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16 pages, 2339 KiB

Open AccessArticle

Sink/Source Balance of Leaves Influences Amino Acid Pools and Their Associated Metabolic Fluxes in Winter Oilseed Rape (Brassica napus L.)

by Younès Dellero, Maud Heuillet, Nathalie Marnet, Floriant Bellvert, Pierre Millard and Alain Bouchereau

Metabolites 2020, 10(4), 150; https://doi.org/10.3390/metabo10040150 - 13 Apr 2020

Cited by 14 | Viewed by 3323

Abstract

Nitrogen remobilization processes from source to sink tissues in plants are determinant for seed yield and their implementation results in a complete reorganization of the primary metabolism during sink/source transition. Here, we decided to characterize the impact of the sink/source balance on amino [...] Read more.

Nitrogen remobilization processes from source to sink tissues in plants are determinant for seed yield and their implementation results in a complete reorganization of the primary metabolism during sink/source transition. Here, we decided to characterize the impact of the sink/source balance on amino acid metabolism in the leaves of winter oilseed rape grown at the vegetative stage. We combined a quantitative metabolomics approach with an instationary ¹⁵N-labeling experiment by using [¹⁵N]_L-glycine as a metabolic probe on leaf ranks with a gradual increase in their source status. We showed that the acquisition of the source status by leaves was specifically accompanied by a decrease in asparagine, glutamine, proline and S-methyl-l-cysteine sulphoxide contents and an increase in valine and threonine contents. Dynamic analysis of ¹⁵N enrichment and concentration of amino acids revealed gradual changes in the dynamics of amino acid metabolism with respect to the sink/source status of leaf ranks. Notably, nitrogen assimilation into valine, threonine and proline were all decreased in source leaves compared to sink leaves. Overall, our results suggested a reduction in de novo amino acid biosynthesis during sink/source transition at the vegetative stage. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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11 pages, 2118 KiB

Open AccessArticle

Liquid Chromatography Tandem Mass Spectrometry Quantification of ¹³C-Labeling in Sugars

by Jean-Christophe Cocuron, Zacchary Ross and Ana P. Alonso

Metabolites 2020, 10(1), 30; https://doi.org/10.3390/metabo10010030 - 10 Jan 2020

Cited by 6 | Viewed by 3979

Abstract

Subcellular compartmentation has been challenging in plant ¹³C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are [...] Read more.

Subcellular compartmentation has been challenging in plant ¹³C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the ¹³C-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-¹³C]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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15 pages, 2105 KiB

Open AccessArticle

On the Inverse Correlation of Protein and Oil: Examining the Effects of Altered Central Carbon Metabolism on Seed Composition Using Soybean Fast Neutron Mutants

by Shrikaar Kambhampati, Jose A. Aznar-Moreno, Cooper Hostetler, Tara Caso, Sally R. Bailey, Allen H. Hubbard, Timothy P. Durrett and Doug K. Allen

Metabolites 2020, 10(1), 18; https://doi.org/10.3390/metabo10010018 - 28 Dec 2019

Cited by 38 | Viewed by 4472

Abstract

Protein and oil levels measured at maturity are inversely correlated across soybean lines; however, carbon is in limited supply during maturation resulting in tradeoffs for the production of other reserves including oligosaccharides. During the late stages of seed development, the allocation of carbon [...] Read more.

Protein and oil levels measured at maturity are inversely correlated across soybean lines; however, carbon is in limited supply during maturation resulting in tradeoffs for the production of other reserves including oligosaccharides. During the late stages of seed development, the allocation of carbon for storage reserves changes. Lipid and protein levels decline while concentrations of indigestible raffinose family oligosaccharides (RFOs) increase, leading to a decreased crop value. Since the maternal source of carbon is diminished during seed maturation stages of development, carbon supplied to RFO synthesis likely comes from an internal, turned-over source and may contribute to the reduction in protein and lipid content in mature seeds. In this study, fast neutron (FN) mutagenized soybean populations with deletions in central carbon metabolic genes were examined for trends in oil, protein, sugar, and RFO accumulation leading to an altered final composition. Two lines with concurrent increases in oil and protein, by combined 10%, were identified. A delayed switch in carbon allocation towards RFO biosynthesis resulted in extended lipid accumulation and without compromising protein. Strategies for future soybean improvement using FN resources are described. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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16 pages, 2350 KiB

Open AccessArticle

13C-Metabolic Flux Analysis in Developing Flax (Linum usitatissinum L.) Embryos to Understand Storage Lipid Biosynthesis

by Sébastien Acket, Anthony Degournay, Yannick Rossez, Stéphane Mottelet, Pierre Villon, Adrian Troncoso-Ponce and Brigitte Thomasset

Metabolites 2020, 10(1), 14; https://doi.org/10.3390/metabo10010014 - 24 Dec 2019

Cited by 6 | Viewed by 4012

Abstract

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase [...] Read more.

