RESUMEN
Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Triterpenos , Arabidopsis/genética , Arabidopsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica de las Plantas , Triterpenos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
Plants biosynthesize a broad range of natural products through specialized and species-specific metabolic pathways that are fuelled by core metabolism, together forming a metabolic network. Specialized metabolites have important roles in development and adaptation to external cues, and they also have invaluable pharmacological properties. A growing body of evidence has highlighted the impact of translational, transcriptional, epigenetic and chromatin-based regulation and evolution of specialized metabolism genes and metabolic networks. Here we review the forefront of this research field and extrapolate to medicinal plants that synthetize rare molecules. We also discuss how this new knowledge could help in improving strategies to produce useful plant-derived pharmaceuticals.
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Plantas Medicinales , Plantas Medicinales/genética , Redes y Vías MetabólicasRESUMEN
Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster (BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.
Asunto(s)
Arabidopsis , Triterpenos , Animales , Arabidopsis/genética , Secuencias Reguladoras de Ácidos Nucleicos , Cromatina/genética , Mamíferos/genéticaRESUMEN
Hi-C enables the characterization of the 0conformation of the genome in the three-dimensional nuclear space. This technique has revolutionized our ability to detect interactions between linearly distant genomic sites on a genome-wide scale. Here, we detail a protocol to carry out in situ Hi-C in plants and describe a straightforward bioinformatics pipeline for the analysis of such data, in particular for comparing samples from different organs or conditions.
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Cromatina , Biología Computacional , Núcleo Celular/genética , Biología Computacional/métodos , Genoma , Genómica/métodos , Plantas/genéticaRESUMEN
Chromosomes are the carriers of inheritable traits and define cell function and development. This is not only based on the linear DNA sequence of chromosomes but also on the additional molecular information they are associated with, including the transcription machinery, histone modifications, and their three-dimensional folding. The synergistic application of experimental approaches and computer simulations has helped to unveil how these organizational layers of the genome interplay in various organisms. However, such multidisciplinary approaches are still rarely explored in the plant kingdom. Here, we provide an overview of our current knowledge on plant 3D genome organization and review recent efforts to integrate cutting-edge experiments from microscopy and next-generation sequencing approaches with theoretical models. Building on these recent approaches, we propose possible avenues to extend the application of theoretical modeling in the characterization of the 3D genome organization in plants.
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Cromosomas , Genoma de Planta , Cromatina , Simulación por Computador , Genoma de Planta/genéticaRESUMEN
Chromosomes are dynamic entities in the eukaryotic nucleus. During cell development and in response to biotic and abiotic change, individual sections as well as entire chromosomes re-organise and reposition within the nuclear space. A focal point for these processes is the nuclear envelope (NE) providing both barrier and anchor for chromosomal movement. In plants, positioning of chromosome regions and individual genes at the nuclear envelope has been shown to be associated with distinct transcriptional patterns. Here, we will review recent findings on the interplay between transcriptional activity and gene positioning at the nuclear periphery (NP). We will discuss potential mechanisms of transcriptional regulation at the nuclear envelope and outline future perspectives in this research area.
RESUMEN
The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila. Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O. pumila. It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation. Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC. Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.
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The 3D genome is characterized by a complex organization made of genomic and epigenomic layers with profound implications on gene regulation and cell function. However, the understanding of the fundamental mechanisms driving the crosstalk between nuclear architecture and (epi)genomic information is still lacking. The plant Arabidopsis thaliana is a powerful model organism to address these questions owing to its compact genome for which we have a rich collection of microscopy, chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymer modelling, we investigate the roles of nucleolus formation and epigenomics-driven interactions in shaping the 3D genome of A. thaliana. By validation of several predictions with published data, we demonstrate that self-attracting nucleolar organizing regions and repulsive constitutive heterochromatin are major mechanisms to regulate the organization of chromosomes. Simulations also suggest that interphase chromosomes maintain a partial structural memory of the V-shapes, typical of (sub)metacentric chromosomes in anaphase. Additionally, self-attraction between facultative heterochromatin regions facilitates the formation of Polycomb bodies hosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin are highly-conserved in eukaryotic cells, our findings pave the way for a comprehensive characterization of the generic principles that are likely to shape and regulate the 3D genome in many species.
