RESUMO
Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.
Assuntos
Arabidopsis , Poligalacturonase , Poligalacturonase/genética , Poligalacturonase/metabolismo , Arabidopsis/metabolismo , Pectinas/metabolismo , Proteínas/metabolismo , Parede Celular/metabolismoRESUMO
INTRODUCTION: Bio stimulants are substances and/or microorganisms that are used to improve plant growth and crop yields by modulating physiological processes and metabolism of plants. While research has primarily focused on the broad effects of bio stimulants in crops, understanding their cellular and molecular influences in plants, using metabolomic analysis, could elucidate their effectiveness and offer possibilities for fine-tuning their application. One such bio stimulant containing galacturonic acid as elicitor is used in agriculture to improve wheat vigor and strengthen resistance to lodging. OBJECTIVE: However, whether a metabolic response is evolved by plants treated with this bio stimulant and the manner in which the latter might regulate plant metabolism have not been studied. METHOD: Therefore, the present study used 1H-NMR and LC-MS to assess changes in primary and secondary metabolites in the roots, stems, and leaves of wheat (Triticum aestivum) treated with the bio stimulant. Orthogonal partial least squares discriminant analysis effectively distinguished between treated and control samples, confirming a metabolic response to treatment in the roots, stems, and leaves of wheat. RESULTS: Fold-change analysis indicated that treatment with the bio stimulation solution appeared to increase the levels of hydroxycinnamic acid amides, lignin, and flavonoid metabolism in different plant parts, potentially promoting root growth, implantation, and developmental cell wall maturation and lignification. CONCLUSION: These results demonstrate how non-targeted metabolomic approaches can be utilized to investigate and monitor the effects of new agroecological solutions based on systemic responses.
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Metabolômica , Triticum , Espectrometria de Massa com Cromatografia Líquida , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Triticum/metabolismo , Triticum/efeitos dos fármacosRESUMO
INTRODUCTION: The cacao tree (Theobroma cacao), a perennial crop that serves as a source of cacao beans, can suffer from drastic climate changes such as irregular rainfall and shorter rainy seasons. The search for hybrids which are capable of producing specific metabolites favoring adaptation in new climatic conditions is a challenge in cacao farming. OBJECTIVES: We aimed to (1) analyze the metabolic changes in calli of three cacao genotypes during water deficit induced by incubation with polyethylene glycol and (2) assess their response to water deficit stress with regard to somatic embryo differentiation. METHODS: Metabolic profiling was carried out using 1H-NMR spectroscopy and multivariate data analysis was applied to crude extracts of calli grown in non-stress or water deficit stress conditions. RESULTS: Water deficit stress influences the capacity of calli to produce embryos. The SCA12 genotype exhibited the best conversion capacity under severe conditions and was considered as tolerant to drought, followed by the SCA6 genotype (mid-tolerant) and the MA12 genotype (sensitive). Fifty-four metabolites were identified in the three cacao genotypes and discriminant metabolites were identified. Metabolites involved in water stress tolerance such as fructose, trans-aconitic acid, leucine, and hydroxybenzene derivatives were observed in SCA12, the tolerant genotype. CONCLUSION: These results demonstrate the utility of 1H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao.
Assuntos
Cacau , Secas , Metaboloma , Polietilenoglicóis , Cacau/metabolismo , Polietilenoglicóis/farmacologia , Genótipo , Metabolômica , Estresse Fisiológico , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Prótons por Ressonância Magnética/métodosRESUMO
Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.
