RESUMO
Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is increasingly popular for the non-targeted exploration of complex samples, where tandem mass spectrometry (MS/MS) is used to characterize the structure of unknown compounds. However, mass spectra do not always contain sufficient information to unequivocally identify the correct structure. This study investigated how much additional information can be gained using hydrogen deuterium exchange (HDX) experiments. The exchange of "easily exchangeable" hydrogen atoms (connected to heteroatoms), with predominantly [M+D]+ ions in positive mode and [M-D]- in negative mode was observed. To enable high-throughput processing, new scoring terms were incorporated into the in silico fragmenter MetFrag. These were initially developed on small datasets and then tested on 762 compounds of environmental interest. Pairs of spectra (normal and deuterated) were found for 593 of these substances (506 positive mode, 155 negative mode spectra). The new scoring terms resulted in 29 additional correct identifications (78 vs 49) for positive mode and an increase in top 10 rankings from 80 to 106 in negative mode. Compounds with dual functionality (polar head group, long apolar tail) exhibited dramatic retention time (RT) shifts of up to several minutes, compared with an average 0.04 min RT shift. For a smaller dataset of 80 metabolites, top 10 rankings improved from 13 to 24 (positive mode, 57 spectra) and from 14 to 31 (negative mode, 63 spectra) when including HDX information. The results of standard measurements were confirmed using targets and tentatively identified surfactant species in an environmental sample collected from the river Danube near Novi Sad (Serbia). The changes to MetFrag have been integrated into the command line version available at http://c-ruttkies.github.io/MetFrag and all resulting spectra and compounds are available in online resources and in the Electronic Supplementary Material (ESM). Graphical abstract.
RESUMO
Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.
Assuntos
Cálcio/metabolismo , Dipeptídeos/fisiologia , Concentração de Íons de Hidrogênio , Phytophthora infestans/química , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Arabidopsis/parasitologia , Sinalização do Cálcio , Espectrometria de Massas , Phytophthora infestans/patogenicidade , Plântula/efeitos dos fármacosRESUMO
The abuse of synthetic opioids has become a major threat in recent years. Several clinical reports and fatal case reports exist discussing life-threatening hypoventilation and fatal respiratory depression following the abuse of trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide (U-47700). The reported concentration of U-47700 in peripheral blood varies between 0.01 µg/mL and 1.46 µg/mL. These values depend on the mode of administration and whether the drug was used in combination with other drugs and/or pharmaceuticals. In the past, U-47700 was predominantly insufflated and not injected. The current study presents a non-targeted liquid chromatography/mass spectrometry (LC/MS)-based screening approach of urine and cerebrospinal fluid samples after intravenous injection of U-47700. Furthermore, quantitative values on U-47700 as obtained by liquid chromatography coupled to a linear ion trap (LC/ESI-QTRAPMS) are presented concerning femoral blood (0.29 µg/mL), urine (0.24 µg/mL), gastric contents (0.57 µg/mL), bile fluid (2.3 µg/mL), heart blood (1.25 µg/mL), liver (9.9 µg/g), cerebrospinal fluid (0.4 µg/mL), and hair (0.14 ng/mg). Thereof, concentrations in hair, gastric contents, bile fluid and cerebrospinal fluid have never been reported before. Drug paraphernalia were also analyzed by liquid chromatography coupled to a diode array detector (LC/DAD) and nuclear magnetic resonance spectrometer (NMR). The analyses show that the powder had a relatively high purity and was adulterated to a low degree. This is the first case report which lists concentration distributions of various specimens after intravenous injection. These findings as well as the U-47700 concentration are important to evaluate autopsy cases of U-47700 intoxication in the future.
Assuntos
Benzamidas/análise , Benzamidas/intoxicação , Drogas Ilícitas/análise , Drogas Ilícitas/intoxicação , Abuso de Substâncias por Via Intravenosa/complicações , Bile/química , Cromatografia Líquida , Toxicologia Forense , Conteúdo Gastrointestinal/química , Cabelo/química , Humanos , Injeções Intravenosas , Fígado/química , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Distribuição TecidualRESUMO
A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.
RESUMO
The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
Assuntos
Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Cumarínicos/metabolismo , Oxigênio/metabolismo , Exsudatos de Plantas/metabolismo , Raízes de Plantas/metabolismo , Metaboloma , Metabolômica/métodosRESUMO
Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes.