Asunto(s)
Arabidopsis/genética , Cromosomas de las Plantas , Heterocromatina , Región Organizadora del Nucléolo , Epigenoma , Genoma de Planta , Modelos Moleculares , Polímeros/químicaRESUMEN
While colocalization within a bacterial operon enables coexpression of the constituent genes, the mechanistic logic of clustering of nonhomologous monocistronic genes in eukaryotes is not immediately obvious. Biosynthetic gene clusters that encode pathways for specialized metabolites are an exception to the classical eukaryote rule of random gene location and provide paradigmatic exemplars with which to understand eukaryotic cluster dynamics and regulation. Here, using 3C, Hi-C, and Capture Hi-C (CHi-C) organ-specific chromosome conformation capture techniques along with high-resolution microscopy, we investigate how chromosome topology relates to transcriptional activity of clustered biosynthetic pathway genes in Arabidopsis thaliana Our analyses reveal that biosynthetic gene clusters are embedded in local hot spots of 3D contacts that segregate cluster regions from the surrounding chromosome environment. The spatial conformation of these cluster-associated domains differs between transcriptionally active and silenced clusters. We further show that silenced clusters associate with heterochromatic chromosomal domains toward the periphery of the nucleus, while transcriptionally active clusters relocate away from the nuclear periphery. Examination of chromosome structure at unrelated clusters in maize, rice, and tomato indicates that integration of clustered pathway genes into distinct topological domains is a common feature in plant genomes. Our results shed light on the potential mechanisms that constrain coexpression within clusters of nonhomologous eukaryotic genes and suggest that gene clustering in the one-dimensional chromosome is accompanied by compartmentalization of the 3D chromosome.
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Arabidopsis/genética , Cromosomas de las Plantas/genética , Familia de Multigenes , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Zea mays/genética , Arabidopsis/metabolismo , Cromosomas de las Plantas/metabolismo , Genoma de Planta , Solanum lycopersicum/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/metabolismoRESUMEN
Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, in this commentary we discuss its potential applicability to aspects of TE biology. Based on recent works on the relationship between genome organisation and TE integration, we argue that existing polymer models may be extended to create a predictive framework for the study of TE integration patterns. We suggest that these models may offer orthogonal and generic insights into the integration profiles (or "topography") of TEs across organisms. In addition, we provide simple polymer physics arguments and preliminary molecular dynamics simulations of TEs inserting into heterogeneously flexible polymers. By considering this simple model, we show how polymer folding and local flexibility may generically affect TE integration patterns. The preliminary discussion reported in this commentary is aimed to lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome.
RESUMEN
Plant metabolic gene clusters consist of neighboring genes that are involved in the biosynthesis of secondary or specialized metabolites. The genes within clusters are typically co-regulated, share a common set of chromatin marks, and code for the biosynthesis enzymes of a single metabolic pathway. Here, we describe three essential protocols for the basic analysis of metabolic gene clusters at transcription, histone modification, and metabolite level. The protocols are specified to clusters in the Arabidopsis thaliana genome and are transferable to other plant species.
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Arabidopsis/genética , Cromatina/genética , Familia de Multigenes/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Genoma de Planta/genética , Histonas/genética , Redes y Vías Metabólicas/genéticaRESUMEN
Plant specialized metabolites have ecological functions, yet the presence of numerous uncharacterized biosynthetic genes in plant genomes suggests that many molecules remain unknown. We discovered a triterpene biosynthetic network in the roots of the small mustard plant Arabidopsis thaliana. Collectively, we have elucidated and reconstituted three divergent pathways for the biosynthesis of root triterpenes, namely thalianin (seven steps), thalianyl medium-chain fatty acid esters (three steps), and arabidin (five steps). A. thaliana mutants disrupted in the biosynthesis of these compounds have altered root microbiota. In vitro bioassays with purified compounds reveal selective growth modulation activities of pathway metabolites toward root microbiota members and their biochemical transformation and utilization by bacteria, supporting a role for this biosynthetic network in shaping an Arabidopsis-specific root microbial community.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Microbiota , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Triterpenos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Redes y Vías Metabólicas , Familia de Multigenes , Raíces de Plantas/genéticaRESUMEN
In bacteria, more than half of the genes in the genome are organized in operons. In contrast, in eukaryotes, functionally related genes are usually dispersed across the genome. There are, however, numerous examples of functional clusters of nonhomologous genes for metabolic pathways in fungi and plants. Despite superficial similarities with operons (physical clustering, coordinate regulation), these clusters have not usually originated by horizontal gene transfer from bacteria, and (unlike operons) the genes are typically transcribed separately rather than as a single polycistronic message. This clustering phenomenon raises intriguing questions about the origins of clustered metabolic pathways in eukaryotes and the significance of clustering for pathway function. Here we review metabolic gene clusters from fungi and plants, highlight commonalities and differences, and consider how these clusters form and are regulated. We also identify opportunities for future research in the areas of large-scale genomics, synthetic biology, and experimental evolution.
