The main goal of metabolomics is the comprehensive qualitative and quantitative analysis of the time‐ and space‐resolved distribution of all metabolites present in a given biological system. Because ...metabolite structures, in contrast to transcript and protein sequences, are not directly deducible from the genomic DNA sequence, the massive increase in genomic information is only indirectly of use to metabolomics, leaving compound annotation as a key problem to be solved by the available analytical techniques. Furthermore, as metabolites vary widely in both concentration and chemical behavior, there is no single analytical procedure allowing the unbiased and comprehensive structural elucidation and determination of all metabolites present in a given biological system. In this review the different approaches for targeted and non‐targeted metabolomics analysis will be described with special emphasis on mass spectrometry‐based techniques. Particular attention is given to approaches which can be employed for the annotation of unknown compounds. In the second part, the different experimental approaches aimed at tissue‐specific or subcellular analysis of metabolites are discussed including a range of non‐mass spectrometry based technologies.
The elucidation of complex biological systems requires integration of multiple molecular parameters. Accordingly, high throughput methods like transcriptomics, proteomics, metabolomics and lipidomics ...have emerged to provide the tools for successful system-wide investigations. Unfortunately, optimized analysis of different compounds requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient extraction protocols often only cover a restricted number of compounds due to the different physico-chemical properties of these biological compounds. Consequently, comprehensive analysis of several molecular components like polar primary metabolites next to lipids or proteins require multiple aliquots to enable the specific extraction procedures required to cover these diverse compound classes. This multi-parallel sample handling of different sample aliquots is therefore not only more sample intensive, it also requires more time and effort to obtain the required extracts.
To circumvent large sample amounts, distributed into several aliquots for the comprehensive extraction of most relevant biological compounds, we developed a simple, robust and reproducible two-phase liquid-liquid extraction protocol. This one-step extraction protocol allows for the analysis of polar-, semi-polar and hydrophobic metabolites, next to insoluble or precipitated compounds, including proteins, starch and plant cell wall components, from a single sample. The method is scalable regarding the used sample amounts but also the employed volumes and can be performed in microcentrifuge tubes, enabling high throughput analysis. The obtained fractions are fully compatible with common analytical methods, including spectroscopic, chromatographic and mass spectrometry-based techniques. To document the utility of the described protocol, we used 25 mg of
rosette leaves for the generation of multi-omics data sets, covering lipidomics, metabolomics and proteomics. The obtained data allowed us to measure and annotate more than 200 lipid compounds, 100 primary metabolites, 50 secondary metabolites and 2000 proteins.
The described extraction protocol provides a simple and straightforward method for the efficient extraction of lipids, metabolites and proteins from minute amounts of a single sample, enabling the targeted but also untargeted high-throughput analyses of diverse biological tissues and samples.
Flux phenotypes predicted by constraint‐based methods can be refined by the inclusion of heterogeneous data. While recent advances facilitate the integration of transcriptomics and proteomics data, ...purely stoichiometry‐based approaches for the prediction of flux phenotypes by considering metabolomics data are lacking. Here we propose a constraint‐based method, termed TREM‐Flux, for integrating time‐resolved metabolomics and transcriptomics data. We demonstrate the applicability of TREM‐Flux in the dissection of the metabolic response of Chlamydomonas reinhardtii to rapamycin treatment by integrating the expression levels of 982 genes and the content of 45 metabolites obtained from two growth conditions. The findings pinpoint cysteine and methionine metabolism to be most affected by the rapamycin treatment. Our study shows that the integration of time‐resolved unlabeled metabolomics data in addition to transcriptomics data can specify the metabolic pathways involved in the system's response to a studied treatment.
Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must ...mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.
The Target of Rapamycin (TOR) kinase is a central regulator of growth and metabolism in all eukaryotic organisms, including animals, fungi, and plants. Even though the inputs and outputs of TOR ...signaling are well characterized for animals and fungi, our understanding of the upstream regulators of TOR and its downstream targets is still fragmentary in photosynthetic organisms. In this study, we employed the rapamycin-sensitive green alga Chlamydomonas reinhardtii to elucidate the molecular cause of the amino acid accumulation that occurs after rapamycin-induced inhibition of TOR. Using different growth conditions and stable 13C- and 15N-isotope labeling, we show that this phenotype is accompanied by increased nitrogen (N) uptake, which is induced within minutes of TOR inhibition. Interestingly, this increased N influx is accompanied by increased activities of glutamine synthetase and glutamine oxoglutarate aminotransferase, the main N-assimilating enzymes, which are responsible for the rise in levels of several amino acids, which occurs within a few minutes. Accordingly, we conclude that even though translation initiation and autophagy have been reported to be the main downstream targets of TOR, the upregulation of de novo amino acid synthesis seems to be one of the earliest responses induced after the inhibition of TOR in Chlamydomonas.
Summary
The target of rapamycin (TOR) pathway is a major regulator of growth in all eukaryotes, integrating energy, nutrient and stress signals into molecular decisions. By using large‐scale MS‐based ...metabolite profiling of primary, secondary and lipid compounds in combination with array‐based transcript profiling, we show that the TOR protein not only regulates growth but also influences nutrient partitioning and central energy metabolism. The study was performed on plants exhibiting conditional down‐regulation of AtTOR expression, revealing strong regulation of genes involved in pathways such as the cell cycle, cell‐wall modifications and senescence, together with major changes in transcripts and metabolites of the primary and secondary metabolism. In agreement with these results, our morphological and metabolic analyses disclosed major metabolic changes leading to massive accumulations of storage lipids and starch. The implications of these data in the context of the general role of TOR in eukaryotic systems are discussed in parallel with the plant‐specific aspects of TOR function. Finally, we propose a role for harnessing the plant TOR pathway by utilizing it as a potent metabolic switch, offering a possible route for biotechnological optimization of plant energy content and carbon partitioning for the production of bioenergy.
