RESUMEN
Autophagy represents a key regulator of aging and metabolism in sensing energy deprivation. We find that fasting in mice activates autophagy in the liver paralleled by activation of hypothalamic AgRP neurons. Optogenetic and chemogenetic activation of AgRP neurons induces autophagy, alters phosphorylation of autophagy regulators, and promotes ketogenesis. AgRP neuron-dependent induction of liver autophagy relies on NPY release in the paraventricular nucleus of the hypothalamus (PVH) via presynaptic inhibition of NPY1R-expressing neurons to activate PVHCRH neurons. Conversely, inhibiting AgRP neurons during energy deprivation abrogates induction of hepatic autophagy and rewiring of metabolism. AgRP neuron activation increases circulating corticosterone concentrations, and reduction of hepatic glucocorticoid receptor expression attenuates AgRP neuron-dependent activation of hepatic autophagy. Collectively, our study reveals a fundamental regulatory principle of liver autophagy in control of metabolic adaptation during nutrient deprivation.
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Hipotálamo , Neuronas , Ratones , Animales , Proteína Relacionada con Agouti/metabolismo , Neuronas/metabolismo , Hipotálamo/metabolismo , Hígado/metabolismo , NutrientesRESUMEN
The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD)-induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.
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Hipotálamo , Proteínas de Unión a Tacrolimus , Autofagia , Dieta Alta en Grasa/efectos adversos , Humanos , Hipotálamo/metabolismo , Obesidad/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.
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Familia 2 del Citocromo P450/genética , Familia 2 del Citocromo P450/metabolismo , Deficiencia de Ácido Fólico/genética , Deficiencia de Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Animales , Biomarcadores/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación/genética , Fenotipo , Proteómica/métodosRESUMEN
Ginger (Zingiber officinale Roscoe) is consumed for health-promoting effects and as a food condiment. Comprehensive phytochemical analysis, other than gingerols and shogaols, has not yet been deeply investigated. Hence, the current research aimed to establish a non-targeted metabolomics approach for the discrimination between fresh ginger rhizome samples collected from four different producing countries, i.e., China, India, Pakistan, and Peru. In addition, lab-dried samples were analyzed to trace drying-induced metabolites. A comprehensive extraction procedure was carried out resulting in production of polar and non-polar fractions. The polar fraction was analyzed by ultra-performance liquid chromatography coupled with Fourier transform tandem mass spectrometry (UPLC-C18-FT-MS/MS) and gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) post derivatization. UPLC-C8-FT-MS/MS was used for analysis of non-polar fraction. Results revealed for identification of a total of 253 metabolites. In addition, multivariate data analysis (MVDA), including principal component analysis (PCA) demonstrated clustering of Asian specimens. Several metabolites with a characteristic pattern for the origin revealing the highest contents of bioactive metabolites in the Peruvian product. Moreover, chemical markers identified, including [6]-gingerol and [6]-shogaol discriminating between fresh and dried samples. Furthermore, abundances of some primary metabolites, including amino acids and cinnamic acid, have confirmed the biosynthetic pathway of gingerols and their transformation upon drying to shogaols. The proposed approach can be applied as a potential candidate for quality assessment of ginger and other medicinal plants.
Asunto(s)
Zingiber officinale , Catecoles , Cromatografía Líquida de Alta Presión , Análisis de Datos , Alcoholes Grasos , Metabolómica , Extractos Vegetales , Espectrometría de Masas en TándemRESUMEN
Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.
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Conducta Animal , ADN (Citosina-5-)-Metiltransferasas/fisiología , Neuronas Dopaminérgicas/metabolismo , Hipotálamo/metabolismo , Actividad Motora , Condicionamiento Físico Animal , Animales , ADN Metiltransferasa 3A , Femenino , Masculino , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl-tert-butyl-ether) phase, eliminated the need for solid-phase extraction for sample clean-up, and therefore reduces both sampling time and cost. As a proof-of-concept analysis, Arabidopsis thaliana plants were subjected to water-deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty-acid derivatives and diverse jasmonates in the course of adaptation to water-deficit stress.
