RESUMEN
Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.
Asunto(s)
Oldenlandia , Oldenlandia/química , Transcriptoma , Metabolómica , Genómica , Ácido UrsólicoRESUMEN
While the structures of plant primary metabolic pathways are generally well defined and highly conserved across species, those defining specialized metabolism are less well characterized and more highly variable across species. In this study, we investigated polyphenolic metabolism in the lycopersicum complex by characterizing the underlying biosynthetic and decorative reactions that constitute the metabolic network of polyphenols across eight different species of tomato. For this purpose, GC-MS- and LC-MS-based metabolomics of different tissues of Solanum lycopersicum and wild tomato species were carried out, in concert with the evaluation of cross-hybridized microarray data for MapMan-based transcriptomic analysis, and publicly available RNA-sequencing data for annotation of biosynthetic genes. The combined data were used to compile species-specific metabolic networks of polyphenolic metabolism, allowing the establishment of an entire pan-species biosynthetic framework as well as annotation of the functions of decoration enzymes involved in the formation of metabolic diversity of the flavonoid pathway. The combined results are discussed in the context of the current understanding of tomato flavonol biosynthesis as well as a global view of metabolic shifts during fruit ripening. Our results provide an example as to how large-scale biology approaches can be used for the definition and refinement of large specialized metabolism pathways.
Asunto(s)
Frutas/metabolismo , Polifenoles/metabolismo , Solanum lycopersicum/metabolismo , Cromatografía Liquida , Flavonoides/metabolismo , Frutas/crecimiento & desarrollo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Variación Genética , Glicosiltransferasas/metabolismo , Solanum lycopersicum/genética , Espectrometría de Masas , Redes y Vías Metabólicas , Metabolómica , Anotación de Secuencia Molecular , Especificidad de la EspecieRESUMEN
Plant cell walls are essential for plant growth and development. The cell walls are traditionally divided into primary walls, which surround growing cells, and secondary walls, which provide structural support to certain cell types and promote their functions. While much information is available about the enzymes and components that contribute to the production of these two types of walls, much less is known about the transition from primary to secondary wall synthesis. To address this question, we made use of a transcription factor system in Arabidopsis (Arabidopsis thaliana) in which an overexpressed master secondary wall-inducing transcription factor, VASCULAR-RELATED NAC DOMAIN PROTEIN7, can be redirected into the nucleus by the addition of dexamethasone. We established the time frame during which primary wall synthesis changed into secondary wall production in dexamethasone-treated seedlings and measured transcript and metabolite abundance at eight time points after induction. Using cluster- and network-based analyses, we integrated the data sets to explore coordination between transcripts, metabolites, and the combination of the two across the time points. We provide the raw data as well as a range of network-based analyses. These data reveal links between hormone signaling and metabolic processes during the formation of secondary walls and provide a framework toward a deeper understanding of how primary walls transition into secondary walls.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Pared Celular/genética , Regulación de la Expresión Génica de las Plantas , Redes y Vías Metabólicas/genética , Factores de Transcripción/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Pared Celular/metabolismo , Cromatografía Liquida , Dexametasona/farmacología , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Metabolómica/métodos , Fotosíntesis/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/genética , Plantones/metabolismo , Espectrometría de Masas en Tándem , Factores de Transcripción/metabolismoRESUMEN
MOTIVATION: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. RESULTS: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. AVAILABILITY AND IMPLEMENTATION: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic CONTACT: usadel@bio1.rwth-aachen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Biología Computacional , Bases de Datos GenéticasRESUMEN
The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.
