RESUMEN
This study reveals striking differences in the content and composition of hydrophilic and lipophilic compounds in blackcurrant buds (Ribes nigrum L., cv. Ben Klibreck) resulting from winter chill or chemical dormancy release following treatment with ERGER, a biostimulant used to promote uniform bud break. Buds exposed to high winter chill exhibited widespread shifts in metabolite profiles relative to buds that experience winter chill by growth under plastic. Specifically, extensive chilling resulted in significant reductions in storage lipids and phospholipids, and increases in galactolipids relative to buds that experienced lower chill. Similarly, buds exposed to greater chill exhibited higher levels of many amino acids and dipeptides, and nucleotides and nucleotide phosphates than those exposed to lower chilling hours. Low chill buds (IN) subjected to ERGER treatment exhibited shifts in metabolite profiles similar to those resembling high chill buds that were evident as soon as 3 days after treatment. We hypothesise that chilling induces a metabolic shift which primes bud outgrowth by mobilising lipophilic energy reserves, enhancing phosphate availability by switching from membrane phospholipids to galactolipids and enhancing the availability of free amino acids for de novo protein synthesis by increasing protein turnover. Our results additionally suggest that ERGER acts at least in part by priming metabolism for bud outgrowth. Finally, the metabolic differences presented highlight the potential for developing biochemical markers for dormancy status providing an alternative to time-consuming forcing experiments.
Asunto(s)
Frío , Ribes , Ribes/metabolismo , Ribes/crecimiento & desarrollo , Ribes/química , Latencia en las Plantas/efectos de los fármacos , Metaboloma , Flores/crecimiento & desarrollo , Flores/metabolismo , Aminoácidos/metabolismoRESUMEN
OBJECTIVE: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism. METHODS: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization. To identify potential markers of brown adipose tissue aging, non-targeted proteomic and metabolomic as well as targeted lipid analyses were conducted on young and aged tissue samples. Subsequently, the effects of several candidate lipid classes on brown adipocyte function were examined. RESULTS: Corroborating previous reports of reduced expression of uncoupling protein-1, we observe impaired signaling required for lipid mobilization in aged brown fat after adrenergic stimulation. Omics analyses additionally confirm the age-related impairment of lipid homeostasis and reveal the accumulation of specific lipid classes, including certain sphingolipids, ceramides, and dolichols in aged brown fat. While ceramides as well as enzymes of dolichol metabolism inhibit brown adipogenesis, inhibition of sphingosine 1-phosphate receptor 2 induces brown adipocyte differentiation. CONCLUSIONS: Our functional analyses show that changes in specific lipid species, as observed during aging, may contribute to reduced thermogenic potential. They thus uncover potential biomarkers of aging as well as molecular mechanisms that could contribute to the degradation of brown adipocytes, thereby providing potential treatment strategies of age-related metabolic conditions.
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Adipocitos Marrones/metabolismo , Envejecimiento/metabolismo , Metabolismo de los Lípidos , Adipocitos Marrones/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Dolicoles/metabolismo , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Proteoma/genética , Proteoma/metabolismo , Esfingolípidos/metabolismoRESUMEN
Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein-Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein-small molecule and of protein-protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein-protein (PPIs) and protein-metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr-Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatografía en Gel/métodos , Metaboloma , Proteoma/metabolismo , Proteómica/métodos , Programas Informáticos , Unión Proteica , Proteoma/aislamiento & purificaciónRESUMEN
Metabolic genome-wide association studies (mGWAS), whereupon metabolite levels are regarded as traits, can help unravel the genetic basis of metabolic networks. A total of 309 Arabidopsis accessions were grown under two independent environmental conditions (control and stress) and subjected to untargeted LC-MS-based metabolomic profiling; levels of the obtained hydrophilic metabolites were used in GWAS. Our two-condition-based GWAS for more than 3000 semi-polar metabolites resulted in the detection of 123 highly resolved metabolite quantitative trait loci (p ≤ 1.0E-08), 24.39% of which were environment-specific. Interestingly, differently from natural variation in Arabidopsis primary metabolites, which tends to be controlled by a large number of small-effect loci, we found several major large-effect loci alongside a vast number of small-effect loci controlling variation of secondary metabolites. The two-condition-based GWAS was followed by integration with network-derived metabolite-transcript correlations using a time-course stress experiment. Through this integrative approach, we selected 70 key candidate associations between structural genes and metabolites, and experimentally validated eight novel associations, two of them showing differential genetic regulation in the two environments studied. We demonstrate the power of combining large-scale untargeted metabolomics-based GWAS with time-course-derived networks both performed under different abiotic environments for identifying metabolite-gene associations, providing novel global insights into the metabolic landscape of Arabidopsis.