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Hongos/genética , Redes y Vías Metabólicas/genética , Familia de Multigenes/genética , Plantas/genética , Eucariontes/genética , Eucariontes/metabolismo , Hongos/metabolismo , Transferencia de Gen Horizontal , Genoma/genética , Operón/genética , Plantas/metabolismoAsunto(s)
Botánica , Investigadores , Evolución Biológica , Desarrollo de la Planta , Plantas/genéticaRESUMEN
Contents 771 I. 771 II. 772 III. 780 IV. 781 V. 786 786 References 786 SUMMARY: Plant natural products are of great value for agriculture, medicine and a wide range of other industrial applications. The discovery of new plant natural product pathways is currently being revolutionized by two key developments. First, breakthroughs in sequencing technology and reduced cost of sequencing are accelerating the ability to find enzymes and pathways for the biosynthesis of new natural products by identifying the underlying genes. Second, there are now multiple examples in which the genes encoding certain natural product pathways have been found to be grouped together in biosynthetic gene clusters within plant genomes. These advances are now making it possible to develop strategies for systematically mining multiple plant genomes for the discovery of new enzymes, pathways and chemistries. Increased knowledge of the features of plant metabolic gene clusters - architecture, regulation and assembly - will be instrumental in expediting natural product discovery. This review summarizes progress in this area.
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Genómica , Plantas/genética , Plantas/metabolismo , Productos Biológicos/metabolismo , Vías Biosintéticas , Familia de MultigenesRESUMEN
Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromatina/química , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Histonas/genética , Familia de Multigenes , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Avena/genética , Avena/metabolismo , Cromatina/metabolismo , Mapeo Cromosómico , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Redes y Vías Metabólicas , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantones/genética , Plantones/metabolismo , Triterpenos/metabolismo , Zea mays/genética , Zea mays/metabolismoAsunto(s)
Bacterias/genética , Biología Computacional/normas , Hongos/genética , Familia de Multigenes , Plantas/genética , Biosíntesis de Proteínas , Alcaloides/biosíntesis , Bacterias/metabolismo , Bases de Datos Genéticas , Hongos/metabolismo , Marcadores Genéticos , Cooperación Internacional , Metagenoma , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptidos/metabolismo , Plantas/metabolismo , Policétidos/metabolismo , Polisacáridos/biosíntesis , Terminología como Asunto , Terpenos/metabolismoRESUMEN
Recent discoveries have revealed that the genes for the biosynthesis of a variety of plant specialized metabolites are organized in operon-like clusters within plant genomes. Here we identify a regulatory process that is required for normal expression of metabolic gene clusters in Arabidopsis thaliana. Comparative gene expression analysis of a representative clustered gene was performed in a set of chromatin mutant lines. Subsequently, metabolite levels were analysed by GC-MS and the local chromatin structure was investigated by chromatin immunoprecipitation and nucleosome positioning. We show that the transcript levels of genes within two metabolic clusters are coordinately reduced in an arp6 and h2a.z background. We demonstrate that H2A.Z enrichment in the clusters is positively correlated with active cluster expression. We further show that nucleosome stability within the cluster regions is higher in the arp6 background compared with the wild-type. These results implicate ARP6 and H2A.Z in the regulation of metabolic clusters in Arabidopsis thaliana through localized chromatin modifications that enable the coordinate expression of groups of contiguous genes. These findings shed light on the complex process of cluster regulation, an area that could in the future open up new opportunities for the discovery and manipulation of specialized metabolic pathways in plants.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Inmunoprecipitación de Cromatina , Genoma de Planta , Histonas/metabolismo , Histonas/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/fisiología , Familia de MultigenesRESUMEN
The fungal genus Armillaria is unique in that it is the only natural source of melleolide antibiotics, i.e., protoilludene alcohols esterified with orsellinic acid or its derivatives. This class of natural products is known to exert antimicrobial and cytotoxic effects. Here, we present a refined relationship between the structure and the antimicrobial activity of the melleolides. Using both agar diffusion and broth dilution assays, we identified the Δ(2,4)-double bond of the protoilludene moiety as a key structural feature for antifungal activity against Aspergillus nidulans, Aspergillus flavus, and Penicillium notatum. These findings contrast former reports on cytotoxic activities and may indicate a different mode of action towards susceptible fungi. We also report the isolation and structure elucidation of five melleolides (6'-dechloroarnamial, 6'-chloromelleolide F, 10-hydroxy-5'-methoxy-6'-chloroarmillane, and 13-deoxyarmellides A and B), along with the finding that treatment with an antifungal melleolide impacts transcription of A. nidulans natural product genes.
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Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Armillaria/química , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Antibacterianos/química , Antiinfecciosos/química , Antifúngicos/química , Aspergillus/efectos de los fármacos , Hongos/efectos de los fármacos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Resorcinoles , Sesquiterpenos/química , Relación Estructura-ActividadRESUMEN
Physically linked clusters of genes that encode the enzymatic information for the synthesis of specialized metabolites are a well-established feature of microbial secondary metabolism. In contrast, the biosynthesis of plant specialized metabolites has until recently been thought to be almost exclusively encoded by genes that are randomly scattered in the genome. However, recent reports highlight the growing number of examples of gene clusters for specialized metabolic pathways in plants. Numerous gene clusters that encode for the biosynthesis of different classes of metabolite have now been discovered in a variety of plant species. Comparison of these characterized clusters now enables us to begin to define their salient features and to exploit plant biosynthetic gene clusters for synthetic biology applications.