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Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography–mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.

A novel bacterium, designated strain Msb3T, was recently isolated from leaves of the yam family plant Dioscorea bulbifera (Dioscoreaceae). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Paraburkholderia with Paraburkholderia xenovorans as nearest validly named neighbour taxon (99.3 % sequence similarity towards the P. xenovorans type strain). Earlier genome sequence analysis revealed a genome of 8.35 Mb in size with a G+C content of 62.5 mol%, which was distributed over two chromosomes and three plasmids. Here, we confirm that strain Msb3T represents a novel Paraburkholderia species. In silico DNA–DNA hybridization and average nucleotide identity (OrthoANIu) analyses towards P. xenovorans LB400T yielded 58.4 % dDDH and 94.5 % orthoANIu. Phenotypic and metabolic characterization revealed growth at 15 °C on tryptic soy agar, growth in the presence of 1 % NaCl and the lack of assimilation of phenylacetic acid as distinctive features. Together, these data demonstrate that strain Msb3T represents a novel species of the genus Paraburkholderia, for which we propose the name Paraburkholderia dioscoreae sp. nov. The type strain is Msb3T (=LMG 31881T, DSM 111632T, CECT 30342T).

Shifts in the duration and intensity of ambient temperature impair plant development and reproduction, particularly male gametogenesis. Stress exposure causes meiotic defects or premature spore abortion in male reproductive organs, leading to male sterility. However, little is known about the mechanisms underlying stress and male sterility. To elucidate these mechanisms, we imposed a moderate transient heat stress on maize (Zea mays) plants at the tetrad stage of pollen development. After completion of pollen development at optimal conditions, stress responses were assessed in mature pollen. Transient heat stress resulted in reduced starch content, decreased enzymatic activity, and reduced pollen germination, resulting in sterility. A transcriptomic comparison pointed toward misregulation of starch, lipid, and energy biosynthesis-related genes. Metabolomic studies showed an increase of Suc and its monosaccharide components, as well as a reduction in pyruvate. Lipidomic analysis showed increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids. In contrast, the majority of genes involved in developmental processes such as those required for auxin and unfolded protein responses, signaling, and cell wall biosynthesis remained unaltered. It is noteworthy that changes in the regulation of transcriptional and metabolic pathway genes, as well as heat stress proteins, remained altered even though pollen could recover during further development at optimal conditions. In conclusion, our findings demonstrate that a short moderate heat stress during the highly susceptible tetrad stage strongly affects basic metabolic pathways and thus generates germination-defective pollen, ultimately leading to severe yield losses in maize.

Roots secrete a vast array of low molecular weight compounds into the soil broadly referred to as root exudates. It is a key mechanism by which plants and soil microbes interact in the rhizosphere. The effect of drought stress on the exudation process and composition is rarely studied, especially in cereal crops. This study focuses on comparative metabolic profiling of the exudates from sensitive and tolerant genotypes of pearl millet after a period of drought stress. We employed a combined platform of gas and liquid chromatography coupled to mass spectrometry to cover both primary and secondary metabolites. The results obtained demonstrate that both genotype and drought stress have a significant impact on the concentration and composition of root exudates. The complexity and function of these differential root exudates are discussed. To reveal the potential effect of root exudates on the soil microbial community after a period of drought stress, we also tested for biological nitrification inhibition (BNI) activity. The analysis revealed a genotype-dependent enhancement of BNI activity after a defined period of drought stress. In parallel, we observed a genotype-specific relation of elongated root growth and root exudation under drought stress. These data suggest that the drought stress-dependent change in root exudation can manipulate the microbial soil communities to adapt and survive under harsh conditions.The online version contains supplementary material available at 10.1007/s00374-021-01578-w.

Experiments involving food restriction are common practice in metabolic research. Under fasted conditions, mice supplement their diet with cage bedding. We aimed at identifying metabolic and microbiota-related parameters affected by the bedding type. We exposed mice housed with wooden, cellulose, or corncob cage beddings to ad libitum feeding, caloric restriction (CR), or over-night (ON) fasting. Additionally, two subgroups of the ON fast group were kept without any bedding or on a metal grid preventing coprophagy. Mice under CR supplemented their diet substantially with bedding; however, the amount varied depending on the kind of bedding. Bedding-related changes in body weight loss, fat loss, cecum size, stomach weight, fecal output, blood ghrelin levels as well as a response to glucose oral tolerance test were recorded. As fiber is fermented by the gut bacteria, the type of bedding affects gut bacteria and fecal metabolites composition of CR mice. CR wood and cellulose groups showed distinct cecal metabolite and microbiome profiles when compared to the CR corncob group. While all ad libitum fed animal groups share similar profiles. We show that restriction-related additional intake of bedding-derived fiber modulates multiple physiological parameters. Therefore, the previous rodent studies on CR, report the combined effect of CR and increased fiber consumption.

