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1.
Artigo em Inglês | MEDLINE | ID: mdl-34542391

RESUMO

A novel bacterium, designated strain Msb3T, was recently isolated from leaves of the yam family plant Dioscorea bulbifera (Dioscoreaceae). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Paraburkholderia with Paraburkholderia xenovorans as nearest validly named neighbour taxon (99.3 % sequence similarity towards the P. xenovorans type strain). Earlier genome sequence analysis revealed a genome of 8.35 Mb in size with a G+C content of 62.5 mol%, which was distributed over two chromosomes and three plasmids. Here, we confirm that strain Msb3T represents a novel Paraburkholderia species. In silico DNA-DNA hybridization and average nucleotide identity (OrthoANIu) analyses towards P. xenovorans LB400T yielded 58.4 % dDDH and 94.5 % orthoANIu. Phenotypic and metabolic characterization revealed growth at 15 °C on tryptic soy agar, growth in the presence of 1 % NaCl and the lack of assimilation of phenylacetic acid as distinctive features. Together, these data demonstrate that strain Msb3T represents a novel species of the genus Paraburkholderia, for which we propose the name Paraburkholderia dioscoreae sp. nov. The type strain is Msb3T (=LMG 31881T, DSM 111632T, CECT 30342T).


Assuntos
Florestas , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Burkholderiaceae , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona
2.
Plant Physiol ; 181(2): 683-700, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31378720

RESUMO

Shifts in the duration and intensity of ambient temperature impair plant development and reproduction, particularly male gametogenesis. Stress exposure causes meiotic defects or premature spore abortion in male reproductive organs, leading to male sterility. However, little is known about the mechanisms underlying stress and male sterility. To elucidate these mechanisms, we imposed a moderate transient heat stress on maize (Zea mays) plants at the tetrad stage of pollen development. After completion of pollen development at optimal conditions, stress responses were assessed in mature pollen. Transient heat stress resulted in reduced starch content, decreased enzymatic activity, and reduced pollen germination, resulting in sterility. A transcriptomic comparison pointed toward misregulation of starch, lipid, and energy biosynthesis-related genes. Metabolomic studies showed an increase of Suc and its monosaccharide components, as well as a reduction in pyruvate. Lipidomic analysis showed increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids. In contrast, the majority of genes involved in developmental processes such as those required for auxin and unfolded protein responses, signaling, and cell wall biosynthesis remained unaltered. It is noteworthy that changes in the regulation of transcriptional and metabolic pathway genes, as well as heat stress proteins, remained altered even though pollen could recover during further development at optimal conditions. In conclusion, our findings demonstrate that a short moderate heat stress during the highly susceptible tetrad stage strongly affects basic metabolic pathways and thus generates germination-defective pollen, ultimately leading to severe yield losses in maize.


Assuntos
Resposta ao Choque Térmico , Infertilidade das Plantas , Pólen/crescimento & desenvolvimento , Zea mays/fisiologia , Metabolismo Energético , Gametogênese Vegetal , Regulação da Expressão Gênica de Plantas , Lipídeos/biossíntese , Meiose , Pólen/enzimologia , Fatores de Transcrição/metabolismo
3.
Ecol Lett ; 22(1): 159-169, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30556313

RESUMO

Climate warming affects plant physiology through genetic adaptation and phenotypic plasticity, but little is known about how these mechanisms influence ecosystem processes. We used three elevation gradients and a reciprocal transplant experiment to show that temperature causes genetic change in the sedge Eriophorum vaginatum. We demonstrate that plants originating from warmer climate produce fewer secondary compounds, grow faster and accelerate carbon dioxide (CO2 ) release to the atmosphere. However, warmer climate also caused plasticity in E. vaginatum, inhibiting nitrogen metabolism, photosynthesis and growth and slowing CO2 release into the atmosphere. Genetic differentiation and plasticity in E. vaginatum thus had opposing effects on CO2 fluxes, suggesting that warming over many generations may buffer, or reverse, the short-term influence of this species over carbon cycle processes. Our findings demonstrate the capacity for plant evolution to impact ecosystem processes, and reveal a further mechanism through which plants will shape ecosystem responses to climate change.


Assuntos
Ciclo do Carbono , Plásticos , Carbono , Dióxido de Carbono , Mudança Climática , Ecossistema , Plantas
4.
Plant J ; 87(3): 318-32, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27136060

RESUMO

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.


Assuntos
Cacau/metabolismo , Polifenóis/metabolismo , Sementes/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia
5.
J Exp Bot ; 66(3): 863-78, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25392479

RESUMO

Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.


