As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms.

Rice bran (RB) is a major by-product of rice polishing and a rich source of bioactive compounds. Here, we investigated the anti-colitis effect of diet supplementation with fermented rice bran (FRB) in a murine model of ulcerative colitis. FRB was prepared by dual fermentation of RB using fungi and lactic acid bacteria. Colitis was induced in C57Bl/6N male mice (n = 8/group) by dextran sodium sulfate (DSS). Body weight change, disease activity index (DAI), histopathology score, tissue myeloperoxidase (MPO) activity, cytokine and chemokine transcript levels, and the production of short-chain fatty acids (SCFAs) and mucin in the colonic tissue were monitored. Based on histopathology scores, DSS induced severe mucosal inflammation, with an increased loss of crypts, and inflammatory cell infiltration in the control and RB groups, but not in the FRB group. MPO activity, thiobarbituric acid-reactive substance levels, and pro-inflammatory cytokine transcript (Tnf-α, Il-1β, Il-6, and Il-17) levels were significantly higher in the control and RB groups than in the FRB group. Thus, dietary FRB attenuated intestinal inflammation owing to elevated SCFAs and tryptamine production, which might regulate tight junction barrier integrity and intestinal homeostasis. These results suggest that FRB could comprise an effective potential preventive agent for ulcerative colitis.

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

Aflatoxin contamination of crops is a serious problem worldwide. Utilization of aflatoxin production inhibitors is attractive, as the elucidation of their modes of action contributes to clarifying the mechanism of aflatoxin production. Here, we identified mitochondrial protease ClpP as the target of dioctatin, an inhibitor of aflatoxin production of Aspergillus flavus. Dioctatin conferred uncontrolled caseinolytic capacity on ClpP of A. flavus and Escherichia coli. Dioctatin-bound ClpP selectively degraded mitochondrial energy-related proteins in vitro, including a subunit of respiratory chain complex V, which was also reduced by dioctatin in a ClpP-dependent manner in vivo. Dioctatin enhanced glycolysis and alcohol fermentation while reducing tricarboxylic acid cycle metabolites. These disturbances were accompanied by reduced histone acetylation and reduced expression of aflatoxin biosynthetic genes. Our results suggest that dioctatin inhibits aflatoxin production by inducing ClpP-mediated degradation of mitochondrial energy-related components, and that mitochondrial energy metabolism functions as a key determinant of aflatoxin production.

Durian is an economically important fruit of Southeast Asia. There is, however, a lack of in-depth information on the alteration of its metabolic networks during ripening. Here, we annotated 94 ripening-associated metabolites from the pulp of durian cv. Monthong fruit at unripe and ripe stages, using capillary electrophoresis- and gas chromatography- time-of-flight mass spectrometry, specifically focusing on taste-related metabolites. During ripening, sucrose content increased. Change in raffinose-family oligosaccharides are reported herein for the first time. The malate and succinate contents increased, while those of citrate, an abundant organic acid, were unchanged. Notably, most amino acids increased, including isoleucine, leucine, and valine, whereas aspartate decreased, and glutamate was unchanged. Furthermore, transcriptomic analysis was performed to analyze the dynamic changes in sugar metabolism, glycolysis, TCA cycle, and amino acid pathways to identify key candidate genes. Taken together, our results elucidate the fundamental taste-related metabolism of durian, which can be exploited to develop durian metabolic and genetic markers in the future.

Soy protein β-conglycinin has serum lipid-lowering and anti-obesity effects. We showed that single ingestion of β-conglycinin after fasting alters gene expression in mouse liver. A sharp increase in fibroblast growth factor 21 (FGF21) gene expression, which is depressed by normal feeding, resulted in increased postprandial circulating FGF21 levels along with a significant decrease in adipose tissue weights. Most increases in gene expressions, including FGF21, were targets for the activating transcription factor 4 (ATF4), but not for peroxisome proliferator-activated receptor α. Overexpression of a dominant-negative form of ATF4 significantly reduced β-conglycinin-induced increases in hepatic FGF21 gene expression. In FGF21-deficient mice, β-conglycinin effects were partially abolished. Methionine supplementation to the diet or primary hepatocyte culture medium demonstrated its importance for activating liver or hepatocyte ATF4-FGF21 signaling. Thus, dietary β-conglycinin intake can impact hepatic and systemic metabolism by increasing the postprandial circulating FGF21 levels.

