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Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (−S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under –S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency., Sulfur Deficiency Induced1 (SDI1) upregulates S-poor seed storage proteins in favor of S-rich seed storage proteins.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography– and gas chromatography–mass spectrometry-based metabolomics-derived data.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin–Benson–Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.

An excess of nitrogen (N) is used in agriculture endangering the environment and food quality. One approach to circumvent this is to generate crops with a stable or even increased productivity under limited N. Here, we studied the effect of reduced N availability on potato (Solanum tuberosum) tuber yield and quality traits using five varieties: the wild Andigena and the commercial cultivars Désirée, Milva, Saturna and Alegria. Growth on limited N resulted in less tubers with a reduced weight except for Andigena. Tubers from low N-grown plants contained more starch, less sucrose and were delayed in sprouting. Some of the trait differences can be explained by changes in hormone levels between cultivars and N conditions. In general, Saturna and Alegria performed better under limited N making them excellent breeding candidates. Our results suggest that wild species more flexibly adapt to limited N, a trait lost in commercial potatoes.

Aging is accompanied by a general decline in the function of many cellular pathways. However, whether these are causally or functionally interconnected remains elusive. Here, we study the effect of mitochondrial–nuclear communication on stem cell aging. We show that aged mesenchymal stem cells exhibit reduced chromatin accessibility and lower histone acetylation, particularly on promoters and enhancers of osteogenic genes. The reduced histone acetylation is due to impaired export of mitochondrial acetyl-CoA, owing to the lower levels of citrate carrier (CiC). We demonstrate that aged cells showed enhanced lysosomal degradation of CiC, which is mediated via mitochondrial-derived vesicles. Strikingly, restoring cytosolic acetyl-CoA levels either by exogenous CiC expression or via acetate supplementation, remodels the chromatin landscape and rescues the osteogenesis defects of aged mesenchymal stem cells. Collectively, our results establish a tight, age-dependent connection between mitochondrial quality control, chromatin and stem cell fate, which are linked together by CiC. Aged mesenchymal stem cells exhibit decreased osteogenesis. Pouikli et al. link impaired MSC differentiation to histone hypoacetylation caused by lower mitochondrial acetyl-CoA export due to enhanced lysosomal degradation of the citrate carrier Slc25a1. Restoring histone acetylation to youthful levels rescues osteogenesis.

International audience; Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenicmechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, lossof which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomicstudies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels asputative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We alsoconfirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disruptsmitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in ourmouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites in oxidative phosphorylation and purine biosynthesis are consistently present in lower concentrations in the brains of humans. In particular, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution is responsible for much or all of the reduction of de novo synthesis of purines in humans.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an acute threat to public health and the world economy, especially because no approved specific drugs or vaccines are available. Pharmacological modulation of metabolism-dependent cellular pathways such as autophagy reduced propagation of highly pathogenic Middle East respiratory syndrome (MERS)-CoV.Here we show that SARS-CoV-2 infection limits autophagy by interfering with multiple metabolic pathways and that compound-driven interventions aimed at autophagy induction reduce SARS-CoV-2 propagation in vitro. In-depth analyses of autophagy signaling and metabolomics indicate that SARS-CoV-2 reduces glycolysis and protein translation by limiting activation of AMP-protein activated kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). Infection also downregulates autophagy-inducing spermidine, and facilitates AKT1/SKP2-dependent degradation of autophagy-initiating Beclin-1 (BECN1). Targeting of these pathways by exogenous administration of spermidine, AKT inhibitor MK-2206, and the Beclin-1 stabilizing, antihelminthic drug niclosamide inhibited SARS-CoV-2 propagation by 85, 88, and >99%, respectively. In sum, SARS-CoV-2 infection causally diminishes autophagy. A clinically approved and well-tolerated autophagy-inducing compound shows potential for evaluation as a treatment against SARS-CoV-2.