Assuntos
Parede Celular/enzimologia , Chenopodium/genética , Secas , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Solanum lycopersicum/fisiologia , beta-Frutofuranosidase/genética , Chenopodium/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , beta-Frutofuranosidase/metabolismo
6.
New Phytol ; 201(2): 476-485, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24117492

RESUMO

The enzyme myo-inositol oxygenase is the key enzyme of a pathway leading from myo-inositol to UDP-glucuronic acid. In Arabidopsis, myo-inositol oxygenase is encoded by four genes. All genes are strongly expressed in syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Here, we studied the effect of a quadruple myo-inositol oxygenase mutant on nematode development. We performed metabolite profiling of syncytia induced in roots of the myo-inositol oxygenase quadruple mutant. The role of galactinol in syncytia was studied using Arabidopsis lines with elevated galactinol levels and by supplying galactinol to wild-type seedlings. The quadruple myo-inositol oxygenase mutant showed a significant reduction in susceptibility to H. schachtii, and syncytia had elevated myo-inositol and galactinol levels and an elevated expression level of the antimicrobial thionin gene Thi2.1. This reduction in susceptibility could also be achieved by exogenous application of galactinol to wild-type seedlings. The primary function of myo-inositol oxygenase for syncytium development is probably not the production of UDP-glucuronic acid as a precursor for cell wall polysaccharides, but the reduction of myo-inositol levels and thereby a reduction in the galactinol level to avoid the induction of defence-related genes.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Inositol Oxigenase/fisiologia , Inositol/metabolismo , Nematoides/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inositol Oxigenase/genética , Inositol Oxigenase/metabolismo , Raízes de Plantas/metabolismo
7.
Plant Physiol ; 162(4): 1822-33, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23660836

RESUMO

Investigation of the metabolome and the transcriptome of pollen of lily (Lilium longiflorum) gave a comprehensive overview of metabolic pathways active during pollen germination and tube growth. More than 100 different metabolites were determined simultaneously by gas chromatography coupled to mass spectrometry, and expressed genes of selected metabolic pathways were identified by next-generation sequencing of lily pollen transcripts. The time-dependent changes in metabolite abundances, as well as the changes after inhibition of the mitochondrial electron transport chain, revealed a fast and dynamic adaption of the metabolic pathways in the range of minutes. The metabolic state prior to pollen germination differed clearly from the metabolic state during pollen tube growth, as indicated by principal component analysis of all detected metabolites and by detailed observation of individual metabolites. For instance, the amount of sucrose increased during the first 60 minutes of pollen culture but decreased during tube growth, while glucose and fructose showed the opposite behavior. Glycolysis, tricarbonic acid cycle, glyoxylate cycle, starch, and fatty acid degradation were activated, providing energy during pollen germination and tube growth. Inhibition of the mitochondrial electron transport chain by antimycin A resulted in an immediate production of ethanol and a fast rearrangement of metabolic pathways, which correlated with changes in the amounts of the majority of identified metabolites, e.g. a rapid increase in γ-aminobutyric acid indicated the activation of a γ-aminobutyric acid shunt in the tricarbonic acid cycle, while ethanol fermentation compensated the reduced ATP production after inhibition of the oxidative phosphorylation.


Assuntos
Germinação/fisiologia , Lilium/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Adaptação Fisiológica/fisiologia , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Antimicina A/farmacologia , Metabolismo dos Carboidratos , Transporte de Elétrons , Enzimas/genética , Enzimas/metabolismo , Etanol/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Germinação/efeitos dos fármacos , Lilium/efeitos dos fármacos , Lilium/crescimento & desenvolvimento , Redes e Vias Metabólicas/genética , Fosforilação Oxidativa , Análise de Componente Principal , Sacarose/metabolismo , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismo
8.
Plant Genome ; 17(1): e20337, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37165696

RESUMO

Drought is one of the major constraints limiting chickpea productivity. To unravel complex mechanisms regulating drought response in chickpea, we generated transcriptomics, proteomics, and metabolomics datasets from root tissues of four contrasting drought-responsive chickpea genotypes: ICC 4958, JG 11, and JG 11+ (drought-tolerant), and ICC 1882 (drought-sensitive) under control and drought stress conditions. Integration of transcriptomics and proteomics data identified enriched hub proteins encoding isoflavone 4'-O-methyltransferase, UDP-d-glucose/UDP-d-galactose 4-epimerase, and delta-1-pyrroline-5-carboxylate synthetase. These proteins highlighted the involvement of pathways such as antibiotic biosynthesis, galactose metabolism, and isoflavonoid biosynthesis in activating drought stress response mechanisms. Subsequently, the integration of metabolomics data identified six metabolites (fructose, galactose, glucose, myoinositol, galactinol, and raffinose) that showed a significant correlation with galactose metabolism. Integration of root-omics data also revealed some key candidate genes underlying the drought-responsive "QTL-hotspot" region. These results provided key insights into complex molecular mechanisms underlying drought stress response in chickpea.