The growing problem with the irreproducibility of the results of metabolic research urged us to analyse potential critical factors contributing to the disparities. Microbiota composition constitutes crucial variable in animal research. Gut flora changes depending on multiple factors including fibre intake. Mice derive fibre from diet but, especially under fasted conditions, also from bedding ingestion. Our project aims at assessing the impact of cage bedding on several aspects of metabolic research. Mice were divided into an overnight (ON) fasted, caloric restricted (CR) and ad libitum groups. In order to evaluate the effects of different beddings, mice were housed in cages with the most commonly used beddings: wooden, corncob or cellulose. Additionally, ON-fasted mice were kept without bedding or on metal grid preventing coprophagy. Mice bedding consumption and faeces production were measured. Oral glucose tolerance test (OGTT) was performed. Metabolites composition and microbial diversity were analysed in caecum content. Mice organ weight and gene expression measurements were conducted. Mice show preference towards wooden bedding and consume it in highest amount followed by corncob and cellulose beddings. As a result of the bedding ingestion, caecum weight increases in the CR compared to ad libitum fed mice. CR leads to a reduced faeces production and it is the lowest in the CR wood and CR cellulose groups. Housing on metal grid results in greater body weight loss and decreases stomach and caecum size. ON-fasted mice housed without bedding or on grids show lower glucose levels when submitted to OGTT. CR influences the caecal bacterial diversity as well as the caecal metabolic profile. In the CR group, mice on the corncob bedding show a distinct caecal microbial composition and caecal metabolic profile compared to wooden and cellulose groups. CR is associated with increased abundance of Marinifilaceaefrom the phylum Bacteroidales, and Erysipelotrichaceaefrom the phylum Firmicutes and with a decreased abundance of Lachnospiraceaefrom the phylum Firmicutes. The main metabolites effected by CR are organic acids, fructose, sucrose and glutamine. The type of bedding exerts the strongest effect on valeric acid, lysine, threonine and organic acids. CR- and ON-fasting-associated weight loss, bedding consumption and fibre accumulation in the caecum depends on the bedding type. CR mice housed on wooden and cellulose beddings show similarities and are distinguishable from CR corncob group. Thus, depending on the composition and consumed amount, bedding intake affects numerous parameters and shapes the microbiome.

Climate warming affects plant physiology through genetic adaptation and phenotypic plasticity, but little is known about how these mechanisms influence ecosystem processes. We used three elevation gradients and a reciprocal transplant experiment to show that temperature causes genetic change in the sedge Eriophorum vaginatum. We demonstrate that plants originating from warmer climate produce fewer secondary compounds, grow faster and accelerate carbon dioxide (CO2) release to the atmosphere. However, warmer climate also caused plasticity in E. vaginatum, inhibiting nitrogen metabolism, photosynthesis and growth and slowing CO2 release into the atmosphere. Genetic differentiation and plasticity in E. vaginatum thus had opposing effects on CO2 fluxes, suggesting that warming over many generations may buffer, or reverse, the short‐term influence of this species over carbon cycle processes. Our findings demonstrate the capacity for plant evolution to impact ecosystem processes, and reveal a further mechanism through which plants will shape ecosystem responses to climate change. ISSN:1461-023X ISSN:1461-0248

Metabolite changes and senescence behaviour after mechanical phloem girdling were studied in leaf tissue of Quercus pubescens. Sugar accumulation is not only considered to be an important part of several developmental signalling pathways, but is also seen as one of the basic triggers for senescence induction, or at least an obligatory accessory phenomenon. Our survey showed that an accumulation of the soluble sugars, glucose and fructose, was not on its own obligatorily connected with the induction of leaf senescence, since no indication or even an onset of senescence could be observed during the course of the experiment. Instead, we observed an inhibition of leaf development with a decrease of photosynthesis and a slow-down of development in nearly all chlorophyll a fluorescence analysis parameters using the JIP-test. We detected a change of metabolites linked to oxidative stress, possibly due to an overexcitation of the developmentally inhibited photosynthetic apparatus.

We investigated the response, adaptation and tolerance mechanisms of the temperate seagrass Zostera marina to water column anoxia. We exposed Z. marina to a diurnal light/dark cycle under anoxia and assessed the metabolic response by measuring the metabolome with gas chromatography coupled to mass spectrometry (GC–MS). During anoxia and light exposure the roots showed an altered metabolome whereas the leaves were only marginally affected, indicating that photosynthetically derived oxygen could satisfy the oxygen demand in the leaves but not in the roots. Nocturnal anoxia caused a biphasic shift in the metabolome of roots and leaves. The first phase, after 15 h under anoxia and 3 h of darkness showed a fast increase of lactate, pyruvate, GABA (γ-aminobutyric acid), succinate, alanine and a decrease in glutamate and glutamine. The second phase, after 21 h under anoxia and 9 h of darkness showed a decrease in lactate and pyruvate and an increase in alanine, GABA and succinate. This reprogramming of the metabolome after 21 h under anoxia indicates a possible mitigation mechanism to avoid the toxic effects of anoxia. A pathway enrichment analysis proposes the alanine shunt, the GABA shunt and the 2-oxoglutarate shunt as such mitigation mechanisms that alleviate pyruvate levels and lead to carbon and nitrogen storage during anoxia. This work demonstrates the applicability of metabolomics to assess low oxygen stress responses of Z. marina and allows us to propose an anoxia recovery model.