Vegetative growth requires the systemic coordination of numerous cellular processes, which are controlled by regulatory proteins that monitor extracellular and intracellular cues and translate them into growth decisions. In eukaryotes, one of the central factors regulating growth is the serine/threonine protein kinase Target of Rapamycin (TOR), which forms complexes with regulatory proteins. To understand the function of one such regulatory protein, Regulatory-Associated Protein of TOR 1B (RAPTOR1B), in plants, we analyzed the effect of raptor1b mutations on growth and physiology in Arabidopsis (Arabidopsis thaliana) by detailed phenotyping, metabolomic, lipidomic, and proteomic analyses. Mutation of RAPTOR1B resulted in a strong reduction of TOR kinase activity, leading to massive changes in central carbon and nitrogen metabolism, accumulation of excess starch, and induction of autophagy. These shifts led to a significant reduction of plant growth that occurred nonlinearly during developmental stage transitions. This phenotype was accompanied by changes in cell morphology and tissue anatomy. In contrast to previous studies in rice (Oryza sativa), we found that the Arabidopsis raptor1b mutation did not affect chloroplast development or photosynthetic electron transport efficiency; however, it resulted in decreased CO2 assimilation rate and increased stomatal conductance. The raptor1b mutants also had reduced abscisic acid levels. Surprisingly, abscisic acid feeding experiments resulted in partial complementation of the growth phenotypes, indicating the tight interaction between TOR function and hormone synthesis and signaling in plants.

Ginger (Zingiber officinale Roscoe) is consumed for health-promoting effects and as a food condiment. Comprehensive phytochemical analysis, other than gingerols and shogaols, has not yet been deeply investigated. Hence, the current research aimed to establish a non-targeted metabolomics approach for the discrimination between fresh ginger rhizome samples collected from four different producing countries, i.e., China, India, Pakistan, and Peru. In addition, lab-dried samples were analyzed to trace drying-induced metabolites. A comprehensive extraction procedure was carried out resulting in production of polar and non-polar fractions. The polar fraction was analyzed by ultra-performance liquid chromatography coupled with Fourier transform tandem mass spectrometry (UPLC-C18-FT-MS/MS) and gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) post derivatization. UPLC-C8-FT-MS/MS was used for analysis of non-polar fraction. Results revealed for identification of a total of 253 metabolites. In addition, multivariate data analysis (MVDA), including principal component analysis (PCA) demonstrated clustering of Asian specimens. Several metabolites with a characteristic pattern for the origin revealing the highest contents of bioactive metabolites in the Peruvian product. Moreover, chemical markers identified, including [6]-gingerol and [6]-shogaol discriminating between fresh and dried samples. Furthermore, abundances of some primary metabolites, including amino acids and cinnamic acid, have confirmed the biosynthetic pathway of gingerols and their transformation upon drying to shogaols. The proposed approach can be applied as a potential candidate for quality assessment of ginger and other medicinal plants.

Summary Target of rapamycin (TOR) is a positive regulator of growth and development in all eukaryotes, which positively regulates anabolic processes like protein synthesis, while repressing catabolic processes, including autophagy. To better understand TOR function we decided to analyze its role in seed development and germination. We therefore performed a detailed phenotypic analysis using mutants of the REGULATORY-ASSOCIATED PROTEIN OF TOR (RAPTOR1B), a conserved TOR-interactor, acting as a scaffold protein, which recruits substrates for the TOR kinase. Our results show that raptor1b plants produced seeds, which were delayed in germination and less resistant to stresses, leading to decreased vitaliy. These physiological phenotypes were accompanied by morphological changes including decreased seed coat pigmentation and reduced production of seed coat mucilage. A detailed molecular analysis revealed that many of these morphological changes were associated with significant changes of the metabolic content of raptor1b seeds, including elevated levels of free amino acids, as well as reduced levels of protective secondary metabolites and storage proteins. Most of these observed changes, were accompanied by significantly altered phytohormone levels in the raptor1b seeds with increases in abscisic acid, auxin and jasmonic acid, which are known to inhibit germination. Delayed germination and seedling growth, observed in the raptor1b seeds, could be partially restored by the exogenous supply of gibberellic acid, indicating that TOR is in the center of a regulatory hub controlling seed metabolism, maturation and germination. This article is protected by copyright. All rights reserved.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with yet incompletely uncovered molecular determinants. Alterations in the abundance of low molecular weight compounds (metabolites) in ASD could add to our understanding of the disease. Indeed, such alterations take place in the urine, plasma and cerebellum of ASD individuals. In this work, we investigated mass-spectrometric signal intensities of 1,366 metabolites in the prefrontal cortex grey matter of 32 ASD and 40 control individuals. 15% of these metabolites showed significantly different intensities in ASD and clustered in 16 metabolic pathways. Of them, ten pathways were altered in urine and blood of ASD individuals (Fisher test, p, Kurochkin, Khrameeva et al. show differences in metabolite intensities within the prefrontal cortex of individuals with autism spectrum disorders (ASD) that converge into 16 cellular pathways. Parallel measurements conducted in primates also revealed unique metabolite intensity differences unique to humans, highlighting unique evolutionary cortical mechanism in ASD.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