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase in seed yield via marker-assisted selection. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryo development, a comprehensive analysis of metabolic flux in flax is still lacking. In this context, we have utilized in vitro cultured developing embryos of flax and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer-aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism and 12 pseudo-fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our hands, glucose was determined to be the main source of carbon in flax embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insights into the flax embryo metabolic processes involved in storage lipid biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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16 pages, 4631 KiB

Open AccessArticle

Lipidomic Analysis of Plastidial Octanoyltransferase Mutants of Arabidopsis thaliana

by Raquel Martins-Noguerol, Antonio Javier Moreno-Pérez, Sebastien Acket, Salim Makni, Rafael Garcés, Adrián Troncoso-Ponce, Joaquín J. Salas, Brigitte Thomasset and Enrique Martínez-Force

Metabolites 2019, 9(10), 209; https://doi.org/10.3390/metabo9100209 - 29 Sep 2019

Cited by 6 | Viewed by 3451

Abstract

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate [...] Read more.

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO₂, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography–electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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18 pages, 1302 KiB

Open AccessArticle

Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures

by Edward N. Smith, James S. O. McCullagh, R. George Ratcliffe and Nicholas J. Kruger

Metabolites 2019, 9(10), 205; https://doi.org/10.3390/metabo9100205 - 28 Sep 2019

Cited by 7 | Viewed by 3908

Abstract

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH [...] Read more.

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-²H]glucose or D₂O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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16 pages, 1401 KiB

Open AccessArticle

Untargeted Metabolomics Approach Reveals Diverse Responses of Pastinaca Sativa to Ozone and Wounding Stresses

by Gianni Galati, Anthony Gandin, Yves Jolivet, Romain Larbat and Alain Hehn

Metabolites 2019, 9(7), 153; https://doi.org/10.3390/metabo9070153 - 23 Jul 2019

Cited by 2 | Viewed by 4595

Abstract

Stresses such as wounding or atmospheric pollutant exposure have a significant impact on plant fitness. Since it has been widely described that the metabolome directly reflects plant physiological status, a way to assess this impact is to perform a global metabolomic analysis. In [...] Read more.

Stresses such as wounding or atmospheric pollutant exposure have a significant impact on plant fitness. Since it has been widely described that the metabolome directly reflects plant physiological status, a way to assess this impact is to perform a global metabolomic analysis. In this study, we investigated the effect of two abiotic stresses (mechanical wounding and ozone exposure) on parsnip metabolic balance using a liquid chromatography-mass spectrometry-based untargeted metabolomic approach. For this purpose, parsnip leaves were submitted to an acute ozone exposure or were mechanically wounded and sampled 24, 48, and 72 h post-treatment. Multivariate and univariate statistical analyses highlighted numerous differentially-accumulated metabolic features as a function of time and treatment. Mechanical wounding led to a more differentiated response than ozone exposure. We found that the levels of coumarins and fatty acyls increased in wounded leaves, while flavonoid concentration decreased in the same conditions. These results provide an overview of metabolic destabilization through differentially-accumulated compounds and provide a better understanding of global plant metabolic changes in defense mechanisms. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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Review

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23 pages, 1227 KiB

Open AccessEditor’s ChoiceReview

Metabolomics as an Emerging Tool for the Study of Plant–Pathogen Interactions

by Fernanda R. Castro-Moretti, Irene N. Gentzel, David Mackey and Ana P. Alonso

Metabolites 2020, 10(2), 52; https://doi.org/10.3390/metabo10020052 - 29 Jan 2020

Cited by 143 | Viewed by 13090

Abstract

Plants defend themselves from most microbial attacks via mechanisms including cell wall fortification, production of antimicrobial compounds, and generation of reactive oxygen species. Successful pathogens overcome these host defenses, as well as obtain nutrients from the host. Perturbations of plant metabolism play a [...] Read more.

Plants defend themselves from most microbial attacks via mechanisms including cell wall fortification, production of antimicrobial compounds, and generation of reactive oxygen species. Successful pathogens overcome these host defenses, as well as obtain nutrients from the host. Perturbations of plant metabolism play a central role in determining the outcome of attempted infections. Metabolomic analyses, for example between healthy, newly infected and diseased or resistant plants, have the potential to reveal perturbations to signaling or output pathways with key roles in determining the outcome of a plant–microbe interaction. However, application of this -omic and its tools in plant pathology studies is lagging relative to genomic and transcriptomic methods. Thus, it is imperative to bring the power of metabolomics to bear on the study of plant resistance/susceptibility. This review discusses metabolomics studies that link changes in primary or specialized metabolism to the defense responses of plants against bacterial, fungal, nematode, and viral pathogens. Also examined are cases where metabolomics unveils virulence mechanisms used by pathogens. Finally, how integrating metabolomics with other -omics can advance plant pathology research is discussed. Full article

(This article belongs to the Special Issue Metabolomic and Flux Analysis in Plants)

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