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3. Massive production of butanediol during plant infection by phytopathogenic bacteria of the genera Dickeya and Pectobacterium

Author

Effantin, Géraldine, Rivasseau, Corinne, Gromova, Marina, Bligny, Richard, Hugouvieux-Cotte-Pattat, Nicole, Trafic et signalisation membranaires chez les bactéries (MTSB), Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Magnetic Resonance Spectroscopy, Virulence Factors, Pectobacterium, metabolite, Carbohydrates, plant, Nuclear magnetic resonance, butanediol biosynthesis, phytopathogen, Enterobacteriaceae, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acids, Dickeya, bacteria, Butylene Glycols, Plant Diseases, Virulence, Gene Expression Profiling, fungi, food and beverages, Gene Expression Regulation, Bacterial, Plants, NMR, Genes, Bacterial, gene expression, Metabolome, budA, budB, budC, physiopathology, metabolism, Gene Deletion
Abstract

International audience; Plant pathogenic bacteria of the genera Dickeya and Pectobacterium are broad-host-range necrotrophs which cause soft-rot diseases in important crops. A metabolomic analysis, based on (13) C-NMR spectroscopy, was used to characterize the plant-bacteria interaction. Metabolic profiles revealed a decline in plant sugars and amino acids during infection and the concomitant appearance of a compound identified as 2,3-butanediol. Butanediol is the major metabolite found in macerated tissues of various host plants. It is accumulated during the symptomatic phase of the disease. Different species of Dickeya or Pectobacterium secrete high levels of butanediol during plant infection. Butanediol has been described as a signalling molecule involved in plant/bacterium interactions and, notably, able to induce plant systemic resistance. The bud genes, involved in butanediol production, are conserved in the phytopathogenic enterobacteria of the genera Dickeya, Pectobacterium, Erwinia, Pantoea and Brenneria. Inactivation of the bud genes of Dickeya dadantii revealed that the virulence of budA, budB and budR mutants was clearly reduced. The genes budA, budB and budC are highly expressed during plant infection. These data highlight the importance of butanediol metabolism in limiting acidification of the plant tissue during the development of the soft-rot disease caused by pectinolytic enterobacteria.

Published
2011
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4. Measurement of carbon flux through the MEP pathway for isoprenoid synthesis by (31)P-NMR spectroscopy after specific inhibition of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate reductase. Effect of light and temperature

Author

Mongélard, Gaëlle, Seemann, Myriam, Boisson, Anne-Marie, Rohmer, Michel, Bligny, Richard, Rivasseau, Corinne, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie de Strasbourg, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Magnetic Resonance Spectroscopy, Buxus sempervirens, methylerythritol 4-phosphate, Mahonia, Mevalonic Acid, plant, Mahonia aquifolium, Carbon Cycle, isoprenoid biosynthesis, mevalonate, Spinacia oleracea, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Enzyme Inhibitors, Buxus, Pisum sativum, Terpenes, Peas, temperature, metabolic pathway, Plants, metabolic flux, nuclear magnetic resonance, Erythritol, Oxidoreductases, light, Phosphorus Radioisotopes, metabolism, Metabolic Networks and Pathways, Cadmium
Abstract

International audience; The methylerythritol 4-phosphate (MEP) and the mevalonate pathways are the unique synthesis routes for the precursors of all isoprenoids. An original mean to measure the carbon flux through the MEP pathway in plants is proposed by using cadmium as a total short-term inhibitor of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) reductase (GcpE) and measuring the accumulation rate of its substrate MEcDP by (31) P-NMR spectroscopy. The MEP pathway metabolic flux was determined in spinach (Spinacia oleracea), pea (Pisum sativum), Oregon grape (Mahonia aquifolium) and boxwood (Buxus sempervirens) leaves. In spinach, flux values were compared with the synthesis rate of major isoprenoids. The flux increases with light intensity (fourfold in the 200-1200 µmol m(-2) s(-1) PPFR range) and temperature (sevenfold in the 25-37 °C range). The relationship with the light and the temperature dependency of isoprenoid production downstream of the MEP pathway is discussed.