Assuntos
Arabidopsis/metabolismo , Extratos Vegetais/química , Arabidopsis/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica , Raízes de Plantas/metabolismo , Metabolismo Secundário , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana's roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Basidiomycota/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Arabidopsis/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Dipeptídeos/análise , Glucosinolatos/análise , Glucosinolatos/metabolismo , Nucleosídeos/análise , Reguladores de Crescimento de Plantas/análise , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray , SimbioseRESUMO
Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities.
Assuntos
Arabidopsis/metabolismo , Genômica , Metabolômica , Exsudatos de Plantas/análise , Arabidopsis/genética , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Ácidos Cumáricos/química , Variação Genética , Glucosinolatos/metabolismo , Hidrólise , Metaboloma , Raízes de Plantas/metabolismo , Rizosfera , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.
Assuntos
Arabidopsis/metabolismo , Cumarínicos/metabolismo , Lignina/metabolismo , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Fosfatos/farmacologia , Exsudatos de Plantas/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arginina/metabolismo , Ácido Cítrico/metabolismo , Análise por Conglomerados , Hidroponia , Malatos/metabolismo , Meristema/efeitos dos fármacos , Meristema/metabolismo , Floroglucinol/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimentoRESUMO
The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.
Assuntos
Arabidopsis/metabolismo , Doenças das Plantas , Solanum lycopersicum/metabolismo , Verticillium/patogenicidade , Arabidopsis/genética , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Redes e Vias Metabólicas/genética , Micoses/genética , Micoses/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Microbiologia do Solo , Verticillium/crescimento & desenvolvimentoRESUMO
RATIONALE: Gas chromatography (GC) coupled to atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (APCI-QTOFMS) is an emerging technology in metabolomics. Reference spectra for GC/APCI-MS/MS barely exist; therefore, in silico fragmentation approaches and structure databases are prerequisites for annotation. To expand the limited coverage of derivatised structures in structure databases, in silico derivatisation procedures are required. METHODS: A cheminformatics workflow has been developed for in silico derivatisation of compounds found in KEGG and PubChem, and validated on the Golm Metabolome Database (GMD). To demonstrate this workflow, these in silico generated databases were applied together with MetFrag to APCI-MS/MS spectra acquired from GC/APCI-MS/MS profiles of Arabidopsis thaliana and Solanum tuberosum. The Metabolite-Likeness of the original candidate structure was included as additional scoring term aiming at candidate structures of natural origin. RESULTS: The validation of our in silico derivatisation workflow on the GMD showed a true positive rate of 94%. MetFrag was applied to two datasets. In silico derivatisation of the KEGG and PubChem database served as a candidate source. For both datasets the Metabolite-Likeness score improved the identification performance. The derivatised data sources have been included into the MetFrag web application for the annotation of GC/APCI-MS/MS spectra. CONCLUSIONS: We demonstrated that MetFrag can support the identification of components from GC/APCI-MS/MS profiles, especially in the (common) case where reference spectra are not available. This workflow can be easily adapted to other types of derivatisation and is freely accessible together with the generated structure databases.
Assuntos
Curadoria de Dados/métodos , Bases de Dados de Compostos Químicos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Software , Espectrometria de Massas em Tandem/métodos , Simulação por Computador , Internet , Modelos Químicos , Extratos Vegetais/análise , Extratos Vegetais/química , Reprodutibilidade dos TestesRESUMO
During a compatible interaction, the sebacinoid root-associated fungi Piriformospora indica and Sebacina vermifera induce modification of root morphology and enhance shoot growth in Arabidopsis thaliana. The genomic traits common in these two fungi were investigated and compared with those of other root-associated fungi and saprotrophs. The transcriptional responses of the two sebacinoid fungi and of Arabidopsis roots to colonization at three different symbiotic stages were analyzed by custom-designed microarrays. We identified key genomic features characteristic of sebacinoid fungi, such as expansions for gene families involved in hydrolytic activities, carbohydrate-binding and protein-protein interaction. Additionally, we show that colonization of Arabidopsis correlates with the induction of salicylic acid catabolism and accumulation of jasmonate and glucosinolates (GSLs). Genes involved in root developmental processes were specifically induced by S. vermifera at later stages during interaction. Using different Arabidopsis indole-GSLs mutants and measurement of secondary metabolites, we demonstrate the importance of the indolic glucosinolate pathway in the growth restriction of P. indica and S. vermifera and we identify indole-phytoalexins and specifically indole-carboxylic acids derivatives as potential key players in the maintenance of a mutualistic interaction with root endophytes.