Assuntos
Aciltransferases , Cichorium intybus , Aciltransferases/genética , Aciltransferases/metabolismo , Alcenos , Compostos Aza , Cichorium intybus/genética , Cichorium intybus/metabolismo , Espermidina/metabolismo , Espermina/metabolismoRESUMO
Leaves of Pistacia lentiscus were collected from two Algerian sites in the mountains and the littoral of the Tizi-Ouzou region. The harvest was conducted in four consecutive seasons on the same selected set of trees. Essential oils (EOs) were extracted by hydrodistillation; then, they were analyzed by gas chromatography coupled mass spectrometry (GC-MS). Forty-seven constituents could be detected and quantified, including α-pinene (2-13%), ß-caryophyllene (8-25%), ß-myrcene (0.3-19%), bornyl acetate (0.8-7%), δ-cadinene (3-8%), bisabolol (1-9%), ß-pinene (0.9-7%), caryophyllene oxide (4-9%), and α-cadinol (3-11%). Antioxidant (AOx) activities of the EOs were assessed by ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assays. Significant differences in EO composition and AOx activities appeared dependent on the season and the site. Variations of AOx activities were significant for the FRAP and ABTS tests but not for DPPH. Characterization of the leaf fatty acyl (FA) profiles was performed by GC-MS. Variability appeared according to season and altitude. Polyunsaturated fatty acids levels were high (27-55%) at the coldest date and place. The levels of linolenic acyl in the leaves were significantly correlated with bisabolol levels in the EOs (Spearman's correlation coefficient: 0.818). Such results will be useful for the sustainable local valorization of wild P. lentiscus. These data also open new routes for further studies on terpenoid biosynthesis using correlation networks and fluxomic approaches.
Assuntos
Óleos Voláteis , Pistacia , Argélia , Antioxidantes/química , Óleos Voláteis/química , Pistacia/química , Folhas de Planta/químicaRESUMO
In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant-pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2-10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.
Assuntos
Quitosana , Linho , Parede Celular/metabolismo , Quitosana/farmacologia , Linho/metabolismo , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Metabolismo Secundário , Plântula/metabolismoRESUMO
Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)-the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.
Assuntos
Cromatografia Líquida , Linho/metabolismo , Lignanas/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Pressão Osmótica , Linho/química , FenótipoRESUMO
INTRODUCTION: Biotransformation constitutes an important aspect of the drug discovery process, to mimic human metabolism of active principal ingredient but also to generate new chemical entities. Several microorganisms such as fungi are well adapted to transform drug, whether at the stage of screening or for large-scale production. OBJECTIVES: Due to the high chemical complexity of the biotransformation media, it seems attractive to develop new analytical strategies in order to guarantee an adequate monitoring and optimize the production of targeted metabolites or drug candidates. METHODS: The model designed for this purpose concerns the biotransformation of a potential histamine H3 antagonist (S38093) in order to produce phase I metabolites. MS, NMR and chemometrics tools were used to monitor biotransformation reactions. RESULTS: First, a screening of eleven filamentous fungi was carried out by UHPLC-UV-MS and principal component analysis to select the best candidates. Subsequently, MS (tR, m/z) and NMR (1H, JRES) fingerprints associated with Consensus OPLS-DA multiblock approach were used to better understand the bioreaction mechanisms in terms of nutrient consumption and hydroxylated metabolites production. Then an experimental design was set up to optimize the production conditions (pH, kinetic) of these target metabolites. CONCLUSION: This study demonstrates how NMR and MS acquisitions combined with chemometric methods offer an innovative analytical strategy to have a grasp of functionalization mechanisms, and identify metabolites and other compounds (amino acids, nutrients, etc.) in complex biotransformation mixtures.
Assuntos
Fungos/metabolismo , Antagonistas dos Receptores Histamínicos H3/metabolismo , Metabolômica , Biotransformação , Fungos/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H3/química , Antagonistas dos Receptores Histamínicos H3/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Análise de Componente PrincipalRESUMO
INTRODUCTION: Proton nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic profiling has a range of applications in plant sciences. OBJECTIVES: The aim of the present work is to provide advice for minimizing uncontrolled variability in plant sample preparation before and during NMR metabolomic profiling, taking into account sample composition, including its specificity in terms of pH and paramagnetic ion concentrations, and NMR spectrometer performances. METHODS: An automation of spectrometer preparation routine standardization before NMR acquisition campaign was implemented and tested on three plant sample sets (extracts of durum wheat spikelet, Arabidopsis leaf and root, and flax leaf, root and stem). We performed 1H-NMR spectroscopy in three different sites on the wheat sample set utilizing instruments from two manufacturers with different probes and magnetic field strengths. The three collections of spectra were processed separately with the NMRProcFlow web tool using intelligent bucketing, and