogenesis, growth retardation, suppression of plastid division, and spontaneous light intensity-dependent localized cell death. A suppressor screen conducted on mutagenized crl mutants revealed that a missense mutation in OEP80 suppresses the pleiotropic defects of crl. Furthermore, we found that OEP80 complex formation is compromised in crl. Additionally, we demonstrated that CRL interacts with OEP80 in vivo and that a portion of CRL is present at the same molecular weight as the OEP80 complex. Our results suggest that CRL interacts with OEP80 to facilitate its complex formation. CRL is involved in plastid protein import; therefore, the pleiotropic defects in crl are likely due to the combined effects of decreased plastid protein import and altered membrane integration of ß-barrel proteins in the outer envelope membrane. This study sheds light on the mechanisms that allow ß-barrel protein integration into the plastid outer envelope membrane and the importance of this finding for plant cellular processes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Plastidios/genética , Plastidios/metabolismo , Transporte de ProteínasRESUMEN
The 'Competing interests' statement of this Article has been updated; see accompanying Amendment for further details.
RESUMEN
Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.
Asunto(s)
Arabidopsis , ADN Mitocondrial , Arabidopsis/genética , Arabidopsis/metabolismo , Puntos de Control del Ciclo Celular/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Plantas/genética , Recombinación GenéticaRESUMEN
In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.
RESUMEN
The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.
Asunto(s)
Reparación del ADN/genética , Endospermo/genética , Proteínas de Plantas/genética , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Sistemas CRISPR-Cas , Muerte Celular/genética , Roturas del ADN de Doble Cadena , Replicación del ADN/genética , Endospermo/citología , Inestabilidad Genómica , Mutación , Células Vegetales , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Semillas/citología , Semillas/genética , Semillas/crecimiento & desarrollo , Zea mays/citología , Zea mays/crecimiento & desarrolloRESUMEN
CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Lipoma/tratamiento farmacológico , Lipoma/enzimología , Terapia Molecular Dirigida , Anomalías Musculoesqueléticas/tratamiento farmacológico , Anomalías Musculoesqueléticas/enzimología , Nevo/tratamiento farmacológico , Nevo/enzimología , Tiazoles/uso terapéutico , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/enzimología , Adulto , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Células HeLa , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Masculino , Ratones , Fenotipo , Escoliosis/complicaciones , Escoliosis/tratamiento farmacológico , Sirolimus/uso terapéutico , Síndrome , Neoplasias Vasculares/complicaciones , Neoplasias Vasculares/tratamiento farmacológicoRESUMEN
In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.
Asunto(s)
Arabidopsis , Cucumis melo , Cucurbitaceae , Arabidopsis/genética , Cucumis melo/genética , Cucumis melo/metabolismo , Cucurbitaceae/genética , Regulación de la Expresión Génica de las Plantas/genética , Histonas/metabolismo , Desarrollo de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants' viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants' responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , ADN Polimerasa II/metabolismo , Reparación del ADN , Replicación del ADN , ADN de Plantas/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Polimerasa II/genética , ADN Polimerasa Dirigida por ADN/genética , Inestabilidad Genómica , Genotipo , Meristema/genética , Meristema/fisiología , Modelos Biológicos , Mutación , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Estrés Fisiológico , ADN Polimerasa thetaRESUMEN
The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Homeostasis , Lisina/metabolismo , Ácido Salicílico/metabolismo , Regiones no Traducidas 5'/genética , Acetilación , Arabidopsis/inmunología , Histonas/química , Lisina/química , Inmunidad de la Planta/genética , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Arabidopsis/genética , Complejo del Señalosoma COP9/genética , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Proteínas Asociadas a Microtúbulos/genética , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Puntos de Control del Ciclo Celular/genética , División Celular/genética , Proliferación Celular/genética , Drosophila/genética , Nicotiana/genética , UbiquitinaRESUMEN
Methylations at position N6 of internal adenosines (m6As) are the most abundant and widespread mRNA modifications. These modifications play crucial roles in reproduction, growth, and development by controlling gene expression patterns at the posttranscriptional level. Their function is decoded by readers that share the YTH domain, which forms a hydrophobic pocket that directly accommodates the m6A residues. While the physiological and molecular functions of YTH readers have been extensively studied in animals, little is known about plant readers, even though m6As are crucial for plant survival and development. Viridiplantae contains high numbers of YTH domain proteins. Here, we performed comprehensive evolutionary analysis of YTH domain proteins and demonstrated that they are highly likely to be actual readers with redundant as well as specific functions. We also show that the ECT2 protein from Arabidopsis thaliana binds to m6A-containing RNAs in vivo and that this property relies on the m6A binding pocket carried by its YTH domain. ECT2 is cytoplasmic and relocates to stress granules upon heat exposure, suggesting that it controls mRNA fate in the cytosol. Finally, we demonstrate that ECT2 acts to decode the m6A signal in the trichome and is required for their normal branching through controlling their ploidy levels.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Tricomas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Unión ProteicaRESUMEN