Assuntos
Arabidopsis/microbiologia , Basidiomycota/fisiologia , Endófitos/fisiologia , Imunidade Inata , Imunidade Vegetal , Raízes de Plantas/fisiologia , Simbiose/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Basidiomycota/genética , Endófitos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Genoma Fúngico , Glucosinolatos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Indóis/farmacologia , Metaboloma/efeitos dos fármacos , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Estrutura Terciária de Proteína , Sesquiterpenos/farmacologia , FitoalexinasRESUMO
To explore the chemical composition of root exudates of the model plant Arabidopsis thaliana a workflow for nontargeted metabolite profiling of the semipolar fraction of root exudates was developed. It comprises hydroponic plant cultivation and sampling of root exudates under sterile conditions, sample preparation by solid-phase extraction and analysis by reversed-phase UPLC/ESI-QTOFMS. Following the established workflow, root exudates of six-week-old plants were profiled and a set of reproducibly occurring molecular features was compiled. To structurally elucidate the corresponding metabolites, accurate mass tandem mass spectrometry and on-line hydrogen/deuterium exchange were applied. Currently, a total of 103 compounds were detected and annotated by elemental composition of which more than 90 were structurally characterized or classified. Among them, 42 compounds were rigorously identified using an authenticated standard. The compounds identified so far include nucleosides, deoxynucleosides, aromatic amino acids, anabolites and catabolites of glucosinolates, dipeptides, indolics, salicylic and jasmonic acid catabolites, coumarins, mono-, di- and trilignols, hydroxycinnamic acid derivatives and oxylipins and exemplify the high chemical diversity of plant root exudates.
Assuntos
Arabidopsis/metabolismo , Aminoácidos Aromáticos/química , Ciclopentanos/metabolismo , Dipeptídeos/química , Glucosinolatos/metabolismo , Indóis/química , Estrutura Molecular , Nucleosídeos/química , Oxilipinas/metabolismo , Raízes de Plantas/química , Ácido Salicílico/metabolismo , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Lipídeos/biossíntese , Proteínas de Plantas/fisiologia , Solanum tuberosum/metabolismo , Transportadores de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Solanum tuberosum/genéticaRESUMO
Chlorine substituted hexaarylbisimidazole (o-Cl-HABI) efficiently initiates radical polymerization of multifunctional acrylic esters in the presence of a heterocyclic mercapto compound if the latter can form its tautomeric thione. Exposure of o-Cl-HABI results in lophyl radicals, which efficiently add to the thione in the first step while the second step releases a highly reactive thiyl radical from this intermediate. LC-MS and CID-MS measurements support this reaction scheme. Furthermore, photo-DSC experiments applying UV light between 320 and 380 nm showed that mercaptotriazole and phenylmercaptotriazole exhibited the best reactivity in the monomer 1,6-hexanediol diacrylate (HDDA) while alkyl substituted mercaptotriazoles showed less reactivity. Change of the triazole heterocycle by mercaptoimidazole resulted in a significant decrease of photoinitiation efficiency. This heterocycle does not form the corresponding thione in HDDA as shown by NMR measurements. Replacement of mercaptotriazole by an alkylthiol leads to a system showing the lowest photoinitiation efficiency in this series. Formation of thione structure in the case of heterocyclic mercapto compounds may cause higher reactivity of the heterocyclic mercapto compounds with the lophyl radical in the monomer chosen.