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Lípidos/análisis , Reguladores del Crecimiento de las Plantas/análisis , Hojas de la Planta/química , Proteínas de Plantas/análisis , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análisis , Cromatografía Líquida de Alta Presión , Deshidratación , Espectrometría de Masas , Metaboloma , Extractos Vegetales/química , ProteómicaRESUMEN
The allocation of nutrient resources to growth and metabolism is an essential function for controlling biomass accumulation in photoautotrophic organisms. One essential protein complex involved in this process is the target of rapamycin (TOR) kinase. It has been shown that the inhibition of TOR leads to a considerable upsurge in the amino acid levels. This molecular phenotype relies mainly on the availability of light, carbon (C) and nitrogen (N). To validate the time-resolved response of C and N metabolites, we used a targeted gas chromatography mass spectrometery (GC-MS)-based metabolomic approach, where we examined the response of Chlamydomonas reinhardtii upon TOR inhibition under C-limited condition, namely extended darkness. Contrary to C-supplemented conditions, the rapid increase in the amino acid levels is suppressed almost completely 4 h after TOR inhibition, confirming that C supply is essential to raise the amino acid levels mediated by their de novo synthesis. An exception to this observation was the levels of aspartate, which is presumably synthesized via the anaplerotic pathway. In agreement with previous reports, TOR repression, under these C-limited conditions, leads to a significant reduction in the C/N ratio, corroborating with the crucial role of the pathway in maintaining the metabolic balance of the cells and consequently propelling growth.
Asunto(s)
Carbono/metabolismo , Chlamydomonas reinhardtii/metabolismo , Homeostasis , Nitrógeno/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Aminoácidos/metabolismoRESUMEN
An excess of nitrogen (N) is used in agriculture endangering the environment and food quality. One approach to circumvent this is to generate crops with a stable or even increased productivity under limited N. Here, we studied the effect of reduced N availability on potato (Solanum tuberosum) tuber yield and quality traits using five varieties: the wild Andigena and the commercial cultivars Désirée, Milva, Saturna and Alegria. Growth on limited N resulted in less tubers with a reduced weight except for Andigena. Tubers from low N-grown plants contained more starch, less sucrose and were delayed in sprouting. Some of the trait differences can be explained by changes in hormone levels between cultivars and N conditions. In general, Saturna and Alegria performed better under limited N making them excellent breeding candidates. Our results suggest that wild species more flexibly adapt to limited N, a trait lost in commercial potatoes.
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Productos Agrícolas/metabolismo , Nitrógeno/metabolismo , Tubérculos de la Planta/metabolismo , Solanum tuberosum/metabolismo , Disponibilidad Biológica , Productos Agrícolas/crecimiento & desarrollo , Fenotipo , Fitomejoramiento , Solanum tuberosum/crecimiento & desarrollo , Almidón , SacarosaRESUMEN
A large-scale metabolic quantitative trait loci (mQTL) analysis was performed on the well-characterized Solanum pennellii introgression lines to investigate the genomic regions associated with secondary metabolism in tomato fruit pericarp. In total, 679 mQTLs were detected across the 76 introgression lines. Heritability analyses revealed that mQTLs of secondary metabolism were less affected by environment than mQTLs of primary metabolism. Network analysis allowed us to assess the interconnectivity of primary and secondary metabolism as well as to compare and contrast their respective associations with morphological traits. Additionally, we applied a recently established real-time quantitative PCR platform to gain insight into transcriptional control mechanisms of a subset of the mQTLs, including those for hydroxycinnamates, acyl-sugar, naringenin chalcone, and a range of glycoalkaloids. Intriguingly, many of these compounds displayed a dominant-negative mode of inheritance, which is contrary to the conventional wisdom that secondary metabolite contents decreased on domestication. We additionally performed an exemplary evaluation of two candidate genes for glycolalkaloid mQTLs via the use of virus-induced gene silencing. The combined data of this study were compared with previous results on primary metabolism obtained from the same material and to other studies of natural variance of secondary metabolism.