Lipids are essential to brain functions, yet they remain largely unexplored. Here we investigated the lipidome composition of prefrontal cortex gray matter in 396 cognitively healthy individuals with ...ages spanning 100 years, as well as 67 adult individuals diagnosed with autism (ASD), schizophrenia (SZ), and Down syndrome (DS). Of the 5024 detected lipids, 95% showed significant age-dependent concentration differences clustering into four temporal stages, and resulting in a gradual increase in membrane fluidity in individuals ranging from newborn to nonagenarian. Aging affects 14% of the brain lipidome with late-life changes starting predominantly at 50-55 years of age-a period of general metabolic transition. All three diseases alter the brain lipidome composition, leading-among other things-to a concentration decrease in glycerophospholipid metabolism and endocannabinoid signaling pathways. Lipid concentration decreases in SZ were further linked to genetic variants associated with disease, indicating the relevance of the lipidome changes to disease progression.
Astrocytes represent central regulators of brain glucose metabolism and neuronal function. They have recently been shown to adapt their function in response to alterations in nutritional state ...through responding to the energy state-sensing hormones leptin and insulin. Here, we demonstrate that glucagon-like peptide (GLP)-1 inhibits glucose uptake and promotes β-oxidation in cultured astrocytes. Conversely, postnatal GLP-1 receptor (GLP-1R) deletion in glial fibrillary acidic protein (GFAP)-expressing astrocytes impairs astrocyte mitochondrial integrity and activates an integrated stress response with enhanced fibroblast growth factor (FGF)21 production and increased brain glucose uptake. Accordingly, central neutralization of FGF21 or astrocyte-specific FGF21 inactivation abrogates the improvements in glucose tolerance and learning in mice lacking GLP-1R expression in astrocytes. Collectively, these experiments reveal a role for astrocyte GLP-1R signaling in maintaining mitochondrial integrity, and lack of GLP-1R signaling mounts an adaptive stress response resulting in an improvement of systemic glucose homeostasis and memory formation.
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•GLP-1 inhibits glucose uptake and promotes β-oxidation in cultured astrocytes•Lack of astrocyte GLP-1R in vivo activates a stress response and increases FGF21•Adaptations to astrocyte GLP-1R deletion improve glucose metabolism and memory
Astrocytes regulate brain glucose metabolism and neuronal function. Timper et al. describe a role for astrocyte GLP-1R signaling in maintaining mitochondrial integrity and demonstrate that lack of astrocyte GLP-1R signaling mounts an adaptive stress response to increase astrocyte FGF21 expression resulting in an improvement of systemic glucose homeostasis and memory formation.
Developmental senescence is a coordinated physiological process in plants and is critical for nutrient redistribution from senescing leaves to newly formed sink organs, including young leaves and ...developing seeds. Progress has been made concerning the genes involved and the regulatory networks controlling senescence. The resulting complex metabolome changes during senescence have not been investigated in detail yet. Therefore, we conducted a comprehensive profiling of metabolites, including pigments, lipids, sugars, amino acids, organic acids, nutrient ions, and secondary metabolites, and determined approximately 260 metabolites at distinct stages in leaves and siliques during senescence in Arabidopsis (Arabidopsis thaliana). This provided an extensive catalog of metabolites and their spatiotemporal cobehavior with progressing senescence. Comparison with silique data provides clues to source-sink relations. Furthermore, we analyzed the metabolite distribution within single leaves along the basipetal sink-source transition trajectory during senescence. Ceramides, lysolipids, aromatic amino acids, branched chain amino acids, and stressinduced amino acids accumulated, and an imbalance of asparagine/aspartate, glutamate/glutamine, and nutrient ions in the tip region of leaves was detected. Furthermore, the spatiotemporal distribution of tricarboxylic acid cycle intermediates was already changed in the presenescent leaves, and glucosinolates, raffinose, and galactinol accumulated in the base region of leaves with preceding senescence. These results are discussed in the context of current models of the metabolic shifts occurring during developmental and environmentally induced senescence. As senescence processes are correlated to crop yield, the metabolome data and the approach provided here can serve as a blueprint for the analysis of traits and conditions linking crop yield and senescence.
Summary
Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the ...adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl‐tert‐butyl‐ether) phase, eliminated the need for solid‐phase extraction for sample clean‐up, and therefore reduces both sampling time and cost. As a proof‐of‐concept analysis, Arabidopsis thaliana plants were subjected to water‐deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty‐acid derivatives and diverse jasmonates in the course of adaptation to water‐deficit stress.
Significance Statement
We report on an important methodological improvement for the systemic analysis of plant samples, enabling us to obtain maximal information from a single sample aliquot. The improved extraction method enables the comprehensive analysis of lipids, proteins, metabolites and phytohormones from a single sample of water deficit‐stressed leaf tissue. This analysis allowed us not only to confirm the previously reported water deficit‐induced molecular changes but more importantly to uncover novel regulatory interactions during water deficit.
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