Published
2011
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5. Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Author

Stefanovic, Aleksandra, Arpat, A Bulak, Bligny, Richard, Gout, Elisabeth, Vidoudez, Charles, Bensimon, Michaël, Poirier, Yves, Department of Plant Molecular Biology, Biophore, Université de Lausanne = University of Lausanne (UNIL), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de géologie de l'ingénieur et de l'environnement (GEOLEP), Ecole Polytechnique Fédérale de Lausanne (EPFL), Université de Lausanne (UNIL), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de geologie de l'ingénieur et de l'environnement, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Cytoplasm, Higher-Plant Cells, Murine Leukemia Viruses, Arabidopsis thaliana, Nuclear-Magnetic-Resonance, Nuclear Magnetic Resonance, Gene Family, Arabidopsis, Transport, translocation, plant, xylem, Plant Roots, Phosphates, Gene Expression Regulation, Plant, Thaliana, Homeostasis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell-Surface Receptor, phosphate, Arabidopsis Proteins, Protoplasts, fungi, food and beverages, Plants, Genetically Modified, efflux, NMR, Plant Leaves, Pho1, Vacuoles, gene expression, Deficiency, metabolism, Plant Shoots
Abstract

International audience; Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

Published
2011
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6. Early response of plant cell to carbon deprivation: in vivo 31P-NMR spectroscopy shows a quasi-instantaneous disruption on cytosolic sugars, phosphorylated intermediates of energy metabolism, phosphate partitioning, and intracellular pHs

Author

Gout, Elisabeth, Bligny, Richard, Douce, Roland, Boisson, Anne-Marie, Rivasseau, Corinne, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), UMR [5168], Institut de Recherche en Technologies et Sciences pour le Vivant, CEA-Grenoble, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Hexose-P, Time Factors, Nuclear Magnetic Resonance, intracellular pH, Acer, Inorganic phosphate, Acer pseudoplatanus, Hydrogen-Ion Concentration, Cytosoi/vacuole, Carbon, Phosphates, Cytosol, metabolic changes, Carbohydrate Metabolism, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphorylation, Energy Metabolism, metabolism, sugar starvation, Nuclear Magnetic Resonance, Biomolecular, plant cell
Abstract

International audience; • In plant cells, sugar starvation triggers a cascade of effects at the scale of 1-2 days. However, very early metabolic response has not yet been investigated. • Soluble phosphorus (P) compounds and intracellular pHs were analysed each 2.5 min intervals in heterotrophic sycamore (Acer pseudoplatanus) cells using in vivo phosphorus nuclear magnetic resonance ((31)P-NMR). • Upon external-sugar withdrawal, the glucose 6-P concentration dropped in the cytosol, but not in plastids. The released inorganic phosphate (Pi) accumulated transiently in the cytosol before influx into the vacuole; nucleotide triphosphate concentration doubled, intracellular pH increased and cell respiration decreased. It was deduced that the cytosolic free-sugar concentration was low, corresponding to only 0.5 mM sucrose in sugar-supplied cells. • The release of sugar from the vacuole and from plastids is insufficient to fully sustain the cell metabolism during starvation, particularly in the very short term. Similarly to Pi-starvation, the cell's first response to sugar starvation occurs in the cytosol and is of a metabolic nature. Unlike the cytoplasm, cytosolic homeostasis is not maintained during starvation. The important metabolic changes following cytosolic sugar exhaustion deliver early endogenous signals that may contribute to trigger rescue metabolism.