Target of Rapamycin (TOR) regulates multiple growth- and metabolic-related processes in Arabidopsis thaliana as in all other eukaryotes. While several of these processes have been investigated in diverse Arabidopsis growth stages, little is known about hormonal and metabolic regulation of TOR during seed germination. This is mainly due to the fact that Arabidopsis knockout lines of TOR are embryo lethal. Here, we utilized the knockout lines of TOR-interacting protein, REGULATORY-ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), to perform comprehensive hormone profiling during seed germination. We previously reported that RAPTOR1B positively regulates seed germination by maintaining the nutritional and hormonal balance. In the current analysis, dry and imbibed seeds as well as germinated seeds were subjected to detailed hormone analysis. Accordingly, the abscisic acid content of dry and imbibed raptor1b seeds was higher than that of WT, while the amounts of gibberellins were comparable after stratification. Further analysis showed that raptor1b seeds maintained higher levels of indole-3-acetic acid and jasmonates, namely jasmonic acid (JA) and 12-oxo-phytodienoic acid, even after stratification. The combination of this hormonal perturbation seems to be the driving factor for the observed delayed germination phenotypes in raptor1b seeds.

The allocation of nutrient resources to growth and metabolism is an essential function for controlling biomass accumulation in photoautotrophic organisms. One essential protein complex involved in this process is the target of rapamycin (TOR) kinase. It has been shown that the inhibition of TOR leads to a considerable upsurge in the amino acid levels. This molecular phenotype relies mainly on the availability of light, carbon (C) and nitrogen (N). To validate the time-resolved response of C and N metabolites, we used a targeted gas chromatography mass spectrometery (GC-MS)-based metabolomic approach, where we examined the response of Chlamydomonas reinhardtii upon TOR inhibition under C-limited condition, namely extended darkness. Contrary to C-supplemented conditions, the rapid increase in the amino acid levels is suppressed almost completely 4 h after TOR inhibition, confirming that C supply is essential to raise the amino acid levels mediated by their de novo synthesis. An exception to this observation was the levels of aspartate, which is presumably synthesized via the anaplerotic pathway. In agreement with previous reports, TOR repression, under these C-limited conditions, leads to a significant reduction in the C/N ratio, corroborating with the crucial role of the pathway in maintaining the metabolic balance of the cells and consequently propelling growth.

The Target of Rapamycin (TOR) complex (TORC) regulates plant growth and development by modulation of metabolism in response to environmental cues. TORC contains in its core the TOR kinase and two interacting partners, namely; regulatory-associated partner of TOR (RAPTOR) and lethal with sec thirteen protein 8 (LST8). RAPTOR is described to act as a scaffold protein which recruits substrates for phosphorylation to the TOR kinase. In the current manuscript we show that mutation of Arabidopsis RAPTOR1B leads to significantly decreased levels of free jasmonic acid (JA), jasmonoyl-(L)-isoleucine (JA-Ile) as well as its biosynthetic precursor 12-oxo-phytodienoic acid (OPDA). Although raptor1b leaves showed decreased basic JA level compared to WT, the mutant responded substantially to wounding stress by producing the same amount of JA as WT. Furthermore, we could show that the chemical inhibition of TOR by AZD-8055 led to an opposite response. AZD-treated WT and raptor1b leaves accumulated high JA levels. These results strongly imply that the TOR signaling pathway is responding differentially to the inhibition of the TOR kinase as compared to the inhibition of the scaffold protein RAPTOR.