In metabolomics researches using mass spectrometry (MS), systematic searching of high-resolution mass data against compound databases is often the first step of metabolite annotation to determine elemental compositions possessing similar theoretical mass numbers. However, incorrect hits derived from errors in mass analyses will be included in the results of elemental composition searches. To assess the quality of peak annotation information, a novel methodology for false discovery rates (FDR) evaluation is presented in this study. Based on the FDR analyses, several aspects of an elemental composition search, including setting a threshold, estimating FDR, and the types of elemental composition databases most reliable for searching are discussed.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

Metabolites in the fruits of edible plants include sweet sugars, visually appealing pigments, various products with human nutritional value, and biologically active plant hormones. Although quantities of these metabolites vary during fruit development and ripening because of cell division and enlargement, there are few reports describing the actual dynamics of these changes. Therefore, we applied multiple metabolomic techniques to identify the changes in metabolite levels during the development and ripening of pear fruits (Pyrus communis L. 'La France'). We quantified and classified over 250 metabolites into six groups depending on their specific patterns of variation during development and ripening. Approximately half the total number of metabolites, including histidine and malate, accumulated transiently around the blooming period, during which cells are actively dividing, and then decreased either rapidly or slowly. Furthermore, the amounts of sulfur-containing amino acids also increased in pear fruits around 3-4 months after the blooming period, when fruit cells are enlarging, but virtually disappeared from ripened fruits. Some metabolites, including the plant hormone abscisic acid, accumulated particularly in the receptacle prior to blooming and/or fruit ripening. Our results show several patterns of variation in metabolite levels in developing and ripening pear fruits, and provide fundamental metabolomic data that is useful for understanding pear fruit physiology and enhancing the nutritional traits of new cultivars.

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase-a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-d-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops.

Phosphorus (P) is one of the most important macronutrients limiting plant growth and development, particularly in forest ecosystems such as temperate beech (Fagus sylvatica) forests in Central Europe. Efficient tree internal P cycling during annual growth is an important strategy of beech trees to adapt to low soil-P. Organic P (Porg) is thought to play a decisive role in P cycling, but the significance of individual compounds and processes has not been elucidated. To identify processes and metabolites involved in P cycling of beech trees, polar-metabolome and lipidome profiling was performed during annual growth with twig tissues from a sufficient (Conventwald, Con) and a low-soil-P (Tuttlingen, Tut) forest. Autumnal phospholipid degradation in leaves and P export from senescent leaves, accumulation of phospholipids and glucosamine-6-phosphate (GlcN6P) in the bark, storage of N-acetyl-D-glucosamine-6-phosphate (GlcNAc6P) in the wood, and establishing of a phospholipid "start-up capital" in buds constitute main processes involved in P cycling that were enhanced in beech trees on low-P soil of the Tut forest. In spring, mobilization of P from storage pools in the bark contributed to an effective P cycling. Due to the higher phospholipid "start-up capital" in buds of Tut beeches, the P metabolite profile in developing leaves in spring was similar in beech trees of both forests. During summer, leaves of Tut beeches meet their phosphate (Pi) needs by replacing phospholipids by galacto- and sulfolipids. Thus, several processes contribute to adequate Pi supply on P impoverished soil thereby mediating similar growth of beech at low and sufficient soil-P availability.

Shoot formation is accompanied by active cell proliferation and expansion, requiring that metabolic state adapts to developmental control. Despite the importance of such metabolic reprogramming, it remains unclear how development and metabolism are integrated. Here, we show that disruption of ANGUSTIFOLIA3 orthologs (PpAN3s) compromises gametophore shoot formation in the moss Physcomitrium patens due to defective cell proliferation and expansion. Trans-omics analysis reveals that the downstream activity of PpAN3 is linked to arginine metabolism. Elevating arginine level by chemical treatment leads to stunted gametophores and causes Ppan3 mutant-like transcriptional changes in the wild-type plant. Furthermore, ectopic expression of AtAN3 from Arabidopsis thaliana ameliorates the defective arginine metabolism and promotes gametophore formation in Ppan3 mutants. Together, these findings indicate that arginine metabolism is a key pathway associated with gametophore formation and provide evolutionary insights into the establishment of the shoot system in land plants through the integration of developmental and metabolic processes.