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Patrón de Herencia/genética , Sitios de Carácter Cuantitativo/genética , Metabolismo Secundario/genética , Solanum/genética , Solanum/metabolismo , Vías Biosintéticas/genética , Cromatografía Liquida , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estudios de Asociación Genética , Endogamia , Espectrometría de Masas , Metaboloma/genética , Metabolómica , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Reproducibilidad de los Resultados , Solanum/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
Hakea prostrata (Proteaceae) is adapted to severely phosphorus-impoverished soils and extensively replaces phospholipids during leaf development. We investigated how polar lipid profiles change during leaf development and in response to external phosphate supply. Leaf size was unaffected by a moderate increase in phosphate supply. However, leaf protein concentration increased by more than 2-fold in young and mature leaves, indicating that phosphate stimulates protein synthesis. Orthologs of known lipid-remodeling genes in Arabidopsis (Arabidopsis thaliana) were identified in the H. prostrata transcriptome. Their transcript profiles in young and mature leaves were analyzed in response to phosphate supply alongside changes in polar lipid fractions. In young leaves of phosphate-limited plants, phosphatidylcholine/phosphatidylethanolamine and associated transcript levels were higher, while phosphatidylglycerol and sulfolipid levels were lower than in mature leaves, consistent with low photosynthetic rates and delayed chloroplast development. Phosphate reduced galactolipid and increased phospholipid concentrations in mature leaves, with concomitant changes in the expression of only four H. prostrata genes, GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE1, N-METHYLTRANSFERASE2, NONSPECIFIC PHOSPHOLIPASE C4, and MONOGALACTOSYLDIACYLGLYCEROL3. Remarkably, phosphatidylglycerol levels decreased with increasing phosphate supply and were associated with lower photosynthetic rates. Levels of polar lipids with highly unsaturated 32:x (x = number of double bonds in hydrocarbon chain) and 34:x acyl chains increased. We conclude that a regulatory network with a small number of central hubs underpins extensive phospholipid replacement during leaf development in H. prostrata. This hard-wired regulatory framework allows increased photosynthetic phosphorus use efficiency and growth in a low-phosphate environment. This may have rendered H. prostrata lipid metabolism unable to adjust to higher internal phosphate concentrations.
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Metabolismo de los Lípidos , Fosfatos/metabolismo , Fósforo/metabolismo , Hojas de la Planta/metabolismo , Proteaceae/metabolismo , Cloroplastos/metabolismo , Lípidos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/metabolismo , Fosfolípidos/metabolismo , Fotosíntesis , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteaceae/genética , Proteaceae/crecimiento & desarrollo , Biosíntesis de ProteínasRESUMEN
Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus ('delayed greening'), with young leaves having very low levels of chlorophyll and Calvin-Benson cycle enzymes. In mature leaves, soluble protein and Calvin-Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin-Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.
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Fósforo/metabolismo , Proteaceae/fisiología , ARN de Planta/metabolismo , ARN Ribosómico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Clorofila/metabolismo , Glucosa-6-Fosfato/metabolismo , Fotosíntesis , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/metabolismo , Proteaceae/genética , Proteaceae/metabolismo , Proteínas Ribosómicas/metabolismo , Almidón/metabolismoRESUMEN
Drosophila light-dependent channels, TRP and TRPL, reside in the light-sensitive microvilli of the photoreceptor's rhabdomere. Phospholipase C mediates TRP/TRPL opening, but the gating process remains unknown. Controversial evidence has suggested diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs, a DAG metabolite), phosphatidylinositol bisphosphate (PIP2), and H(+) as possible channel activators. We tested each of them directly in inside-out TRP-expressing patches excised from the rhabdomere, making use of mutants and pharmacology. When patches were excised in darkness TRP remained closed, while when excised under illumination it stayed constitutively active. TRP was opened by DAG and silenced by ATP, suggesting DAG-kinase (DGK) involvement. The ATP effect was abolished by inhibiting DGK and in the rdgA mutant, lacking functional DGK, implicating DGK. DAG activated TRP even in the presence of a DAG-lipase inhibitor, inconsistent with a requirement of PUFAs in opening TRP. PIP2 had no effect and acidification, pH 6.4, activated TRP irreversibly, unlike the endogenous activator. Complementary liquid-chromatography/mass-spectrometry determinations of DAG and PUFAs in membranes enriched in rhabdomere obtained from light- and dark-adapted eyes showed light-dependent increment in six DAG species and no changes in PUFAs. The results strongly support DAG as the endogenous TRP agonist, as some of its vertebrate TRPC homologs of the same channel family.
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Diglicéridos/farmacología , Proteínas de Drosophila/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Microvellosidades/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Adaptación Ocular , Adenosina Trifosfato/farmacología , Animales , Oscuridad , Diacilglicerol Quinasa/metabolismo , Activación Enzimática/efectos de los fármacos , Ácidos Grasos Insaturados/metabolismo , Luz , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Membranas/fisiología , ProtonesRESUMEN
Although Neanderthals are extinct, fragments of their genomes persist in contemporary humans. Here we show that while the genome-wide frequency of Neanderthal-like sites is approximately constant across all contemporary out-of-Africa populations, genes involved in lipid catabolism contain more than threefold excess of such sites in contemporary humans of European descent. Evolutionally, these genes show significant association with signatures of recent positive selection in the contemporary European, but not Asian or African populations. Functionally, the excess of Neanderthal-like sites in lipid catabolism genes can be linked with a greater divergence of lipid concentrations and enzyme expression levels within this pathway, seen in contemporary Europeans, but not in the other populations. We conclude that sequence variants that evolved in Neanderthals may have given a selective advantage to anatomically modern humans that settled in the same geographical areas.