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.

Alternating temperatures require fast and coordinated adaptation responses of plants. Cold acclimation has been extensively investigated and results in increased freezing tolerance in Arabidopsis thaliana. Here, we show that the two Arabidopsis accessions, Col-0 and N14, which differ in their freezing tolerance, showed memory of cold acclimation, that is, cold priming. Freezing tolerance was higher in plants exposed to cold priming at 4°C, a lag phase at 20°C, and a second triggering cold stress (4°C) than in plants that were only cold primed. To our knowledge, this is the first report on cold memory improving plant freezing tolerance. The triggering response was distinguishable from the priming response at the levels of gene expression (RNA-Seq), lipid (ultraperformance liquid chromatography-mass spectrometry), and metabolite composition (gas chromatography-mass spectrometry). Transcriptomic responses pointed to induced lipid, secondary metabolism, and stress in Col-0 and growth-related functions in N14. Specific accumulation of lipids included arabidopsides with possible functions as signalling molecules or precursors of jasmonic acid. Whereas cold-induced metabolites such as raffinose and its precursors were maintained in N14 during the lag phase, they were strongly accumulated in Col-0 after the cold trigger. This indicates genetic differences in the transcriptomic and metabolic patterns during cold memory.

Chronic exposure to ocean acidification and elevated sea-surface temperatures pose significant stress to marine ecosystems. This in turn necessitates costly acclimation responses in corals in both the symbiont and host, with a reorganization of cell metabolism and structure. A large-scale untargeted metabolomics approach comprising gas chromatography mass spectrometry (GC-MS) and ultraperformance liquid chromatography coupled to high resolution mass spectrometry (UPLC-MS) was applied to profile the metabolite composition of the soft coral Sarcophyton ehrenbergi and its dinoflagellate symbiont. Metabolite profiling compared ambient conditions with response to simulated climate change stressors and with the sister species, S. glaucum. Among ∼300 monitored metabolites, 13 metabolites were modulated. Incubation experiments providing four selected upregulated metabolites (alanine, GABA, nicotinic acid, and proline) in the culturing water failed to subside the bleaching response at temperature-induced stress, despite their known ability to mitigate heat stress in plants or animals. Thus, the results hint to metabolite accumulation (marker) during heat stress. This study provides the first detailed map of metabolic pathways transition in corals in response to different environmental stresses, accounting for the superior thermal tolerance of S. ehrenbergi versus S. glaucum, which can ultimately help maintain a viable symbiosis and mitigate against coral bleaching.

Upon illumination, etiolated seedlings experience a transition from heterotrophic to photoautotrophic growth. During this process, the tetrapyrrole biosynthesis pathway provides chlorophyll for photosynthesis. This pathway has to be tightly controlled to prevent the accumulation of photoreactive metabolites and to provide stoichiometric amounts of chlorophyll for its incorporation into photosynthetic protein complexes. Therefore, plants have evolved regulatory mechanisms to synchronize the biosynthesis of chlorophyll and chlorophyll-binding proteins. Two phytochrome-interacting factors (PIF1 and PIF3) and the DELLA proteins, which are controlled by the gibberellin pathway, are key regulators of this process. Here, we show that impairment of TARGET OF RAPAMYCIN (TOR) activity in Arabidopsis (Arabidopsis thaliana), either by mutation of the TOR complex component RAPTOR1B or by treatment with TOR inhibitors, leads to a significantly reduced accumulation of the photoreactive chlorophyll precursor protochlorophyllide in darkness but an increased greening rate of etiolated seedlings after exposure to light. Detailed profiling of metabolic, transcriptomic, and physiological parameters revealed that the TOR-repressed lines not only grow slower, they grow in a nutrient-saving mode, which allows them to resist longer periods of low nutrient availability. Our results also indicated that RAPTOR1B acts upstream of the gibberellin-DELLA pathway and its mutation complements the repressed greening phenotype of pif1 and pif3 after etiolation.

Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.

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 receptacle of the strawberry (Fragaria × ananassa) fruit accounts for the main properties of the ripe fruit for human consumption. As it ripens, it undergoes changes similar to other fruits in sugar : acid ratio, volatile production and cell wall softening. However, the main regulators of this process have not yet been reported. The white stage marks the initiation of the ripening process, and we had previously reported a peak of expression for a FaGAMYB gene. Transient silencing of FaGAMYB using RNAi and further determination of changes in global gene expression by RNAseq, and composition of primary and secondary metabolites have been used to investigate the role played by this gene during the development of the receptacle. Down-regulation of FaGAMYB caused an arrest in the ripening of the receptacle and inhibited colour formation. Consistent with this, several transcription factors associated with the regulation of flavonoid biosynthetic pathway showed altered expression. FaGAMYB silencing also caused a reduction of ABA biosynthesis and sucrose content. Interestingly, exogenous ABA application to the RNAI-transformed receptacle reversed most defects caused by FaGAMYB down-regulation. The study assigns a key regulatory role to FaGAMYB in the initiation of strawberry receptacle ripening and acting upstream of the known regulator ABA.

The paper presents the ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometric analysis and comparison of total lipid profiles of two green algal species, Chlamydomonas reinhardtii and Scenedesmus (Acutodesmus) obliquus. The targeted UPLC-mass spectroscopy (MS) analysis revealed that both the green algae showed the presence of almost similar types of lipids. However, there were differences in the presence of three triacylglycerol (TAG) species (TAG 54:4, TAG 54:5 and TAG 54:8) and two diacylglycerol (DAG) species (DAG 36:3 and DAG 36:4) in C. reinhardtii that were found to be completely absent in Scenedesmus obliquus. The triacylglycerol content in S. obliquus was five times more than that in C. reinhardtii. In addition, amount of diacylglycerol-O-(N,N,N-trimethyl) homoserine, a characteristic algal lipid, in S. obliquus was only half of that in C. reinhardtii. The paper also discusses the metabolic roles of the lipids produced by these algal species with reference to the lipids identified by UPLC-MS analysis.

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants.

Summary Trehalose-6-phosphate is a signal of sucrose status in plants and forms part of a homeostatic mechanism that maintains sucrose levels within a range that is appropriate for the cell type and stage of development., Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, has a profound influence on plant metabolism, growth, and development. It has been proposed that Tre6P acts as a signal of sugar availability and is possibly specific for sucrose status. Short-term sugar-feeding experiments were carried out with carbon-starved Arabidopsis thaliana seedlings grown in axenic shaking liquid cultures. Tre6P increased when seedlings were exogenously supplied with sucrose, or with hexoses that can be metabolized to sucrose, such as glucose and fructose. Conditional correlation analysis and inhibitor experiments indicated that the hexose-induced increase in Tre6P was an indirect response dependent on conversion of the hexose sugars to sucrose. Tre6P content was affected by changes in nitrogen status, but this response was also attributable to parallel changes in sucrose. The sucrose-induced rise in Tre6P was unaffected by cordycepin but almost completely blocked by cycloheximide, indicating that de novo protein synthesis is necessary for the response. There was a strong correlation between Tre6P and sucrose even in lines that constitutively express heterologous trehalose-phosphate synthase or trehalose-phosphate phosphatase, although the Tre6P:sucrose ratio was shifted higher or lower, respectively. It is proposed that the Tre6P:sucrose ratio is a critical parameter for the plant and forms part of a homeostatic mechanism to maintain sucrose levels within a range that is appropriate for the cell type and developmental stage of the plant.

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 stress-induced 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.