In angiosperms, the gynoecium is the last structure to develop within the flower due to the determinate fate of floral meristem (FM) stem cells. The maintenance of stem cell activity before its arrest at the stage called FM termination affects the number of carpels that develop. The necessary inhibition at this stage of WUSCHEL (WUS), which is responsible for stem cell maintenance, involves a two-step mechanism. Direct repression mediated by the MADS domain transcription factor AGAMOUS (AG), followed by indirect repression requiring the C2H2 zinc-finger protein KNUCKLES (KNU), allow for the complete termination of floral stem cell activity. Here, we show that Arabidopsis thaliana MINI ZINC FINGER2 (AtMIF2) and its homolog in tomato (Solanum lycopersicum), INHIBITOR OF MERISTEM ACTIVITY (SlIMA), participate in the FM termination process by functioning as adaptor proteins. AtMIF2 and SlIMA recruit AtKNU and SlKNU, respectively, to form a transcriptional repressor complex together with TOPLESS and HISTONE DEACETYLASE19. AtMIF2 and SlIMA bind to the WUS and SlWUS loci in the respective plants, leading to their repression. These results provide important insights into the molecular mechanisms governing (FM) termination and highlight the essential role of AtMIF2/SlIMA during this developmental step, which determines carpel number and therefore fruit size.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Flores/metabolismo , Meristema/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Acetilación , Arabidopsis/genética , Secuencia de Bases , Proteínas de Unión al ADN , Flores/genética , Frutas , Regulación de la Expresión Génica de las Plantas , Sitios Genéticos , Meristema/genética , Especificidad de Órganos/genética , Fenotipo , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Polycomb repressive complexes (PRCs) have been traditionally associated with the regulation of developmental processes in various organisms, including higher plants. However, similar to other epigenetic regulators, there is accumulating evidence for their role in the regulation of stress and immune-related pathways. In the current study we show that the PRC1 protein LHP1 is required for the repression of the MYC2 branch of jasmonic acid (JA)/ethylene (ET) pathway of immunity. Loss of LHP1 induces the reduction in H3K27me3 levels in the gene bodies of ANAC019 and ANAC055, as well as some of their targets, leading to their transcriptional upregulation. Consistently, increased expression of these two transcription factors leads to the misregulation of several of their genomic targets. The lhp1 mutant mimics the MYC2, ANAC019, and ANAC055 overexpressers in several of their phenotypes, including increased aphid resistance, abscisic acid (ABA) sensitivity and drought tolerance. In addition, like the MYC2 and ANAC overexpressers, lhp1 displays reduced salicylic acid (SA) content caused by a deregulation of ICS1 and BSMT1, as well as increased susceptibility to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. Together, our results indicate that LHP1 regulates the expression of stress-responsive genes as well as the homeostasis and responses to the stress hormones SA and ABA. This protein emerges as a key chromatin player fine tuning the complex balance between developmental and stress-responsive processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Inmunidad de la Planta/inmunología , Factores de Transcripción/metabolismo , Ácido Abscísico/metabolismo , Animales , Áfidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclopentanos , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Oxilipinas , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/fisiología , Proteínas del Grupo Polycomb , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidad , Ácido Salicílico/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.
Asunto(s)
Arabidopsis/genética , Genoma de Planta/genética , Pirimidinas/metabolismo , Timidina Quinasa/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Daño del ADN , Nucleótidos/metabolismo , Timidina/metabolismo , Timidina Quinasa/genéticaRESUMEN
In recent years, we have witnessed a significant increase in studies addressing the three-dimensional (3D) chromatin organization of the plant nucleus. Important advances in chromatin conformation capture (3C)-derived and related techniques have allowed the exploration of the nuclear topology of plants with large and complex genomes, including various crops. In addition, the increase in their resolution has permitted the depiction of chromatin compartmentalization and interactions at the gene scale. These studies have revealed the highly complex mechanisms governing plant nuclear architecture and the remarkable knowledge gaps in this field. Here we discuss the state-of-the-art in plant chromosome architecture, including our knowledge of the hierarchical organization of the genome in 3D space and regarding other nuclear components. Furthermore, we highlight the existence in plants of topologically associated domain (TAD)-like structures that display striking differences from their mammalian counterparts, proposing the concept of ICONS-intergenic condensed spacers. Similarly, we explore recent advances in the study of chromatin loops and R-loops, and their implication in the regulation of gene activity. Finally, we address the impact that polyploidization has had on the chromatin topology of modern crops, and how this is related to phenomena such as subgenome dominance and biased gene retention in these organisms.
Asunto(s)
Cromatina , Genoma , Animales , Núcleo Celular/genética , Cromatina/genética , Cromosomas de las Plantas , Plantas/genéticaRESUMEN
Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10-15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Plantas/enzimología , Plantas/genética , Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/química , Epigenómica/métodos , Genoma de Planta , Meiosis , Subunidades de Proteína , Origen de Réplica , Transducción de Señal , Estrés FisiológicoRESUMEN
Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ADN Polimerasa II/genética , Replicación del ADN , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , ADN Polimerasa II/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Hidroxiurea/farmacología , Microscopía Fluorescente , Modelos Genéticos , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Plantas Modificadas Genéticamente , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.