Mickaël Maucourt

Quantitative metabolic profiling by 1-dimensional 1H-NMR analyses: application to plant genetics and functional genomics

Metabolic profiling by 1-dimensional (1-D) 1H-nuclear magnetic resonance (NMR) was tested for absolute quantification of soluble sugars, organic acids, amino acids and some secondary metabolites in fruit, roots and leaves. The metabolite responsible for each peak of the 1H-NMR spectra was identified from spectra of pure compounds. Peak identity was confirmed by the addition of a small amount of commercially-available pure substance. 1H-NMR spectra acquisition was automated. 1H-NMR absolute quantification was performed with a synthesised electronic reference signal and validated by comparison with enzymatic or HPLC analyses; the correlation coefficients between 1H-NMR data and enzymatic or HPLC data were highly significant. Depending on the species and tissues, 14-17 metabolites could be quantified with 15-25 min acquisition time. The detection limit was approximately 1-9 µg in the NMR tube, depending on the compound. Quantitative data were used for (1) a genetic study of strawberry fruit quality, (2) a functional study of tomato transformants overexpressing hexokinase and (3) a study of Arabidopsis phosphoenolpyruvate carboxylase transformants with several lines showing decreased activity of the enzyme. Biochemical phenotyping of the fruits of a strawberry offspring allowed the detection of quantitative trait loci (QTL) controlling fruit quality. Comparison of the roots of wild types and hexokinase tomato transformants using principal component analysis of metabolic profiles revealed that environmental factors, i.e. culture conditions, can significantly modify the metabolic status of plants and thus hide or emphasise the expression of a given genetic background. The decrease in phosphoenolpyruvate carboxylase activity (up to 75%) in Arabidopsis transformants impacted on the metabolic profiles without compromising plant growth, thus supporting the idea that the enzyme has a low influence on the carbon flux through the anaplerotic pathway.

1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: Application to spatial metabolite analysis in melon

A metabolomics approach combining (1)H NMR and gas chromatography-electrospray ionization time-of-flight mass spectrometry (GC-EI-TOFMS) profiling was employed to characterize melon (Cucumis melo L.) fruit. In a first step, quantitative (1)H NMR of polar extracts and principal component analyses (PCA) of the corresponding data highlighted the major metabolites in fruit flesh, including sugars, organic acids, and amino acids. In a second step, the spatial localization of metabolites was investigated using both analytical techniques. Direct (1)H NMR profiling of juice or GC-EI-TOFMS profiling of tissue extracts collected from different locations in the fruit flesh provided information on advantages and drawbacks of each technique for the analysis of a sugar-rich matrix such as fruit. (1)H NMR and GC-EI-TOFMS data sets were compared using independently performed PCA and multiblock hierarchical PCA (HPCA), respectively. In addition a correlation-based multiblock HPCA was used for direct comparison of both analytical data sets. These data analyses revealed several gradients of metabolites in fruit flesh which can be related with differences in metabolism and indicated the suitability of multiblock HPCA for correlation of data from two (or potentially more) metabolomics platforms.

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 R(2) = 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.

A DEMETER-like DNA demethylase governs tomato fruit ripening

Significance This work shows that active DNA demethylation governs ripening, an important plant developmental process. Our work defines a molecular mechanism, which has until now been missing, to explain the correlation between genomic DNA demethylation and fruit ripening. It demonstrates a direct cause-and-effect relationship between active DNA demethylation and induction of gene expression in fruits. The importance of these findings goes far beyond understanding the developmental biology of ripening and provides an innovative strategy for its fine control through fine modulation of epimarks in the promoters of ripening related genes. Our results have significant application for plant breeding especially in species with limited available genetic variation. In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a 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 knockdown 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 tomato.

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.

Sugar Import and Phytopathogenicity of Spiroplasma citri: Glucose and Fructose Play Distinct Roles

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.

Metabolic acclimation to hypoxia revealed by metabolite gradients in melon fruit.

A metabolomics approach using (1)H NMR and GC-MS profiling of primary metabolites and quantification of adenine nucleotides with luciferin bioluminescence was employed to investigate the spatial changes of metabolism in melon fruit. Direct (1)H NMR profiling of juice collected from different locations in the fruit flesh revealed several gradients of metabolites, e.g. sucrose, alanine, valine, GABA or ethanol, with increase in concentrations from the periphery to the center of the fruit. GC-MS profiling of ground samples revealed gradients for metabolites not detected using (1)H NMR, including pyruvic and fumaric acids. The quantification of adenine nucleotides highlighted a strong decrease in both ATP and ADP ratios and the adenylate energy charge from the periphery to the center of the fruit. These concentration patterns are consistent with an increase in ethanol fermentation due to oxygen limitation and were confirmed by observed changes in alanine and GABA concentrations, as well as other markers of hypoxia in plants. Ethanol content in melon fruit can affect organoleptic properties and consumer acceptance. Understanding how and when fermentation occurred can help to manage the culture and limit ethanol production.