Assuntos
Cicer , Cicer/genética , Multiômica , Raízes de Plantas/genética , Secas , Galactose/metabolismo , Difosfato de Uridina/metabolismo
9.
J Exp Bot ; 64(14): 4193-206, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24064926

RESUMO

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/farmacologia , Metaboloma/efeitos dos fármacos , Proteoma/metabolismo , Regulação para Cima/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Cromatografia Líquida , Dexametasona/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/efeitos dos fármacos , Hordeum/metabolismo , Espectrometria de Massas , Metaboloma/genética , Metabolômica , Plantas Geneticamente Modificadas , Proteômica , Plântula/efeitos dos fármacos , Plântula/genética , Regulação para Cima/genética
10.
Biomolecules ; 12(2)2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35204826

RESUMO

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography-mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.


Assuntos
Doenças Neurodegenerativas , Neurodegeneração Associada a Pantotenato-Quinase , Acetilcoenzima A , Citratos , Ácido Cítrico , Eritrócitos/metabolismo , Humanos , Ferro/metabolismo , Mutação , Neurodegeneração Associada a Pantotenato-Quinase/genética , Neurodegeneração Associada a Pantotenato-Quinase/patologia , Fosfotransferases (Aceptor do Grupo Álcool) , Isoformas de Proteínas/genética
11.
Biol Fertil Soils ; 58(3): 291-306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35399158

RESUMO

Roots secrete a vast array of low molecular weight compounds into the soil broadly referred to as root exudates. It is a key mechanism by which plants and soil microbes interact in the rhizosphere. The effect of drought stress on the exudation process and composition is rarely studied, especially in cereal crops. This study focuses on comparative metabolic profiling of the exudates from sensitive and tolerant genotypes of pearl millet after a period of drought stress. We employed a combined platform of gas and liquid chromatography coupled to mass spectrometry to cover both primary and secondary metabolites. The results obtained demonstrate that both genotype and drought stress have a significant impact on the concentration and composition of root exudates. The complexity and function of these differential root exudates are discussed. To reveal the potential effect of root exudates on the soil microbial community after a period of drought stress, we also tested for biological nitrification inhibition (BNI) activity. The analysis revealed a genotype-dependent enhancement of BNI activity after a defined period of drought stress. In parallel, we observed a genotype-specific relation of elongated root growth and root exudation under drought stress. These data suggest that the drought stress-dependent change in root exudation can manipulate the microbial soil communities to adapt and survive under harsh conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s00374-021-01578-w.

12.
Front Mol Biosci ; 8: 683671, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395523

RESUMO

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

14.
Front Microbiol ; 11: 581, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32373084

RESUMO

The genus Paraburkholderia includes a variety of species with promising features for sustainable biotechnological solutions in agriculture through increasing crop productivity. Here, we present a novel Paraburkholderia isolate, a permanent and predominant member of the Dioscoreae bulbifera (yam family, Dioscoreaceae) phyllosphere, making up to 25% of the microbial community on leaf acumens. The 8.5 Mbp genome of isolate Msb3 encodes an unprecedented combination of features mediating a beneficial plant-associated lifestyle, including biological nitrogen fixation (BNF), plant hormone regulation, detoxification of various xenobiotics, degradation of aromatic compounds and multiple protein secretion systems including both T3SS and T6SS. The isolate exhibits significant growth promotion when applied to agriculturally important plants such as tomato, by increasing the total dry biomass by up to 40%. The open question about the "beneficial" nature of this strain led us to investigate ecological and generic boundaries in Burkholderia sensu lato. In a refined phylogeny including 279 Burkholderia sensu lato isolates strain Msb3 clusters within Clade I Paraburkholderia, which also includes few opportunistic strains that can potentially act as pathogens, as revealed by our ecological meta-data analysis. In fact, we demonstrate that all genera originating from the "plant beneficial and environmental" (PBE) Burkholderia species cluster include opportunists. This indicates that further functional examinations are needed before safe application of these strains in sustainable agricultural settings can be assured.

15.
Cell Rep ; 30(5): 1542-1552.e7, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32023468

RESUMO

Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.