The metabolome of an organism represents the readout of its biochemistry comprising numerous and tightly regulated metabolic pathways. Experimental analysis of the metabolome and its interpretation in a biochemically and physiologically meaningful context is focused by the research field of metabolomics which has become an integral part of many systems biological studies. Pollen development, germination and tube growth comprise numerous steps of metabolic regulation resulting in significant metabolome dynamics. To unravel involved regulatory molecular processes and to promote the understanding of developmental reprogramming and stress tolerance mechanisms in pollen, it is crucial to quantitatively resolve dynamics in the pollen metabolome. Since these dynamics affect various substance groups with different physico-chemical properties, different experimental platforms are needed for robust compound identification and quantification. It has been shown that developmentally and stress-induced metabolic reprogramming in pollen significantly affects the redox homeostasis as well as metabolism of carbohydrates, amino acids, lipids, polyamines, flavonoids and phytohormones. In this chapter, mechanisms of metabolic reprogramming are summarized and discussed in the context of pollen development and stress exposure. Finally, it is discussed how these metabolome dynamics can be resolved methodologically in order to unravel molecular physiological mechanisms of pollen development.

© The Author 2014. Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.

Investigation of the metabolome and the transcriptome of pollen of lily (Lilium longiflorum) gave a comprehensive overview of metabolic pathways active during pollen germination and tube growth. More than 100 different metabolites were determined simultaneously by gas chromatography coupled to mass spectrometry, and expressed genes of selected metabolic pathways were identified by next-generation sequencing of lily pollen transcripts. The time-dependent changes in metabolite abundances, as well as the changes after inhibition of the mitochondrial electron transport chain, revealed a fast and dynamic adaption of the metabolic pathways in the range of minutes. The metabolic state prior to pollen germination differed clearly from the metabolic state during pollen tube growth, as indicated by principal component analysis of all detected metabolites and by detailed observation of individual metabolites. For instance, the amount of sucrose increased during the first 60 minutes of pollen culture but decreased during tube growth, while glucose and fructose showed the opposite behavior. Glycolysis, tricarbonic acid cycle, glyoxylate cycle, starch, and fatty acid degradation were activated, providing energy during pollen germination and tube growth. Inhibition of the mitochondrial electron transport chain by antimycin A resulted in an immediate production of ethanol and a fast rearrangement of metabolic pathways, which correlated with changes in the amounts of the majority of identified metabolites, e.g. a rapid increase in γ-aminobutyric acid indicated the activation of a γ-aminobutyric acid shunt in the tricarbonic acid cycle, while ethanol fermentation compensated the reduced ATP production after inhibition of the oxidative phosphorylation.

Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development.

Flavescence dorée, caused by the quarantine phytoplasma FDp, represents the most devastating of the grapevine yellows diseases in Europe. In an integrated study we have explored the FDp-grapevine interaction in infected grapevines of cv. "Modra frankinja" under natural conditions in the vineyard. In FDp-infected leaf vein-enriched tissues, the seasonal transcriptional profiles of 14 genes selected from various metabolic pathways showed an FDp-specific plant response compared to other grapevine yellows and uncovered a new association of the SWEET17a vacuolar transporter of fructose with pathogens. Non-targeted metabolome analysis from leaf vein-enriched tissues identified 22 significantly changed compounds with increased levels during infection. Several metabolites corroborated the gene expression study. Detailed investigation of the dynamics of carbohydrate metabolism revealed significant accumulation of sucrose and starch in the mesophyll of FDp-infected leaves, as well as significant up-regulation of genes involved in their biosynthesis. In addition, infected leaves had high activities of ADP-glucose pyrophosphorylase and, more significantly, sucrose synthase. The data support the conclusion that FDp infection inhibits phloem transport, resulting in accumulation of carbohydrates and secondary metabolites that provoke a source-sink transition and defense response status.

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramat...

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.

Paenibacillus sp. P22 is a Gram-negative facultative anaerobic endospore-forming bacterium isolated from poplar hybrid 741 (♀[ Populus alba × ( P. davidiana + P. simonii ) × P. tomentosa ]). This bacterium shows strong similarities to Paenibacillus humicus , and important growth-promoting effects on in vitro grown explants of poplar hybrid 741 have been described.