The Grapevine fleshless berry Mutation. A Unique Genotype to Investigate Differences between Fleshy and Nonfleshy Fruit

In flowering plants, fruit morphogenesis is a distinct process following fertilization resulting in the formation of a specialized organ associated with seeds. Despite large variations in types and shapes among species, fleshy fruits share common characteristics to promote seed dispersal by animals such as organ growth and metabolite accumulation to attract animal feeding. The molecular biology of fruit ripening has received considerable attention, but little is known about the determinism of early fruit morphogenesis and why some fruits are fleshy while others lack flesh. We have identified in grapevine (Vitis vinifera) a mutation we have named fleshless berry (flb) that reduces by 20 times the weight of the pericarp at ripening without any effect on fertility or seed size and number. The flb mutation strongly impaired division and differentiation of the most vacuolated cells in the inner mesocarp. The timing of ripening was not altered by the mutation although the accumulation of malic acid in the green stage was noticeably reduced while sucrose content (instead of hexoses) increased during ripening. The mutation segregates as a single dominant locus. These results indicate that the Flb− mutant is suitable material to advance our understanding of the genetic and developmental processes involved in the differentiation of an ovary into a fruit.

Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis

BackgroundHolistic profiling and systems biology studies of nutrient availability are providing more and more insight into the mechanisms by which gene expression responds to diverse nutrients and metabolites. Less is known about the mechanisms by which gene expression is affected by endogenous metabolites, which can change dramatically during development. Multivariate statistics and correlation network analysis approaches were applied to non-targeted profiling data to investigate transcriptional and metabolic states and to identify metabolites potentially influencing gene expression during the heterotrophic to autotrophic transition of seedling establishment.ResultsMicroarray-based transcript profiles were obtained from extracts of Arabidopsis seeds or seedlings harvested from imbibition to eight days-old. 1H-NMR metabolite profiles were obtained for corresponding samples. Analysis of transcript data revealed high differential gene expression through seedling emergence followed by a period of less change. Differential gene expression increased gradually to day 8, and showed two days, 5 and 7, with a very high proportion of up-regulated genes, including transcription factor/signaling genes. Network cartography using spring embedding revealed two primary clusters of highly correlated metabolites, which appear to reflect temporally distinct metabolic states. Principle Component Analyses of both sets of profiling data produced a chronological spread of time points, which would be expected of a developmental series. The network cartography of the transcript data produced two distinct clusters comprising days 0 to 2 and days 3 to 8, whereas the corresponding analysis of metabolite data revealed a shift of day 2 into the day 3 to 8 group. A metabolite and transcript pair-wise correlation analysis encompassing all time points gave a set of 237 highly significant correlations. Of 129 genes correlated to sucrose, 44 of them were known to be sucrose responsive including a number of transcription factors.ConclusionsMicroarray analysis during germination and establishment revealed major transitions in transcriptional activity at time points potentially associated with developmental transitions. Network cartography using spring-embedding indicate that a shift in the state of nutritionally important metabolites precedes a major shift in the transcriptional state going from germination to seedling emergence. Pair-wise linear correlations of transcript and metabolite levels identified many genes known to be influenced by metabolites, and provided other targets to investigate metabolite regulation of gene expression during seedling establishment.

Proton NMR quantitative profiling for quality assessment of greenhouse-grown tomato fruit

Tomato is an essential crop in terms of economic importance and nutritional quality. In France, the third most important region for tomato (Solanum lycopersicum L.) production is Aquitaine where the major part of production is now grown soilless under greenhouse conditions with harvest from March to November. Tomato fruit quality at harvest is a direct function of its metabolite content at that time. The aim of this work was to use a global approach to characterize changes in the fruit organoleptic quality at harvest under commercial culture conditions during an entire season for two varieties and two different fertilization practices (with or without recycling of the nutrient solution) for one variety. Absolute quantification data of 32 major compounds in fruit without seeds were obtained through untargeted (proton nuclear magnetic resonance, 1H-NMR) quantitative profiling. These data were complemented by colorimetric analysis of ascorbate and total phenolics. They were analyzed with chemometric approaches. Principal component analysis (PCA) or partial least square analyses (PLS) revealed more discriminant metabolites for season than for variety and showed that nutrient solution recycling had very little effect on fruit composition. These tendencies were confirmed with univariate analyses. 1H-NMR profiling complemented with colorimetric analyses therefore provided a diagnostic tool to follow the changes in organoleptic and nutritional quality of tomato. In addition the quantitative information generated will help to increase our knowledge on the mechanisms of plant response to environmental modifications.