Assuntos
Polaridade Celular , Genômica , Macrófagos/citologia , Macrófagos/metabolismo , Modelos Biológicos , Fosfoglicerato Desidrogenase/metabolismo , Animais , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Interleucina-4/farmacologia , Ácidos Cetoglutáricos/metabolismo , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Fosfoglicerato Desidrogenase/genética , Análise de Componente Principal , Serina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
16.
Front Plant Sci ; 9: 1556, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30459786

RESUMO

Experimental high-throughput analysis of molecular networks is a central approach to characterize the adaptation of plant metabolism to the environment. However, recent studies have demonstrated that it is hardly possible to predict in situ metabolic phenotypes from experiments under controlled conditions, such as growth chambers or greenhouses. This is particularly due to the high molecular variance of in situ samples induced by environmental fluctuations. An approach of functional metabolome interpretation of field samples would be desirable in order to be able to identify and trace back the impact of environmental changes on plant metabolism. To test the applicability of metabolomics studies for a characterization of plant populations in the field, we have identified and analyzed in situ samples of nearby grown natural populations of Arabidopsis thaliana in Austria. A. thaliana is the primary molecular biological model system in plant biology with one of the best functionally annotated genomes representing a reference system for all other plant genome projects. The genomes of these novel natural populations were sequenced and phylogenetically compared to a comprehensive genome database of A. thaliana ecotypes. Experimental results on primary and secondary metabolite profiling and genotypic variation were functionally integrated by a data mining strategy, which combines statistical output of metabolomics data with genome-derived biochemical pathway reconstruction and metabolic modeling. Correlations of biochemical model predictions and population-specific genetic variation indicated varying strategies of metabolic regulation on a population level which enabled the direct comparison, differentiation, and prediction of metabolic adaptation of the same species to different habitats. These differences were most pronounced at organic and amino acid metabolism as well as at the interface of primary and secondary metabolism and allowed for the direct classification of population-specific metabolic phenotypes within geographically contiguous sampling sites.

17.
Front Mol Biosci ; 4: 84, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29312952

RESUMO

Gestational diabetes mellitus during pregnancy has severe implications for the health of the mother and the fetus. Therefore, early prediction and an understanding of the physiology are an important part of prenatal care. Metabolite profiling is a long established method for the analysis and prediction of metabolic diseases. Here, we applied untargeted and targeted metabolomic protocols to analyze plasma and urine samples of pregnant women with and without GDM. Univariate and multivariate statistical analyses of metabolomic profiles revealed markers such as 2-hydroxybutanoic acid (AHBA), 3-hydroxybutanoic acid (BHBA), amino acids valine and alanine, the glucose-alanine-cycle, but also plant-derived compounds like sitosterin as different between control and GDM patients. PLS-DA and VIP analysis revealed tryptophan as a strong variable separating control and GDM. As tryptophan is biotransformed to serotonin we hypothesized whether serotonin metabolism might also be altered in GDM. To test this hypothesis we applied a method for the analysis of serotonin, metabolic intermediates and dopamine in urine by stable isotope dilution direct infusion electrospray ionization mass spectrometry (SID-MS). Indeed, serotonin and related metabolites differ significantly between control and GDM patients confirming the involvement of serotonin metabolism in GDM. Clustered correlation coefficient visualization of metabolite correlation networks revealed the different metabolic signatures between control and GDM patients. Eventually, the combination of selected blood plasma and urine sample metabolites improved the AUC prediction accuracy to 0.99. The detected GDM candidate biomarkers and the related systemic metabolic signatures are discussed in their pathophysiological context. Further studies with larger cohorts are necessary to underpin these observations.

18.
Biotechnol J ; 11(10): 1262-1267, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27440175

RESUMO

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.


Assuntos
Biocombustíveis/análise , Clorófitas/metabolismo , Ácidos Graxos Insaturados/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Magnoliopsida/metabolismo , Vias Biossintéticas , Chlamydomonas reinhardtii/metabolismo , Chlorella vulgaris/metabolismo , Coffea/metabolismo , Cuscuta/metabolismo , Ácidos Graxos Insaturados/metabolismo , Pisum sativum/metabolismo , Análise de Célula Única
19.
Front Plant Sci ; 7: 750, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27375629

RESUMO

Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development.

20.
PLoS One ; 11(1): e0146135, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26727123

RESUMO

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus-plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed.


Assuntos
Antioxidantes/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Potyvirus/fisiologia , Solanum tuberosum/virologia , Metabolismo dos Carboidratos , Metabolismo Energético , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Redes e Vias Metabólicas , Metaboloma , Fenótipo , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potyvirus/patogenicidade , Espécies Reativas de Oxigênio , Ácido Chiquímico/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Virulência , Replicação Viral
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