Asunto(s)
Arabidopsis/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Pared Celular/metabolismo , Germinación , Pectinas/metabolismo , Semillas/crecimiento & desarrollo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenómenos Biomecánicos , Hidrolasas de Éster Carboxílico/genética , Tamaño de la Célula , Activación Enzimática , Esterificación , Flores/enzimología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Microscopía Electrónica de Rastreo , Fenotipo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Semillas/enzimología , Semillas/ultraestructura , Ácidos Urónicos/metabolismoRESUMEN
The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Especificidad de Órganos , Tallos de la Planta/crecimiento & desarrollo , Arabidopsis/genética , Carbohidratos/análisis , Hidrolasas de Éster Carboxílico/metabolismo , Pared Celular/metabolismo , Fenotipo , Epidermis de la Planta/anatomía & histología , Epidermis de la Planta/crecimiento & desarrollo , Plantas Modificadas GenéticamenteRESUMEN
BACKGROUND: The extensive subcellular compartmentalization of metabolites and metabolism in eukaryotic cells is widely acknowledged and represents a key factor of metabolic activity and functionality. In striking contrast, the knowledge of actual compartmental distribution of metabolites from experimental studies is surprisingly low. However, a precise knowledge of, possibly all, metabolites and their subcellular distributions remains a key prerequisite for the understanding of any cellular function. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe results for the subcellular distribution of 1,117 polar and 2,804 lipophilic mass spectrometric features associated to known and unknown compounds from leaves of the model plant Arabidopsis thaliana. Using an optimized non-aqueous fractionation protocol in conjunction with GC/MS- and LC/MS-based metabolite profiling, 81.5% of the metabolic data could be associated to one of three subcellular compartments: the cytosol (including the mitochondria), vacuole, or plastids. Statistical analysis using a marker-'free' approach revealed that 18.5% of these metabolites show intermediate distributions, which can either be explained by transport processes or by additional subcellular compartments. CONCLUSION/SIGNIFICANCE: Next to a functional and conceptual workflow for the efficient, highly resolved metabolite analysis of the fractionated Arabidopsis thaliana leaf metabolome, a detailed survey of the subcellular distribution of several compounds, in the graphical format of a topological map, is provided. This complex data set therefore does not only contain a rich repository of metabolic information, but due to thorough validation and testing by statistical methods, represents an initial step in the analysis of metabolite dynamics and fluxes within and between subcellular compartments.
Asunto(s)
Arabidopsis/citología , Arabidopsis/metabolismo , Compartimento Celular , Metaboloma , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Biomarcadores/metabolismo , Fraccionamiento Celular , Análisis por Conglomerados , Modelos Biológicos , Modelos Estadísticos , Análisis de Componente Principal , Reproducibilidad de los Resultados , Fracciones Subcelulares/metabolismoRESUMEN
BACKGROUND: High-throughput measurement of transcript intensities using Affymetrix type oligonucleotide microarrays has produced a massive quantity of data during the last decade. Different preprocessing techniques exist to convert the raw signal intensities measured by these chips into gene expression estimates. Although these techniques have been widely benchmarked in the context of differential gene expression analysis, there are only few examples where their performance has been assessed in respect to coexpression-based studies such as sample classification. RESULTS: In the present paper we benchmark the three most used normalization procedures (MAS5, RMA and GCRMA) in the context of inter-array correlation analysis, confirming and extending the finding that RMA and GCRMA consistently overestimate sample similarity upon normalization. We determine that median polish summarization is responsible for generating a large proportion of these over-similarity artifacts. Furthermore, we show that most affected probesets show also internal signal disagreement, and tend to be composed by individual probes hitting different gene transcripts. We finally provide a correction to the RMA/GCRMA summarization procedure that massively reduces inter-array correlation artifacts, without affecting the detection of differentially expressed genes. CONCLUSIONS: We propose tRMA as a modification of RMA to normalize microarray experiments for correlation-based analysis.
Asunto(s)
Algoritmos , Artefactos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Bases de Datos Factuales , Expresión GénicaRESUMEN
Very few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and other metabolic pathways in the fruit, we undertook a fruit systems biology approach. To this end, we have produced tomato transgenic lines altered in ascorbate content and redox ratio by RNAi-targeting several key enzymes involved in ascorbate biosynthesis (2 enzymes) and recycling (2 enzymes). In the VTC (ViTamin C) Fruit project, we then generated phenotypic and genomic (transcriptome, proteome, metabolome) data from wild type and mutant tomato fruit at two stages of fruit development, and developed or implemented statistical and bioinformatic tools as a web application (named VTC Tool box) necessary to store, analyse and integrate experimental data in tomato. By using Kohonen's self-organizing maps (SOMs) to cluster the biological data, pair-wise Pearson correlation analyses and simultaneous visualization of transcript/protein and metabolites (MapMan), this approach allowed us to uncover major relationships between ascorbate and other metabolic pathways.