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Arabidopsis/metabolismo , Metabolómica/métodos , Cromatografía Liquida , Estudio de Asociación del Genoma Completo , Espectrometría de Masas , Sitios de Carácter Cuantitativo/genéticaRESUMEN
RATIONALE: A bottleneck in metabolic profiling of complex biological extracts is confident, non-supervised annotation of ideally all contained, chemically highly diverse small molecules. Recent computational strategies combining sum formula prediction with in silico fragmentation achieve confident de novo annotation, once the correct neutral mass of a compound is known. Current software solutions for automated adduct ion assignment, however, are either publicly unavailable or have been validated against only few experimental electrospray ionization (ESI) mass spectra. METHODS: We here present findMAIN (find Main Adduct IoN), a new heuristic approach for interpreting ESI mass spectra. findMAIN scores MS1 spectra based on explained intensity, mass accuracy and isotope charge agreement of adducts and related ionization products and annotates peaks of the (de)protonated molecule and adduct ions. The approach was validated against 1141 ESI positive mode spectra of chemically diverse standard compounds acquired on different high-resolution mass spectrometric instruments (Orbitrap and time-of-flight). Robustness against impure spectra was evaluated. RESULTS: Correct adduct ion assignment was achieved for up to 83% of the spectra. Performance was independent of compound class and mass spectrometric platform. The algorithm proved highly tolerant against spectral contamination as demonstrated exemplarily for co-eluting compounds as well as systematically by pairwise mixing of spectra. When used in conjunction with MS-FINDER, a state-of-the-art sum formula tool, correct sum formulas were obtained for 77% of spectra. It outperformed both 'brute force' approaches and current state-of-the-art annotation packages tested as potential alternatives. Limitations of the heuristic pertained to poorly ionizing compounds and cationic compounds forming [M]+ ions. CONCLUSIONS: A new, validated approach for interpreting ESI mass spectra is presented, filling a gap in the nontargeted metabolomics workflow. It is freely available in the latest version of R package InterpretMSSpectrum.
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Cromatografía Liquida/métodos , Curaduría de Datos/métodos , Metabolómica/métodos , Metabolómica/normas , Espectrometría de Masa por Ionización de Electrospray/métodos , Reproducibilidad de los ResultadosRESUMEN
Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.
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Senescencia Celular/fisiología , Histona Demetilasas/metabolismo , Proteínas Mitocondriales/metabolismo , Sirtuinas/metabolismo , Células Madre/metabolismo , Trofoblastos/metabolismo , Envejecimiento/metabolismo , Humanos , Redes y Vías Metabólicas/fisiología , Mitocondrias/metabolismo , Oxidación-Reducción , Células Madre/fisiología , Trofoblastos/fisiologíaRESUMEN
Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT-selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1-deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT.
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Tejido Adiposo Pardo/metabolismo , Eliminación de Gen , Histona Demetilasas/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucólisis/genética , Metabolismo de los Lípidos/genética , Ratones Noqueados , Modelos Biológicos , Oxidación-Reducción , Aumento de PesoRESUMEN
Ignorant of the New World, Europeans believed in El Dorado, a hidden city of immense wealth in gold. Many consider the Amazonian forest to be a medicinal treasure chest and potentially the largest drug dispensary in the world. Yet, the quest to obtain drugs from indigenous tropical plants remains elusive. Here, we assess the potential of new technologies to tap into the metabolic diversity of tropical plants. We also consider how regulations affect access to plant resources. We conclude that, although the road to this medicinal El Dorado may be long and arduous, many other smaller but still valuable finds are hidden along the way.
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Bioprospección , Descubrimiento de Drogas , Plantas Medicinales , Bosque Lluvioso , Brasil , Biología Computacional , HumanosRESUMEN
BACKGROUND: Dates are tropical fruits with appreciable nutritional value. Previous attempts at global metabolic characterization of the date metabolome were constrained by small sample size and limited geographical sampling. In this study, two independent large cohorts of mature dates exhibiting substantial diversity in origin, varieties and fruit processing conditions were measured by metabolomics techniques in order to identify major determinants of the fruit metabolome. RESULTS: Multivariate analysis revealed a first principal component (PC1) significantly associated with the dates' countries of production. The availability of a smaller dataset featuring immature dates from different development stages served to build a model of the ripening process in dates, which helped reveal a strong ripening signature in PC1. Analysis revealed enrichment in the dry type of dates amongst fruits with early ripening profiles at one end of PC1 as oppose to an overrepresentation of the soft type of dates with late ripening profiles at the other end of PC1. Dry dates are typical to the North African region whilst soft dates are more popular in the Gulf region, which partly explains the observed association between PC1 and geography. Analysis of the loading values, expressing metabolite correlation levels with PC1, revealed enrichment patterns of a comprehensive range of metabolite classes along PC1. Three distinct metabolic phases corresponding to known stages of date ripening were observed: An early phase enriched in regulatory hormones, amines and polyamines, energy production, tannins, sucrose and anti-oxidant activity, a second phase with on-going phenylpropanoid secondary metabolism, gene expression and phospholipid metabolism and a late phase with marked sugar dehydration activity and degradation reactions leading to increased volatile synthesis. CONCLUSIONS: These data indicate the importance of date ripening as a main driver of variation in the date metabolome responsible for their diverse nutritional and economical values. The biochemistry of the ripening process in dates is consistent with other fruits but natural dryness may prevent degenerative senescence in dates following ripening. Based on the finding that mature dates present varying extents of ripening, our survey of the date metabolome essentially revealed snapshots of interchanging metabolic states during ripening empowering an in-depth characterization of underlying biology.
