An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [1H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [1H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories

A DEMETER-like DNA demethylase protein governs tomato fruit ripening

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DML). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening; an important developmental process unique to plants. RNAi SlDML2 knock-down results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomat

Non-structural carbohydrates in woody plants compared among laboratories

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g⁻¹ for soluble sugars, 6–533 (mean = 94) mg g⁻¹ for starch and 53–649 (mean = 153) mg g⁻¹ for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R² = 0.05–0.12 for soluble sugars, 0.10–0.33 for starch and 0.01–0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g⁻¹ for total NSC, compared with the range of laboratory estimates of 596 mg g⁻¹. Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41–0.91), but less so for total NSC (r = 0.45–0.84) and soluble sugars (r = 0.11–0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Oxford University Press. The published article can be found at: http://treephys.oxfordjournals.org/Keywords: soluble sugars, starch, particle size, reference method, standardization, non-structural carbohydrate chemical analysis, extraction and quantification consistenc

Absolute quantitative metabolomics of plant extracts by fast 2D NMR

Absolute quantitative metabolomics of plant extracts by fast 2D NMR. 56. Experimental Nuclear Magnetic Resonance Conferenc

Aluminium stress disrupts metabolic performance of Plantago almogravensis plantlets transiently.

Little is known about how tolerant plants cope with internalized aluminium (Al). Tolerant plants are known to deploy efficient detoxification mechanisms, however it is not known to what extent the primary and secondary metabolism is affected by Al. The aim of this work was to study the metabolic repercussions of Al stress in the tolerant plant Plantago almogravensis. P. almogravensis is well adapted to acid soils where high concentrations of free Al are found and has been classified as a hyperaccumulator. In vitro reared plantlets were used for this purpose in order to control Al exposure rigorously. The metabolome of P. almogravensis plantlets as well as its metabolic response to the supply of sucrose was characterized. The supply of sucrose leads to an accumulation of amino acids and secondary metabolites and consumption of carbohydrates that result from increased metabolic activity. In Al-treated plantlets the synthesis of amino acids and secondary metabolites is transiently impaired, suggesting that P. almogravensis is able to recover from the Al treatment within the duration of the trials. In the presence of Al the consumption of carbohydrate resources is accelerated. The content of some metabolic stress markers also demonstrates that P. almogravensis is highly adapted to Al stress

Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct roles

International audienc

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceIron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis

EMERODE: nuclEAR MagnEtic Resonance prOfiles DatabasE

La spectrométrie RMN 1D proton est largement utilisée pour caractériser des matrices biologiques lorsd’analyses métabolomiques. Un profil RMN 1D proton de matrice biologique est la signature spécifique d’unematrice biologique, résultant de la combinaison de tous les spectres de molécules composants ce mélange. Parconséquent, l’identification de certains composés n’est parfois basée que sur la visualisation d’un seul déplacementchimique. Elle ne peut être validée qu’en ayant recours à des expériences complémentaires RMN 2D de type HSQCou JRES, selon les directives d’identification des métabolites [1, 2] de la Metabolomics Standards Initiative (MSI).L’identification de composés requiert donc une très bonne connaissance de la matrice étudiée ou la visualisation dematrices biologiques similaires, pour aiguiller l’identification.La Plateforme Métabolome de Bordeaux est utilisée, notamment, pour l’établissement et l’interprétation deprofils métaboliques de matrices végétales, animales, microbiennes ou fongiques selon différents facteurs d’étude.Elle recense près de 90 profils différents, dont les spectres et les annotations de composés connus et inconnus sontarchivés sous forme papier. L’exploitation de ces profils, pour de nouvelles annotations, relève donc d’un processuslaborieux pour les analystes. C’est pourquoi il est nécessaire de pouvoir visualiser, capitaliser et organiser cetteconnaissance.Dans le cadre du projet MetaboHUB IA ANR, nous développons EMERODE (nuclEar MagnEtic ResonanceprOfiles DatabasE), une base de données et son interface web dédiée. La base de données stockera les profilsdécrits selon un minimum d’informations essentielles à leurs caractérisations. L’interface dédiée permettra de (i)charger de manière simple les spectres et leurs annotations, (ii) rechercher des profils de matrices selon différentsfiltres, (iii) visualiser et interagir avec le spectre du profil, (iv) annoter/modifier les profils de RMN 1D 1H et 13C.La visualisation du spectre d’un profil sera possible grâce à un nouvel outil, le Spectra Browser. Celui-ci sebasera sur la libraire SpeckTackle [3] qui permet la visualisation et l’interaction avec le spectre. Les annotationsseront projetées sous forme de symboles sur un panneau de visualisation à l’échelle du spectre, et positionné souscelui-ci. Les métabolites identifiés seront reliés aux composés correspondants dans les autres bases de données(HMDB, Chebi) et plus particulièrement PeakForest, la base de données de spectres de références, développéedans le cadre de MetaboHUB, et dont une partie des composés a été acquise dans les mêmes conditionsd’extraction et d’acquisition. Ainsi, chaque nouvelle annotation viendra enrichir la base de données elle-mêmepour de futures annotations