Asunto(s)
Ácido Ascórbico/metabolismo , Frutas/crecimiento & desarrollo , Genómica/métodos , Solanum lycopersicum/crecimiento & desarrollo , Análisis de Varianza , Ascorbato Oxidasa/genética , Ascorbato Oxidasa/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Solanum lycopersicum/genética , Solanum lycopersicum/efectos de la radiación , Redes y Vías Metabólicas , Metaboloma , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteoma , Integración de SistemasRESUMEN
Indole Acetic Acid 9 (IAA9) is a negative auxin response regulator belonging to the Aux/IAA transcription factor gene family whose downregulation triggers fruit set before pollination, thus giving rise to parthenocarpy. In situ hybridization experiments revealed that a tissue-specific gradient of IAA9 expression is established during flower development, the release of which upon pollination triggers the initiation of fruit development. Comparative transcriptome and targeted metabolome analysis uncovered important features of the molecular events underlying pollination-induced and pollination-independent fruit set. Comprehensive transcriptomic profiling identified a high number of genes common to both types of fruit set, among which only a small subset are dependent on IAA9 regulation. The fine-tuning of Aux/IAA and ARF genes and the downregulation of TAG1 and TAGL6 MADS box genes are instrumental in triggering the fruit set program. Auxin and ethylene emerged as the most active signaling hormones involved in the flower-to-fruit transition. However, while these hormones affected only a small number of transcriptional events, dramatic shifts were observed at the metabolic and developmental levels. The activation of photosynthesis and sucrose metabolism-related genes is an integral regulatory component of fruit set process. The combined results allow a far greater comprehension of the regulatory and metabolic events controlling early fruit development both in the presence and absence of pollination/fertilization.
Asunto(s)
Frutas/metabolismo , Proteínas de Plantas/fisiología , Polinización , ARN Mensajero/metabolismo , Solanum lycopersicum/metabolismo , División Celular/genética , Frutas/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fotosíntesis/genética , Reguladores del Crecimiento de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.
Asunto(s)
Nicotiana/genética , Nicotiana/fisiología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Arabidopsis/genética , Análisis por Conglomerados , Etiquetas de Secuencia Expresada , Eliminación de Gen , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Mutación , Fenotipo , Plantas Modificadas Genéticamente , ARN de Planta/genéticaRESUMEN
BACKGROUND: Genome-wide transcript profiling and analyses of enzyme activities from central carbon and nitrogen metabolism show that transcript levels undergo marked and rapid changes during diurnal cycles and after transfer to darkness, whereas changes in activities are smaller and delayed. In the starchless pgm mutant, where sugars are depleted every night, there are accentuated diurnal changes in transcript levels. Enzyme activities in this mutant do not show larger diurnal changes; instead, they shift towards the levels found in the wild type after several days of darkness. This indicates that enzyme activities change slowly, integrating the changes in transcript levels over several diurnal cycles. RESULTS: To generalize this conclusion, 137 metabolites were profiled using gas and liquid chromatography coupled to mass spectroscopy. The amplitudes of the diurnal changes in metabolite levels in pgm were (with the exception of sugars) similar or smaller than in the wild type. The average levels shifted towards those found after several days of darkness in the wild type. Examples include increased levels of amino acids due to protein degradation, decreased levels of fatty acids, increased tocopherol and decreased myo-inositol. Many metabolite-transcript correlations were found and the proportion of transcripts correlated with sugars increased dramatically in the starchless mutant. CONCLUSION: Rapid diurnal changes in transcript levels are integrated over time to generate quasi-stable changes across large sectors of metabolism. This implies that correlations between metabolites and transcripts are due to regulation of gene expression by metabolites, rather than metabolites being changed as a consequence of a change in gene expression.