Root-to-shoot signaling is used by plants to coordinate shoot development with the conditions experienced by the roots. A mobile and biologically active compound, the bps signal, is over-produced in roots of an Arabidopsis thaliana mutant called bypass1 (bps1), and might also be a normally produced signaling molecule in wild-type plants. Our goal is to identify the bps signal chemically, which will then allow us to assess its production in normal plants. To identify any signaling molecule, a bioassay is required, and here we describe the development of a robust, simple, and quantitative bioassay for the bps signal. The developed bioassay follows the growth-reducing activity of the bps signal using the pCYCB1;1::GUS cell cycle marker. Wild-type plants carrying this marker, and provided the bps signal through either grafts or metabolite extracts, showed reduced cell division. By contrast, control grafts and treatment with control extracts showed no change in pCYCB1;1::GUS expression. To determine the chemical nature of the bps signal, extracts were treated with RNase A, Proteinase K, or heat. None of these treatments diminished the activity of bps1 extracts, suggesting that the active molecule might be a metabolite. This bioassay will be useful for future biochemical fractionation and analysis directed toward bps signal identification.

We conducted a comparative analysis of the root metabolome of six parental maize inbred lines and their 14 corresponding hybrids showing fresh weight heterosis. We demonstrated that the metabolic profiles not only exhibit distinct features for each hybrid line compared with its parental lines, but also separate reciprocal hybrids. Reconstructed metabolic networks, based on robust correlations between metabolic profiles, display a higher network density in most hybrids as compared with the corresponding inbred lines. With respect to metabolite level inheritance, additive, dominant and overdominant patterns are observed with no specific overrepresentation. Despite the observed complexity of the inheritance pattern, for the majority of metabolites the variance observed in all 14 hybrids is lower compared with inbred lines. Deviations of metabolite levels from the average levels of the hybrids correlate negatively with biomass, which could be applied for developing predictors of hybrid performance based on characteristics of metabolite patterns.

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

Although the influence of temperature, particularly cold, on lipid metabolism is well established, previous studies have focused on long-term responses and have largely ignored the influence of other interacting environmental factors. Here, we present a time-resolved analysis of the early responses of the glycerolipidome of Arabidopsis thaliana plants exposed to various temperatures (4, 21 and 32°C) and light intensities (darkness, 75, 150 and 400 μmol m(-2) s(-1)), including selected combinations. Using a UPLC/MS-based lipidomic platform, we reproducibly measured most glycerolipid species reported for Arabidopsis leaves, including the classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). In addition to known lipids, we have identified previously unobserved compounds, such as 36-C PGs and eukaryotic phospholipids containing 16:3 acyl chains. Occurrence of these lipid species implies the action of new biochemical mechanisms. Exposition of Arabidopsis plants to various light and temperature regimes results in two major effects. The first is the dependence of the saturation level of PC and MGDG pools on light intensity, likely arising from light regulation of de novo fatty acid synthesis. The second concerns an immediate decrease in unsaturated species of PG at high-temperature conditions (32°C), which could mark the first stages of adaptation to heat-stress conditions. Observed changes are discussed in the context of current knowledge, and new hypotheses have been formulated concerning the early stages of the plant response to changing light and temperature conditions.

International audience; Plastoglobules, lipid-protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to γ-tocopherol and γ-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C(13) cyclohexenone and C(14) mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

Metabolomics is rapidly becoming an integral part of many life science studies ranging from disease diagnostics to systems biology. However, a number of problems such as the discrimination of biological from non-biological signals, efficient compound annotation, and reliable quantification are still not satisfactorily solved in untargeted LC-MS-based metabolomics research. Extending our previous work on direct infusion-based metabolomics, we here describe a (13)C isotope labeling strategy in combination with an Ultra Performance Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry-based approach (UPLC-FTICR MS) which provides a technological platform offering solutions to a number of the above-mentioned problems. We further demonstrate that the use of a fully labeled metabolome is not only beneficial for high end mass spectrometers, such as that used in this study but also provides a considerable improvement to every other mass spectrometry-based metabolomic platform.