the resulting buckets were subjected to multivariate analysis. RESULTS: Comparability of large- (Arabidopsis) and medium-size (flax) datasets measured at 600 MHz and from the wheat sample set recorded at the three sites (400, 500 and 600 MHz) was exceptionally good in terms of spectral quality. The coefficient of variation of the full width at half maximum (FWHM) and the signal-to-noise ratio (S/N) of two selected peaks was comprised between 5 and 10% depending on the size of sample set and the spectrometer field. EDTA addition improved citrate and malate resonance patterns for wheat sample sets. A collection of 22 samples of wheat spikelet extracts was used as a proof of concept and showed that the data collected at the three sites on instruments of different field strengths and manufacturers yielded the same discrimination pattern of the biological groups. CONCLUSION: Standardization or automation of several steps from extract preparation to data reduction improves data quality for small to large collections of plant samples of different origins.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Extratos Vegetais/isolamento & purificação , Manejo de Espécimes/métodos , Arabidopsis , Automação , Linho , Ensaios de Triagem em Larga Escala/normas , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Folhas de Planta/química , Folhas de Planta/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Padrões de Referência , Manejo de Espécimes/normas , TriticumRESUMO
A global approach that is based on a combination of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data has been developed for a complete and rapid understanding of drug degradation mixtures. We proposed a workflow based on a sample preparation protocol that is compatible to MS and NMR, the selection of the most appropriate experiments for each technique, and the implementation of prediction software and multivariable analysis method for a better interpretation and correlation of MS and NMR spectra. We have demonstrated the efficient quantification of the remaining active pharmaceutical ingredient (API). The unambiguous characterization of degradation products (DPs) was reached while using the potential of ion mobility-mass spectrometry (IM-MS) for fragment ions filtering (HDMSE) and the implementation of two-dimensional (2D) NMR experiments with the non-uniform sampling (NUS) method. We have demonstrated the potential of quantitative NMR (qNMR) for the estimation of low level DPs. Finally, in order to simultaneously monitor multi-samples, the contribution of partial least squares (PLS) regression was evaluated. Our methodology was tested on three indapamide forced degradation conditions (acidic, basic, and oxidative) and it could be easily transposed in the drug development field to assist in the interpretation of complex mixtures (stability studies, impurities profiling, and biotransformation screening).
Assuntos
Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , Indapamida/química , Cromatografia Líquida de Alta Pressão , Humanos , Indapamida/uso terapêutico , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução/efeitos dos fármacosRESUMO
Flax (Linum usitatissimum) is a plant grown in temperate regions either for its fiber or for its seeds, which are rich in the essential fatty acid omega-3. It is also well known as a source of medicinal compounds. The chemical composition of its leaves is currently poorly described. In order to fill this gap, we have conducted a comprehensive analysis of flax leaf metabolome. The exploration of the metabolome allowed the characterization of compounds isolated for the first time in flax leaves. These molecules were isolated by preparative HPLC and then characterized by NMR, LC-MS and standard analysis. This work extended our picture of C-glycosyl-flavonoids and coniferyl alcohol derivatives accumulated in flax. The follow-up of the content of these different metabolites via UPLC-MS revealed significant accumulation differences in spring and winter flax leaves. In particular, two methylated C-glycosylflavonoids (swertisin and swertiajaponin) were the most abundant phenolic compounds in winter flax whereas they were not detected in spring flax. This result suggests that these 2 compounds are involved in cold stress tolerance in flax.
Assuntos
Linho/química , Fenol/química , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/química , Fenóis/química , Folhas de Planta/química , Estações do Ano , Sementes/química , Espectrometria de Massas em Tandem/métodosRESUMO
This report describes a routine method taking less than 20 min to quantify cyanogenic glycosides such as linustatin and neolinustatin from flaxseeds (Linum usitatissimum L.) using 1H nuclear magnetic resonance. After manual dehulling, a higher linustatin content was shown in the almond fraction, while neolinustatin and total cyanogenic glycoside contents were significantly higher in hulls. Linustatin and neolinustatin were quantified in seven cultivars grown in two locations in three different years. Linustatin, neolinustatin, and total cyanogenic glycosides ranged between 91 and 267 mg/100 g, 78-272 mg/100 g, and 198-513 mg/100 g dry weight flaxseeds, respectively. NMR revealed differences of up to 70% between samples with standard deviation variations lower than 6%. This study shows that NMR is a very suitable tool to perform flaxseed varietal selection for the cyanogenic glycoside content. Graphical abstract qNMR can be used to perform flaxseed varietal selection for the cyanogenic glycoside content.