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (Δ13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

Rice varieties that can survive under submergence conditions respond to flooding either by enhancing internode elongation or by quiescence of shoot elongation. Despite extensive efforts to identify key metabolites triggered by complete submergence of rice possessing SUBMERGENCE 1 (SUB1) locus, metabolic responses of internode elongation of deepwater rice governed by the SNORKEL 1 and 2 genes remain elusive. This study investigated specific metabolomic responses under partial submergence (PS) to deepwater- (C9285) and non-deepwater rice cultivars (Taichung 65 (T65)). In addition, we examined the response in a near-isogenic line (NIL-12) that has a C9285 genomic fragment on chromosome 12 introgressed into the genetic background of T65. Under short-term submergence (0-24 h), metabolite profiles of C9285, NIL-12, and T65 were compared to extract significantly changed metabolites in deepwater rice under PS conditions. Comprehensive metabolite and phytohormone profiling revealed increases in metabolite levels in the glycolysis pathway in NIL-12 plants. Under long-term submergence (0-288 h), we found decreased amino acid levels. These metabolomic changes were opposite when compared to those in flood-tolerant rice with SUB1 locus. Auxin conjugate levels related to stress response decreased in NIL-12 lines relative to T65. Our analysis helped clarify the complex metabolic reprogramming in deepwater rice as an escape strategy.

Plants have evolved various mechanisms for low P tolerance, one of which is changing their membrane lipid composition by remodeling phospholipids with non-phospholipids. The objective of this study was to investigate the remodeling of membrane lipids among rice cultivars under P deficiency. Rice (Oryza sativa L.) cultivars (Akamai, Kiyonishiki, Akitakomachi, Norin No. 1, Hiyadateine, Koshihikari, and Netaro) were grown in 0 (-P) and 8 (+P) mg P L-1 solution cultures. Shoots and roots were collected 5 and 10 days after transplanting (DAT) in solution culture and subjected to lipidome profiling using liquid chromatography-mass spectrometry. Phosphatidylcholine (PC)34, PC36, phosphatidylethanolamine (PE)34, PE36, phosphatidylglycerol (PG)34, phosphatidylinositol (PI)34 were the major phospholipids and digalactosyldiacylglycerol (DGDG)34, DGDG36, 1,2-diacyl-3-O-alpha-glucuronosylglycerol (GlcADG)34, GlcADG36, monogalactosyldiacylglycerol (MGDG)34, MGDG36, sulfoquinovosyldiacylglycerol (SQDG)34 and SQDG36 were the major non-phospholipids. Phospholipids were lower in the plants that were grown under -P conditions than that in the plants that were grown under +P for all cultivars at 5 and 10 DAT. The levels of non-phospholipids were higher in -P plants than that in +P plants of all cultivars at 5 and 10 DAT. Decomposition of phospholipids in roots at 5 DAT correlated with low P tolerance. These results suggest that rice cultivars remodel membrane lipids under P deficiency, and the ability of remodeling partly contributes to low P tolerance.

Root exudates improve the nutrient acquisition of plants and affect rhizosphere microbial communities. The plant nutrient status affects the composition of root exudates. The purpose of this study was to examine common bean (Phaseolus vulgaris L.) root exudates under phosphorus (P) deficiency using a metabolite profiling technique. Common bean plants were grown in a culture solution at P concentrations of 0 (P0), 1 (P1) and 8 (P8) mg P L-1 for 1, 10 and 20 days after transplanting (DAT). Root exudates were collected, and their metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). The shoot P concentration and dry weight of common bean plants grown at P0 were lower than those grown at P8. One hundred and fifty-nine, 203 and 212 metabolites were identified in the root exudates, and 16% (26/159), 13% (26/203) and 9% (20/212) of metabolites showed a P0/P8 ratio higher than 2.0 at 1, 10 and 20 DAT, respectively. The relative peak areas of several metabolites, including organic acids and amino acids, in root exudates were higher at P0 than at P8. These results suggest that more than 10% of primary and secondary metabolites are induced to exude from roots of common bean by P deficiency.

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.

TGR5, a G protein-coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle-specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia.

Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.