A new strategy for direct infusion-based metabolite analysis employing a combination of high-resolution mass spectrometry and (13)C-isotope labeling of entire metabolomes is described. Differentially isotope labeled metabolite extracts from otherwise identically grown reference plants were prepared and infused into a Fourier transform ion cyclotron resonance mass spectrometer. The derived accurate mass lists from each extract were searched, using an in-house-developed database search tool, against a number of comprehensive metabolite databases. Comparison of the retrieved chemical formulas from both, the (12)C and (13)C samples, leads to two major advantages compared to nonisotope-based metabolite fingerprinting: first, removal of background contaminations from the result list, due to the (12)C/(13)C peak pairing principle and therefore positive identification of compounds of true biological origin; second, elimination of ambiguity in chemical formula assignment due to the same principle, leading to the clear association of one measured mass to only one chemical formula. Applying this combination of strategies to metabolite extracts of the model plant Arabidopsis thaliana therefore resulted in the reproducible identification of more than 1000 unambiguous chemical sum formulas of biological origin of which more than 80% have not been associated to Arabidopsis before.

The soluble proteins in sieve tube exudate from Brassica napus plants were systematically analyzed by 1-DE and high-resolution 2-DE, partial amino acid sequence determination by MS/MS, followed by database searches. 140 proteins could be identified by their high similarity to database sequences (135 from 2-DE, 5 additional from 1-DE). Most analyzed spots led to successful protein identifications, demonstrating that Brassica napus, a close relative of Arabidopsis thaliana, is a highly suitable model plant for phloem research. None of the identified proteins was formerly known to be present in Brassica napus phloem, but several proteins have been described in phloem sap of other species. The data, which is discussed with respect to possible physiological importance of the proteins in the phloem, further confirms and substantially extends earlier findings and uncovers the presence of new protein functions in the vascular system. For example, we found several formerly unknown phloem proteins that are potentially involved in signal generation and transport, e.g., proteins mediating calcium and G-protein signaling, a set of RNA-binding proteins, and FLOWERING LOCUS T (FT) and its twin sister that might be key components for the regulation of flowering time.

Hakea prostrata (Proteaceae) is adapted to severely phosphorus-impoverished soils and extensively replaces phospholipids during leaf development. We investigated how polar lipid profiles change during leaf development and in response to external phosphate supply. Leaf size was unaffected by a moderate increase in phosphate supply. However, leaf protein concentration increased by more than 2-fold in young and mature leaves, indicating that phosphate stimulates protein synthesis. Orthologs of known lipid-remodeling genes in Arabidopsis (Arabidopsis thaliana) were identified in the H. prostrata transcriptome. Their transcript profiles in young and mature leaves were analyzed in response to phosphate supply alongside changes in polar lipid fractions. In young leaves of phosphate-limited plants, phosphatidylcholine/phosphatidylethanolamine and associated transcript levels were higher, while phosphatidylglycerol and sulfolipid levels were lower than in mature leaves, consistent with low photosynthetic rates and delayed chloroplast development. Phosphate reduced galactolipid and increased phospholipid concentrations in mature leaves, with concomitant changes in the expression of only four H. prostrata genes, GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE1, N-METHYLTRANSFERASE2, NONSPECIFIC PHOSPHOLIPASE C4, and MONOGALACTOSYLDIACYLGLYCEROL3. Remarkably, phosphatidylglycerol levels decreased with increasing phosphate supply and were associated with lower photosynthetic rates. Levels of polar lipids with highly unsaturated 32:x (x = number of double bonds in hydrocarbon chain) and 34:x acyl chains increased. We conclude that a regulatory network with a small number of central hubs underpins extensive phospholipid replacement during leaf development in H. prostrata. This hard-wired regulatory framework allows increased photosynthetic phosphorus use efficiency and growth in a low-phosphate environment. This may have rendered H. prostrata lipid metabolism unable to adjust to higher internal phosphate concentrations.

Accelerated evolution of the human brain and muscle metabolomes reflects our unique cognitive and physical capacities., Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys., Author Summary Physiological processes that maintain our tissues' functionality involve the generation of multiple products and intermediates known as metabolites—small molecules with a weight of less than 1,500 Daltons. Changes in concentrations of these metabolites are thought to be closely related to changes in phenotype. Here, we assessed concentrations of more than 10,000 metabolites in three brain regions and two non-neural tissues (skeletal muscle and kidney) of humans, chimpanzees, macaque monkeys, and mice using mass spectrometry-based approaches. We found that the evolution of the metabolome largely reflects genetic divergence between species and is not greatly affected by environmental factors. In the human lineage, however, we observed an exceptional acceleration of metabolome evolution in the prefrontal cortical region of the brain and in skeletal muscle. Based on additional behavioral tests, we further show that metabolic changes in human muscle seem to be paralleled by a drastic reduction in muscle strength. The observed rapid metabolic changes in brain and muscle, together with the unique human cognitive skills and low muscle performance, might reflect parallel mechanisms in human evolution.