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Evolución Biológica , Metabolismo de los Lípidos , Hombre de Neandertal/genética , Hombre de Neandertal/metabolismo , Población Blanca/genética , Animales , Genoma , Historia Antigua , Humanos , Hombre de Neandertal/clasificación , Pan troglodytes/genética , Pan troglodytes/metabolismo , Filogenia , Población Blanca/clasificación , Población Blanca/historiaRESUMEN
Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.
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Galactolípidos/metabolismo , Lípidos , Fósforo/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Proteaceae/metabolismo , Suelo , Arabidopsis/crecimiento & desarrollo , Lípidos/análisis , Lípidos/química , Fósforo/análisis , Fósforo/deficiencia , Fotosíntesis , Hojas de la Planta/metabolismo , Suelo/análisis , Australia del Sur , Australia OccidentalRESUMEN
The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.
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Arabidopsis/metabolismo , Metabolómica/métodos , Extractos Vegetales/aislamiento & purificación , Proteómica/métodos , Arabidopsis/química , Isótopos de Carbono , Clorofila/análogos & derivados , Clorofila/química , Bases de Datos Factuales , Análisis de Fourier , Marcaje Isotópico , Lípidos/análisis , Espectrometría de Masas , Isótopos de Nitrógeno , Extractos Vegetales/química , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Reproducibilidad de los Resultados , Isótopos de AzufreRESUMEN
We conducted a comparative analysis of the root metabolome of six parental maize inbred lines and their 14 corresponding hybrids showing fresh weight heterosis. We demonstrated that the metabolic profiles not only exhibit distinct features for each hybrid line compared with its parental lines, but also separate reciprocal hybrids. Reconstructed metabolic networks, based on robust correlations between metabolic profiles, display a higher network density in most hybrids as compared with the corresponding inbred lines. With respect to metabolite level inheritance, additive, dominant and overdominant patterns are observed with no specific overrepresentation. Despite the observed complexity of the inheritance pattern, for the majority of metabolites the variance observed in all 14 hybrids is lower compared with inbred lines. Deviations of metabolite levels from the average levels of the hybrids correlate negatively with biomass, which could be applied for developing predictors of hybrid performance based on characteristics of metabolite patterns.
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Metaboloma , Extractos Vegetales/química , Raíces de Plantas/metabolismo , Zea mays/metabolismo , Biomasa , Cruzamientos Genéticos , Genotipo , Vigor Híbrido , Endogamia , Patrón de Herencia , Lípidos/análisis , Redes y Vías Metabólicas , Metabolómica , Modelos Biológicos , Raíces de Plantas/química , Raíces de Plantas/genética , Análisis de Componente Principal , Zea mays/química , Zea mays/genéticaRESUMEN
A new strategy for direct infusion-based metabolite analysis employing a combination of high-resolution mass spectrometry and (13)C-isotope labeling of entire metabolomes is described. Differentially isotope labeled metabolite extracts from otherwise identically grown reference plants were prepared and infused into a Fourier transform ion cyclotron resonance mass spectrometer. The derived accurate mass lists from each extract were searched, using an in-house-developed database search tool, against a number of comprehensive metabolite databases. Comparison of the retrieved chemical formulas from both, the (12)C and (13)C samples, leads to two major advantages compared to nonisotope-based metabolite fingerprinting: first, removal of background contaminations from the result list, due to the (12)C/(13)C peak pairing principle and therefore positive identification of compounds of true biological origin; second, elimination of ambiguity in chemical formula assignment due to the same principle, leading to the clear association of one measured mass to only one chemical formula. Applying this combination of strategies to metabolite extracts of the model plant Arabidopsis thaliana therefore resulted in the reproducible identification of more than 1000 unambiguous chemical sum formulas of biological origin of which more than 80% have not been associated to Arabidopsis before.
Asunto(s)
Arabidopsis/metabolismo , Dióxido de Carbono/química , Isótopos de Carbono , Metabolómica/métodos , Extractos Vegetales/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Arabidopsis/crecimiento & desarrollo , Ciclotrones , Análisis de Fourier , Extractos Vegetales/químicaRESUMEN
Laser microdissection (LM) allows the collection of homogeneous tissue- and cell-specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryo-sectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.