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Frutas/crecimiento & desarrollo , Metaboloma , Phoeniceae/genética , Proteínas de Plantas/genética , Cromatografía Líquida de Alta Presión , Frutas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Metabolómica , Phoeniceae/crecimiento & desarrollo , Phoeniceae/metabolismo , Proteínas de Plantas/metabolismo , TúnezRESUMEN
Diatoms are very efficient in their use of available nutrients. Changes in nutrient availability influence the metabolism and the composition of the cell constituents. Since diatoms are valuable candidates to search for oil producing algae, measurements of diatom-produced compounds can be very useful for biotechnology. In order to explore the diversity of lipophilic compounds produced by diatoms, we describe the results from an analysis of 13 diatom strains. With the help of a lipidomics platform, which combines an UPLC separation with a high resolution/high mass accuracy mass spectrometer, we were able to measure and annotate 142 lipid species. Out of these, 32 were present in all 13 cultures. The annotated lipid features belong to six classes of glycerolipids. The data obtained from the measurements were used to create lipidomic profiles. The metabolomic overview of analysed cultures is amended by the measurement of 96 polar compounds. To further increase the lipid diversity and gain insight into metabolomic adaptation to nitrogen limitation, diatoms were cultured in media with high and low concentrations of nitrate. The growth in nitrogen-deplete or nitrogen-replete conditions affects metabolite accumulation but has no major influence on the species-specific metabolomic profile. Thus, the genetic component is stronger in determining metabolic patterns than nitrogen levels. Therefore, lipid profiling is powerful enough to be used as a molecular fingerprint for diatom cultures. Furthermore, an increase of triacylglycerol (TAG) accumulation was observed in low nitrogen samples, although this trend was not consistent across all 13 diatom strains. Overall, our results expand the current understanding of metabolomics diversity in diatoms and confirm their potential value for producing lipids for either bioenergy or as feed stock.
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Diatomeas/metabolismo , Metabolómica , Nitratos/metabolismo , Adaptación Fisiológica , Diatomeas/fisiología , Nitrógeno/metabolismo , Triglicéridos/metabolismoRESUMEN
A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production.
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Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Proteínas de Plantas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Transcriptoma , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Benzamidas/farmacología , Biocombustibles , Biomasa , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes y Vías Metabólicas/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transcripción GenéticaRESUMEN
Although the plant lipidome show an enormous level of structural and functional diversity, our knowledge about its genetic control and its connection to whole-plant phenotypes is very limited. Here, we profiled 563 lipid species with UPLC-FT-MS in 289 field-grown inbred lines genotyped with 56,110 SNPs. Genome-wide association study identified 174 associations for 76 lipids explaining up to 31.4% of the genetic variance (P-value 8.4 × 10(-18)). Candidate genes were found for lipid synthesis, breakdown, transfer, and protection against peroxidation. The detected SNP-lipid associations could be grouped into associations with 1) individual lipids, 2) lipids from one biochemical class, and 3) lipids from several classes, suggesting a multilevel genetic control architecture. We further found a strong connection between the lipidome and agronomic traits in field-evaluated hybrid progeny. A cross-validated prediction model yielded correlations of up to 0.78 suggesting that the lipidome accurately predicts agronomic traits relevant in hybrid maize breeding.
Asunto(s)
Genoma de Planta/genética , Metabolismo de los Lípidos/genética , Lípidos/genética , Hojas de la Planta/genética , Sitios de Carácter Cuantitativo/genética , Zea mays/genéticaRESUMEN
In the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.
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Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/fisiología , Trichoderma/fisiología , Antibiosis , Arabidopsis/genética , Arabidopsis/microbiología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Raíces de Plantas/microbiologíaRESUMEN
N-Hydroxylated polyamine derivatives were found to decompose during the ionization process of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) experiments. The phenomenon was studied with a model compound, a synthetic N-hydroxylated tetraamine derivative. It was found that reduction, oxidation and water elimination occurred during APCI to generate the corresponding amine, N-oxide, and imine. The investigation further revealed that decomposition of hydroxylamines during APCI depends upon the concentration of the analyte and on the acidity of the solution introduced into the ionization source. The pH-dependence of decomposition was utilized for the development of an MS method that allows for the unambiguous identification of N-OH functionalities. This method was applied for the study of natural products including polyamine toxins from the venom of the spider Agelenopsis aperta and mayfoline, a cyclic polyamine derivative of the shrub Maytenus buxifolia.