Effects of the oxanion sulphate on plant aluminum (Al 3+ ) detoxification mechanisms are not well understood. Therefore, holistic physiological and biochemical modifications induced by progressively increased doses of sulphate fertilization in the presence of long-term Al 3+ stress were investigated in the aluminum sensitive perennial ryegrass ( Lolium perenne L. cvJumbo). Plant growth inhibition induced by Al 3+ was decreased in response to increasing doses of sulphate supply. Aluminum concentrations measured in roots of perennial ryegrass by atomic absorption spectrometry declined significantly with increasing sulphate concentrations. In parallel, we determined a rise of sulphur in shoots and roots of perennial ryegrass. Inclusion of up to 360 μM of sulphate enhanced cysteine and glutathione biosynthesis in Al 3+ (1.07 μM) treated plants. This increase of thiol-containing compounds favored all modifications in the glutathione redox balance, declining lipid peroxidation, decreasing the activity of superoxide dismutase, and modifying the expression of proteins involved in the diminution of Al 3+ toxicity in roots. In particular, proteome analysis by 1D-SDS-PAGE and LC-MS/MS allowed to identify up (e.g. vacuolar proton ATPase, proteosome β subunit, etc) and down (Glyoxilase I, Ascorbate peroxidase, etc.) regulated proteins induced by Al 3+ toxicity symptoms in roots. Although, sulphate supply up to 480 μM caused a reduction in Al 3+ toxicity symptoms, it was not as efficient as compared to 360 μM sulphate fertilization. These results suggest that sulphate fertilization ameliorates Al 3+ toxicity responses in an intracellular specific manner within Lolium perenne .

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.

Every biological organism relies for its proper function on interactions between a multitude of molecular entities like RNA, proteins, and metabolites. The comprehensive measurement and the analysis of all these entities would therefore provide the basis for our functional and mechanistic understanding of most biological processes. Next to their amount and identity, it is most crucial to also gain information about the subcellular distribution and the flux of the measured compounds between the cellular compartments. That is, we want to understand not only the individual functions of cellular components but also their functional implications within the whole organism. While the analysis of macromolecules like DNA, RNA, and proteins is quite established and robust, analytical techniques for small metabolites, which are prone to diffusion and degradation processes, provide a host of unsolved challenges. The major limitations here are the metabolite conversion and relocation processes. In this protocol we describe a methodological workflow which includes a nonaqueous fractionation method, a fractionated two-phase liquid/liquid extraction protocol, and a software package, which together allow extracting and analyzing starch, proteins, and especially polar and lipophilic metabolites from a single sample towards the estimation of their subcellular distributions.

Summary Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.

Human evolution is characterized by the rapid expansion of brain size and drastic increase in cognitive capabilities. It has long been suggested that these changes were accompanied by modifications of brain metabolism. Indeed, human-specific changes on gene expression or amino acid sequence were reported for a number of metabolic genes, but actual metabolite measurements in humans and apes have remained scarce. Here, we investigate concentrations of more than 100 metabolites in the prefrontal and cerebellar cortex in 49 humans, 11 chimpanzees, and 45 rhesus macaques of different ages using gas chromatography–mass spectrometry (GC-MS). We show that the brain metabolome undergoes substantial changes, both ontogenetically and evolutionarily: 88% of detected metabolites show significant concentration changes with age, whereas 77% of these metabolic changes differ significantly among species. Although overall metabolic divergence reflects phylogenetic relationships among species, we found a fourfold acceleration of metabolic changes in prefrontal cortex compared with cerebellum in the human lineage. These human-specific metabolic changes are paralleled by changes in expression patterns of the corresponding enzymes, and affect pathways involved in synaptic transmission, memory, and learning.