Published
2010
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7. On the resilience of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and metabolomic techniques

Author

Bathellier, Camille, Tcherkez, Guillaume, Mauve, Caroline, Bligny, Richard, Gout, Elizabeth, Ghashghaie, Jaleh, Plateforme Métabolisme-Métabolome (IFR87), Université Paris-Sud - Paris 11 (UP11), Ecologie Systématique et Evolution (ESE), Université Paris-Sud - Paris 11 (UP11)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Phaseolus, Carbon Isotopes, Nitrogen Isotopes, carbon primary metabolism, Nitrogen, metabolite, plant, nitrogen assimilation, root, Plant Roots, Gas Chromatography-Mass Spectrometry, NMR, nuclear magnetic resonance, darkness, Isotope Labeling, Metabolomics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, metabolome, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acids, metabolism, Plant Proteins
Abstract

International audience; The response of root metabolism to variations in carbon source availability is critical for whole-plant nitrogen (N) assimilation and growth. However, the effect of changes in the carbohydrate input to intact roots is currently not well understood and, for example, both smaller and larger values of root:shoot ratios or root N uptake have been observed so far under elevated CO(2). In addition, previous studies on sugar starvation mainly focused on senescent or excised organs while an increasing body of data suggests that intact roots may behave differently with, for example, little protein remobilization. Here, we investigated the carbon and nitrogen primary metabolism in intact roots of French bean (Phaseolus vulgaris L.) plants maintained under continuous darkness for 4 days. We combined natural isotopic (15)N/(14)N measurements, metabolomic and (13)C-labeling data and show that intact roots continued nitrate assimilation to glutamate for at least 3 days while the respiration rate decreased. The activity of the tricarboxylic acid cycle diminished so that glutamate synthesis was sustained by the anaplerotic phosphoenolpyruvate carboxylase fixation. Presumably, the pentose phosphate pathway contributed to provide reducing power for nitrate reduction. All the biosynthetic metabolic fluxes were nevertheless down-regulated and, consequently, the concentration of all amino acids decreased. This is the case of asparagine, strongly suggesting that, as opposed to excised root tips, protein remobilization in intact roots remained very low for 3 days in spite of the restriction of respiratory substrates.

Published
2009
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8. Amino acid changes during sunflower infection by the necrotrophic fungus B. cinerea

Author

Dulermo, Thierry, Bligny, Richard, Gout, Elisabeth, Cotton, Pascale, Laboratoire de Génomique Fonctionnelle des Champignons Pathogènes des Plantes (UMR Microbiologie), Université de Lyon, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Botrytis cinerea, pathogenesis, Nuclear Magnetic Resonance, Fungi, food and beverages, plant, Helianthus annuus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, aminoacid, plant-phytopathogen interaction, metabolism, NMR
Abstract

Addendum to: Dulermo T, Rascle C, Chinnici G, Gout E, Bligny R, Cotton P. Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic Fungus Botrytis cinerea: from plant hexoses to mannitol. New Phytol (2009) 183(4): 1149-1162; International audience; Metabolic changes that occur in host tissues during a necrotrophic plant/fungal interaction have been poorly investigated. Whereas carbon metabolism reprogramming and photosynthesis disturbances have been studied,1 data on plant amino acids stores during infection are scarce. Here we report an analysis of sunflower cotyledon amino acid content during infection with the necrotrophic fungus Botrytis cinerea, by using 13C-NMR spectroscopy. A rapid disappearance of plant amino acids was observed, most probably due to fungal assimilation. In order to explore amino acid changes due to host reaction, we investigated the amino acid content in healthy and invaded region of infected leaves. During the course of infection, glutamate and total amino acid stores were affected at distance in the non invaded region. Glutamate depletion was correlated to an enhanced sunflower glutamate dehydrogenase (GDH) transcription level in the area invaded by pathogen. Our data suggest that glutamate could be transferred to the invaded region to supply nitrogen. Such a strategy could delay cell death, and consequently disturb fungal progression in plant tissues.

Published
2009

9. Metabolomic characterisation of the functional division of nitrogen metabolism in variegated leaves G. Tcherkez et al. Variegation and nitrogen metabolism.