Assuntos
Linho/química , Linho/classificação , Glicosídeos/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Abiotic stress is a major cause of yield loss in plant culture. Miscanthus, a perennial C4 grass, is now considered a major source of renewable energy, especially for biofuel production. During the first year of planting in Northern Europe, Miscanthus was exposed to frost temperature, which generated high mortality in young plants and large loss of yield. One strategy to avoid such loss is to apply cold-acclimation, which confers on plants a better resistance to low temperature. OBJECTIVES: The aim of this study is to describe the effect of a cold-acclimation period on the metabolome of two Miscanthus genotypes that vary in their frost sensitivity at the juvenile stage. Miscanthus × giganteus (GIG) is particularly sensitive to frost, whereas Miscanthus sinensis August Feder (AUG) is tolerant. MATERIALS AND METHODS: Polar metabolite extraction was performed on Miscanthus, grown in non-acclimated or cold-acclimated conditions. Extracts were analysed by 1 H-NMR followed by multivariate statistical analysis. Discriminant metabolites were identified. RESULTS: More than 40 metabolites were identified in the two Miscanthus genotypes. GIG and AUG showed a different metabolic background before cold treatment, probably related to their genetic background. After cold-acclimation, GIG and AUG metabolomes remained different. The tolerant genotype showed notably higher levels of accumulation in proline, sucrose and maltose when subjected to cold. CONCLUSION: These two genotypes seem to have a different adaptation strategy in cold conditions. The studied change in the metabolome concerns different types of molecules related to the cold-tolerant behaviour of Miscanthus. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Adaptação Fisiológica , Andropogon/metabolismo , Temperatura Baixa , Genótipo , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética/métodos , Andropogon/genética , Andropogon/fisiologiaRESUMO
The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.
Assuntos
Oxirredutases do Álcool/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Carbono/metabolismo , Nitrogênio/metabolismo , Raízes de Plantas/metabolismo , Alanina/metabolismo , Oxirredutases do Álcool/análise , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Arabidopsis/genética , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Ácido Aspártico/metabolismo , Ciclo do Ácido Cítrico , Perfilação da Expressão Gênica , Ácidos Cetoglutáricos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Folhas de Planta/metabolismo , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismoRESUMO
Tillandsia species are plants from the Bromeliaceae family which display biomonitoring capacities in both active and passive modes. The bioaccumulation potential of Tillandsia aeranthos (Loisiel.) Desf. and Tillandsia bergeri Mez acclimated to Southern/Mediterranean Europe has never been studied. More generally, few studies have detailed the maximum accumulation potential of Tillandsia leaves through controlled experiments. The aim of this study is to evaluate the maximum accumulation values of seven metals (Co, Cu, Mn, Ni, Pb, Pt, and Zn) in T. aeranthos and T. bergeri leaves. Plants were immersed in different mono elemental metallic solutions of Co (II), Cu (II), Mn (II), Ni (II), Pb (II), Pt (IV), and Zn (II) ions at different concentrations. In addition, cocktail solutions of these seven metals at different concentrations were prepared to study the main differences and the potential selectivity between metals. After exposure, the content of these metals in the leaves were measured by inductively coupled plasma-optical emission spectrometry. Data sets were evaluated by a fitted regression hyperbola model and principal component analysis, maximum metal loading capacity, and thermodynamic affinity constant were determined. The results showed important differences between the two species, with T. bergeri demonstrating higher capacity and affinity for metals than T. aeranthos. Furthermore, between the seven metals, Pb and Ni showed higher enrichment factors (EF). T. bergeri might be a better bioaccumulator than T. aeranthos with marked selectivity for Pb and Ni, metals of concern in air quality biomonitoring.