Assuntos
Acrilatos/síntese química , Compostos Heterocíclicos/química , Imidazóis/química , Compostos de Sulfidrila/química , Acrilatos/química , Técnicas Eletroquímicas , Estrutura Molecular , Processos Fotoquímicos , Fotólise , Polimerização , Raios UltravioletaRESUMO
Although iron (Fe) is one of the most abundant elements in the earth's crust, its low solubility in soils restricts Fe uptake by plants. Most plant species acquire Fe by acidifying the rhizosphere and reducing ferric to ferrous Fe prior to membrane transport. However, it is unclear how these plants access Fe in the rhizosphere and cope with high soil pH. In a mutant screening, we identified 2-oxoglutarate-dependent dioxygenase Feruloyl-CoA 6'-Hydroxylase1 (F6'H1) to be essential for tolerance of Arabidopsis (Arabidopsis thaliana) to high pH-induced Fe deficiency. Under Fe deficiency, F6'H1 is required for the biosynthesis of fluorescent coumarins that are released into the rhizosphere, some of which possess Fe(III)-mobilizing capacity and prevent f6'h1 mutant plants from Fe deficiency-induced chlorosis. Scopoletin was the most prominent coumarin found in Fe-deficient root exudates but failed to mobilize Fe(III), while esculetin, i.e. 6,7-dihydroxycoumarin, occurred in lower amounts but was effective in Fe(III) mobilization. Our results indicate that Fe-deficient Arabidopsis plants release Fe(III)-chelating coumarins as part of the strategy I-type Fe acquisition machinery.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cumarínicos/metabolismo , Dioxigenases/metabolismo , Ferro/farmacocinética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA Bacteriano , Dioxigenases/genética , Epiderme/fisiologia , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Rizosfera , Escopoletina/metabolismo , Solo/química , Umbeliferonas/metabolismoRESUMO
Cellular responses to various gels fabricated by photoinitiated crosslinking using acrylated linear and multi-arm poly(ethylene glycol) (PEG)-based and poly(propylene glycol)-b-poly(ethylene glycol) precursors were investigated. While no protein adsorption and cell adhesion were observed on the hydrophilic PEG-based gels, protein adsorption and cell adhesion did occur on the more hydrophobic gel generated from the block copolymer precursor. Murine fibroblast viability on the poly(ethylene glycol)-based gels was studied in the course of 72 h and the results indicated no cytotoxicity. In a systematic study, extra- and intracellular metabolites of the murine fibroblasts cultured on these PEG-based gels were examined by GC-MS. Distinct intra- and extracellular changes in primary metabolism, namely amino acid metabolism, glycolysis and fatty acid metabolism, were observed. Cells cultured on the polymeric gels induced more intense intracellular changes in the metabolite profile by means of higher metabolite intensities with time in comparison to cells cultured on the reference substrate (tissue culture polystyrene). In contrast, extracellular changes of metabolite intensities were comparable.
RESUMO
Gas chromatography coupled to mass spectrometry (GC-MS) is one of the most widespread routine technologies applied to the large scale screening and discovery of novel metabolic biomarkers. However, currently the majority of mass spectral tags (MSTs) remains unidentified due to the lack of authenticated pure reference substances required for compound identification by GC-MS. Here, we accessed the information on reference compounds stored in the Golm Metabolome Database (GMD) to apply supervised machine learning approaches to the classification and identification of unidentified MSTs without relying on library searches. Non-annotated MSTs with mass spectral and retention index (RI) information together with data of already identified metabolites and reference substances have been archived in the GMD. Structural feature extraction was applied to sub-divide the metabolite space contained in the GMD and to define the prediction target classes. Decision tree (DT)-based prediction of the most frequent substructures based on mass spectral features and RI information is demonstrated to result in highly sensitive and specific detections of sub-structures contained in the compounds. The underlying set of DTs can be inspected by the user and are made available for batch processing via SOAP (Simple Object Access Protocol)-based web services. The GMD mass spectral library with the integrated DTs is freely accessible for non-commercial use at http://gmd.mpimp-golm.mpg.de/. All matching and structure search functionalities are available as SOAP-based web services. A XML + HTTP interface, which follows Representational State Transfer (REST) principles, facilitates read-only access to data base entities.
RESUMO
Biomarkers are used to predict phenotypical properties before these features become apparent and, therefore, are valuable tools for both fundamental and applied research. Diagnostic biomarkers have been discovered in medicine many decades ago and are now commonly applied. While this is routine in the field of medicine, it is of surprise that in agriculture this approach has never been investigated. Up to now, the prediction of phenotypes in plants was based on growing plants and assaying the organs of interest in a time intensive process. For the first time, we demonstrate in this study the application of metabolomics to predict agronomic important phenotypes of a crop plant that was grown in different environments. Our procedure consists of established techniques to screen untargeted for a large amount of metabolites in parallel, in combination with machine learning methods. By using this combination of metabolomics and biomathematical tools metabolites were identified that can be used as biomarkers to improve the prediction of traits. The predictive metabolites can be selected and used subsequently to develop fast, targeted and low-cost diagnostic biomarker assays that can be implemented in breeding programs or quality assessment analysis. The identified metabolic biomarkers allow for the prediction of crop product quality. Furthermore, marker-assisted selection can benefit from the discovery of metabolic biomarkers when other molecular markers come to its limitation. The described marker selection method was developed for potato tubers, but is generally applicable to any crop and trait as it functions independently of genomic information.
Assuntos
Biomarcadores/análise , Fenótipo , Plantas/metabolismo , Inteligência Artificial , Solanum tuberosum/metabolismoRESUMO
Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of (13)C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (13)CO(2) and (13)C-acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions.