Changes in plant growth, Cd partitioning and xylem sap composition in two sunflower cultivars exposed to low Cd concentrations in hydroponics.

This study characterizes sunflower response to the levels of Cd encountered in moderately Cd-polluted soils. Two sunflower cultivars differing in their ability to sequestrate Cd in roots were exposed to low concentrations of Cd (0.5 nM or 100 nM) in hydroponics and sampled after 18 days (258 degree-days) when ten leaves were fully expanded. Plant growth, Cd uptake and partitioning among organs were monitored along with the ionomic (ICP-MS) and the metabolic (1H-NMR) composition of the xylem sap. Sunflower tolerance to Cd differed between the two cultivars. The cultivar with the highest ability to sequestrate Cd in roots (Kapllan) was more tolerant to Cd than the one with the lowest ability (ES RICA). The 23% penalization of plant growth observed at 100 nM in cultivar ES RICA was associated with reduced xylem loading fluxes of soluble sugars, perhaps pointing to disruption of carbohydrate metabolism. Retention of Cd in the stem was higher at 100 nM than at 0.5 nM in the Cd-sensitive cultivar ES RICA, which can be seen as a sunflower strategy to restrict the amount of Cd delivered to the leaves under Cd stress. No direct connection was found between the speciation of Cd in the xylem sap and the Cd translocation efficiency, although significant changes in the free ionic fraction of Cd were observed between the two cultivars at 0.5 nM. The relevance of these results in promoting the use of sunflower in phytomanagement of Cd-polluted soils is discussed.

Comparative metabolomics reveals how the severity of predation by the invasive insect Cydalima perspectalis modulates the metabolism re-orchestration of native Buxus sempervirens.

The recent biological invasion of box tree moth Cydalima perspectalis on Buxus trees has a major impact on European boxwood stands through severe defoliation. This can hinder further regrowth and threaten survival of populations. In a mesocosm approach and controlled larval density over a 2-month period, responses of B. sempervirens essential and specialized metabolites were characterized using metabolomics, combining 1H-NMR and LC-MS/MS approaches. This is the first metabolome depiction of major Buxus responses to boxwood moth invasion. Under severe predation, remaining green leaves accumulate free amino acids (with the noticeable exception of proline). The leaf trans-4-hydroxystachydrine and stachydrine reached 10-13% and 2-3% (DW), while root content was lower but also modulated by predation level. Larval predation promoted triterpenoid and (steroidal) alkaloid synthesis and diversification, while flavonoids did not seem to have a relevant role in Buxus resistance. Our results reveal the concomitant responses of central and specialized metabolism, in relation to severity of predation. They also confirm the potential of metabolic profiling using 1H-NMR and LC-MS to detect re-orchestration of metabolism of native boxwood after severe herbivorous predation by the invasive box-tree moth, and thus their relevance for plant-insect relationships and ecometabolomics.

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics.

INTRODUCTION: Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks. OBJECTIVES: The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills. METHODS: NMRProcFlow was developed to be a graphical and interactive 1D NMR (1H & 13C) spectra processing tool. RESULTS: NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment. CONCLUSION: Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

Microclimate influence on mineral and metabolic profiles of grape berries.

The grape berry microclimate is known to influence berry quality. The effects of the light exposure of grape berry clusters on the composition of berry tissues were studied on the "Merlot" variety grown in a vineyard in Bordeaux, France. The light exposure of the fruiting zone was modified using different intensities of leaf removal, cluster position relative to azimuth, and berry position in the cluster. Light exposures were identified and classified by in situ measurements of berry temperatures. Berries were sampled at maturity (>19 Brix) for determination of skin and/or pulp chemical and metabolic profiles based on (1) chemical and physicochemical measurement of minerals (N, P, K, Ca, Mg), (2) untargeted 1H NMR metabolic fingerprints, and HPLC targeted analyses of (3) amino acids and (4) phenolics. Each profile defined by partial least-square discriminant analysis allowed us to discriminate berries from different light exposure. Discriminant compounds between shaded and light-exposed berries were quercetin-3-glucoside, kaempferol-3-glucoside, myricetin-3-glucoside, and isorhamnetin-3-glucoside for the phenolics, histidine, valine, GABA, alanine, and arginine for the amino acids, and malate for the organic acids. Capacities of the different profiling techniques to discriminate berries were compared. Although the proportion of explained variance from the 1H NMR fingerprint was lower compared to that of chemical measurements, NMR spectroscopy allowed us to identify lit and shaded berries. Light exposure of berries increased the skin and pulp flavonols, histidine and valine contents, and reduced the organic acids, GABA, and alanine contents. All the targeted and nontargeted analytical data sets used made it possible to discriminate sun-exposed and shaded berries. The skin phenolics pattern was the most discriminating and allowed us to sort sun from shade berries. These metabolite classes can be used to qualify berries collected in an undetermined environment. The physiological significance of light and temperature effects on berry composition is discussed.

Interactions between pyruvate and lactate metabolism in Propionibacterium freudenreichii subsp. shermanii: in vivo (13)C nuclear magnetic resonance studies.

In vivo (13)C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichii subsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 micromol. min(-1). g(-1) (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 micromol. min(-1). g(-1) when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO(2), succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.

In vivo 13C NMR study of the bidirectional reactions of the Wood-Werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici.

This study used in vitro 13C NMR spectroscopy to directly examine bidirectional reactions of the Wood-Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 micromol min(-1) g(-1) (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood-Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.

Determination of metabolic fluxes during glucose catabolism in Propionibacterium freudenreichii subsp. shermanii.

In vivo 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the pathways of glucose metabolism, non-invasively, in living cell suspensions of Propionibacterium freudenreichii subsp. shermanii. This species is the main ripening flora of the Swiss-type cheeses and is widely used as propionic acid and vitamin B12 industrial producer. The flow of labelled [1-13C]glucose was monitored in living cell suspensions and enrichment was detected in main products like [1-13C]glycogen, [6-13C]lycogen, [1-13C]trehalose, [6-13C]trehalose, [1-13C]propionate, [2-13C]propionate, [3-13C]propionate, [1-13C]acetate, [2-13C]acetate, [1-13C]succinate, [2-13C]succinate and [1-13C]CO2. alpha and beta glucose consumption could be examined separately and were catabolized at the same rate. Three intermediates were also found out, namely [1-13C]glucose-6-phosphate, [6-13C]glucose-6-phosphate and [1-13C]glucose-1-phosphate. From the formation of intermediates such as [6-3C]glucose-6-phosphate and products like [6-13C]glycogen from [1-13C]glucose we concluded the bidirectionality of reactions in the first part of glycolysis and the isomerization at the triose-phosphate level. Comparison of spectra obtained after addition of [1-13C]glucose or [U-12C]glucose revealed production of [1-13C]CO2 which means that pentose phosphates pathway is active under our experimental conditions. From the isotopic pattern of trehalose, it could be postulated that trehalose biosynthesis occurred either by direct condensation of two glucose molecules or by gluconeogenesis. A chemically defined medium was elaborated for the study and trehalose was the main osmolyte found in the intracellular fraction of P. shermanii grown in this medium.