Assuntos
Poluentes Atmosféricos , Monitoramento Ambiental , Metais , Folhas de Planta , Tillandsia , Tillandsia/metabolismo , Folhas de Planta/metabolismo , Poluentes Atmosféricos/metabolismo , Monitoramento Ambiental/métodos , Metais/metabolismo , Espectrofotometria Atômica , Análise de Componente Principal , Análise de Regressão , Bioacumulação , Região do MediterrâneoRESUMO
Pistacia lentiscus L. is a medicinal plant that grows spontaneously throughout the Mediterranean basin and is traditionally used to treat diseases, including diabetes. The aim of this work consists of the evaluation of the α-glucosidase inhibitory effect (i.e., antidiabetic activity in vitro) of different extracts from the leaves, stem barks and fruits of P. lentiscus harvested on mountains and the littoral of Tizi-Ouzou in Algeria. Metabolomic profiling combined with a chemometric approach highlighted the variation of the antidiabetic properties of P. lentiscus according to the plant's part and origin. A multiblock OPLS analysis showed that the metabolites most involved in α-glucosidase inhibition activity were mainly found in the stem bark extracts. The highest inhibitory activity was found for the stem bark extracts, with averaged inhibition percentage values of 84.7% and 69.9% for the harvested samples from the littoral and mountain, respectively. On the other hand, the fruit extracts showed a lower effect (13.6%) at both locations. The UHPLC-ESI-HRMS characterization of the metabolites most likely responsible for the α-glucosidase-inhibitory activity allowed the identification of six compounds: epigallocatechin(4a>8)epigallocatechin (two isomers), (epi)gallocatechin-3'-O-galloyl-(epi)gallocatechin (two isomers), 3,5-O-digalloylquinic acid and dihydroxy benzoic acid pentoside.
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Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by ß-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel ß-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.
Assuntos
Simulação de Dinâmica Molecular , Polissacarídeo-Liases , Polissacarídeo-Liases/química , Glicosídeos , Pectinas/química , Especificidade por SubstratoRESUMO
Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.
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The hop plant (Humulus lupulus L.) has been exploited for a long time for both its brewing and medicinal uses, due in particular to its specific chemical composition. These last years, hop cultivation that was in decline has been experiencing a renewal for several reasons, such as a craze for strongly hopped aromatic beers. In this context, the present work aims at investigating the genetic and chemical diversity of fifty wild hops collected from different locations in Northern France. These wild hops were compared to ten commercial varieties and three heirloom varieties cultivated in the same sampled geographical area. Genetic analysis relying on genome fingerprinting using 11 microsatellite markers showed a high level of diversity. A total of 56 alleles were determined with an average of 10.9 alleles per locus and assessed a significant population structure (mean pairwise FST = 0.29). Phytochemical characterization of hops was based on volatile compound analysis by HS-SPME GC-MS, quantification of the main prenylated phenolic compounds by UHPLC-UV as well as untargeted metabolomics by UHPLC-HRMS and revealed a high level of chemical diversity among the assessed wild accessions. In particular, analysis of volatile compounds revealed the presence of some minor but original compounds, such as aromadendrene, allo-aromadendrene, isoledene, ß-guaiene, α-ylangene and ß-pinene in some wild accessions; while analysis of phenolic compounds showed high content of ß-acids in these wild accessions, up to 2.37% of colupulone. Genetic diversity of wild hops previously observed was hence supported by their chemical diversity. Sample soil analysis was also performed to get a pedological classification of these different collection sites. Results of the multivariate statistical analysis suggest that wild hops constitute a huge pool of chemical and genetic diversity of this species.
Assuntos
Humulus , Humulus/genética , Análise Multivariada , Variação GenéticaRESUMO
VOCs emitted by flowers play an important role in plant ecology. In the past few years, the Tillandsia genus has been scarcely studied according to the VOCs emitted by flowers. Hence, we decided to enlarge the VOCs composition study already undergone in our laboratory on fragrant 3 Tillandsia species to 12 unscented and 2 faint-scented Tillandsia species and hybrids. The headspace solid phase microextraction (HS-SPME) coupled with gas chromatography combined with the mass spectrometry (GC-MS) method was used to explore the chemical diversity of the VOCs. This study allowed the identification of 65 VOCs among the 14 species and between 6 to 25 compounds were identified in each of the species. The aromatic profile of 10 of the species and hybrids are similar to each other's and show 8 predominant compounds: benzaldehyde, benzacetaldehyde, hexanol, hexanal, heptanal, octanal, nonanal, and furan-2-pentyl. Some specific compounds are present only in some unique species such as trans-calamenene, α-muurolene, and α-guaiene trans-ß-bergamotene. The two faint-scented species studied present an original aromatic profile with a high number of monoterpenes or phenylpropanoids/benzenoids. Our studies allow a better understanding of the ecological role and function of these VOCs in the interactions between these plants with their environment.