SummaryBird pollination has evolved repeatedly among flowering plants but is almost exclusively characterized by passive transfer of pollen onto the bird and by nectar as primary reward [1, 2]. Food body rewards are exceedingly rare among eudicot flowering plants and are only known to occur on sterile floral organs [3]. In this study, we report an alternative bird pollination mechanism involving bulbous stamen appendages in the Neotropical genus Axinaea (Melastomataceae). We studied the pollination process by combining pollination experiments, video monitoring, and detailed analyses of stamen structure and metabolomic composition. We show that the bulbous stamen appendages, which are consumed by various species of passerines (Thraupidae, Fringillidae), are bifunctional during the pollination process. First, the appendages work as bellows organs in a unique pollen expulsion mechanism activated by the passerines. As the birds seize an appendage with their beaks in order to remove it from the flower for consumption, air contained in the appendage’s aerenchymatous tissue is pressed into the hollow anther. The resulting air flow causes the expulsion of a pollen jet and the deposition of pollen on the bird’s head and beak. Second, the stamen appendages provide a hexose-rich, highly nutritious (15,100 J/g) food body reward for the pollinating passerines. This discovery expands our knowledge of flowering plant pollination systems and provides the first report of highly specialized bellows organs for active pollen transfer in flowering plants. In addition, this is the only known case of a food body reward associated with reproductive structures in the eudicot clade of flowering plants.PaperFlick

Metabolomics science has emerged as a corner stone technology in functional genomics studies. For explorative and comprehensive metabolomic studies GC-MS developed into a gold standard method in the last decade. In the following chapter we will discuss some aspects of GC-MS based metabolomics.

Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methylcoenzymeM reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymesinvolved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

Potentilla anserina L. (Rosaceae) is known for its beneficial effects of prevention of pre-menstrual syndrome (PMS). For this reason P. anserina is processed into many food supplements and pharmaceutical preparations. Here we analyzed hydroalcoholic reference extracts and compared them with various extracts of different pharmacies using an integrative metabolomics platform comprising GC-MS and LC-MS analysis and software toolboxes for data alignment (MetMAX Beta 1.0) and multivariate statistical analysis (COVAIN 1.0). Multivariate statistics of the integrated GC-MS and LC-MS data showed strong differences between the different plant extract formulations. Different groups of compounds such as chlorogenic acid, kaempferol 3-O-rutinoside, acacetin 7-O-rutinoside, and genistein were reported for the first time in this species. The typical fragmentation pathway of the isoflavone genistein confirmed the identification of this active compound that was present with different abundances in all the extracts analyzed. As a result we have revealed that different extraction procedures from different vendors produce different chemical compositions, e.g. different genistein concentrations. Consequently, the treatment may have different effects. The integrative metabolomics platform provides the highest resolution of the phytochemical composition and a mean to define subtle differences in plant extract formulations. Electronic supplementary material The online version of this article (doi:10.1007/s11306-012-0473-x) contains supplementary material, which is available to authorized users.

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified. Electronic supplementary material The online version of this article (doi:10.1007/s11306-012-0470-0) contains supplementary material, which is available to authorized users.

Resources structure ecological communities and potentially link biodiversity to energy flow. It is commonly believed that functional traits (generalists versus specialists) involved in the exploitation of resources depend on resource availability and environmental fluctuations. The longitudinal nature of stream ecosystems provides changing resources to stream biota with yet unknown effects on microbial functional traits and community structure. We investigated the impact of autochthonous (algal extract) and allochthonous (spruce extract) resources, as they change along alpine streams from above to below the treeline, on microbial diversity, community composition and functions of benthic biofilms. Combining bromodeoxyuridine labelling and 454 pyrosequencing, we showed that diversity was lower upstream than downstream of the treeline and that community composition changed along the altitudinal gradient. We also found that, especially for allochthonous resources, specialisation by biofilm bacteria increased along that same gradient. Our results suggest that in streams below the treeline biofilm diversity, specialisation and functioning are associated with increasing niche differentiation as potentially modulated by divers allochthonous and autochthonous constituents contributing to resources. These findings expand our current understanding on biofilm structure and function in alpine streams.

Summary Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages., Graphical Abstract, Highlights • Metabolomics and inverse modeling reveal a Tsc2/mTORC1-dependent checkpoint in macrophages • M2 macrophages have high Phgdh activity • Phgdh activity promotes M2 polarization • Phgdh activity supports macrophage proliferation, Wilson et al. show that Tsc2, a negative regulator of mTORC1 signaling, critically influences the metabolome of macrophages. Inverse data-driven modeling and multiomics data reveal that Phgdh is an mTORC1-dependent metabolic checkpoint of macrophage proliferation and polarization. Phgdh is required for the expression of key anti-inflammatory molecules and M2 proliferation.