Author

Tcherkez, Guillaume, Guérard, Florence, Gilard, Françoise, Lamothe, Marlène, Mauve, Caroline, Gout, Elisabeth, and Bligny, Richard

Subjects
PLANT metabolism, NITROGEN content of plants, PELARGONIUMS, BIOSYNTHESIS, ARGININE, NUCLEAR magnetic resonance, MASS spectrometry
Abstract

Many horticultural and natural plant species have variegated leaves, that is, patchy leaves with green and non-green or white areas. Specific studies on the metabolism of variegated leaves are scarce and although white (non-green) areas have been assumed to play the role of a 'nitrogen store', there is no specific studies showing the analysis of nitrogenous metabolites and the dynamics of nitrogen assimilation. Here, we examined the metabolism of variegated leaves of Pelargonium x hortorum. We show that white areas have a larger N : C ratio, more amino acids, with a clear accumulation of arginine. Metabolomic analyses revealed clear differences in the chemical composition, suggesting contrasted metabolic commitments such as an enhancement of alkaloid biosynthesis in white areas. Using isotopic labelling followed by nuclear magnetic resonance or liquid chromatography/mass spectrometry, we further showed that in addition to glutamine, tyrosine and tryptophan, N metabolism forms ornithine in green area and huge amounts of arginine in white areas. Fine isotopic measurements with isotope ratio mass spectrometry indicated that white and green areas exchange nitrogenous molecules but nitrogen export from green areas is quantitatively much more important. The biological significance of the metabolic exchange between leaf areas is briefly discussed. Metabolic differences between green and white parts of variegated leaves, which may help understanding nitrogen assimilation and partitioning in horticultural species, are poorly documented. Here, using integrative and isotopic methods, an enhancement of amino acid and alkaloid synthesis in white parts and an exchange of nitrogenous compounds between leaf parts is demonstrated. White parts seem to play the role of an advantageous 'nitrogen reserve' to sustain leaf metabolism. [ABSTRACT FROM AUTHOR]

Published
2012
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10. Resistance to irradiation of micro-algae growing in the storage pools of a nuclear reactor investigated by NMR and neutron spectroscopies.

Author

Rivasseau, Corinne, Farhi, Emmanuel, Gromova, Marina, Ollivier, Jacques, and Bligny, Richard

Subjects
IRRADIATION, MICROALGAE, NUCLEAR reactors, NUCLEAR magnetic resonance, NEUTRONS, GREEN algae, MACROMOLECULES, BACKSCATTERING
Abstract

A green Chlorophycean micro-alga surviving high irradiation doses has been discovered in the storage pools of a nuclear reactor. Investigating the mechanisms sustaining its properties is of peculiar interest. Metabolic impact of irradiation on the micro-alga as well as structural macromolecular impact were investigated using nuclear magnetic resonance and neutron backscattering spectroscopy. Carbohydrate, amino acid and organic acid content studied as a function of the irradiation dose revealed an intense protein repair activity. Resilience was extracted according to irradiation dose and compared to that of other extremophile species. [ABSTRACT FROM AUTHOR]

Published
2010
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11. In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus.

Author

Gauthier, Paul P. G., Bligny, Richard, Gout, Elizabeth, Mah, Aline, Nogués, Salvador, Hodges, Michael, and Tcherkez, Guillaume G. B.

Subjects
*RUTABAGA, *GLUTAMIC acid, *GLUTAMINE, *BRASSICA, *RAPESEED, *GLYCOLYSIS, *NUCLEAR magnetic resonance, *AMINO acids, *METABOLISM
Abstract

•Nitrogen assimilation in leaves requires primary NH2 acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO2 evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. •In this study, we carried out isotopic labelling experiments with 13CO2 and 15N-ammonium nitrate on detached leaves of rapeseed ( Brassica napus), and performed 13C- and 15N-nuclear magnetic resonance analyses. •Our results indicated that the production of 13C-glutamate and 13C-glutamine under a 13CO2 atmosphere was very weak, whereas 13C-glutamate and 13C-glutamine appeared in both the subsequent dark period and the next light period under a 12CO2 atmosphere. Consistently, the analysis of heteronuclear (13C–15N) interactions within molecules indicated that most 15N-glutamate and 15N-glutamine molecules were not 13C labelled after 13C/15N double labelling. That is, recent carbon atoms (i.e. 13C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by 15N incorporation. •We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation. [ABSTRACT FROM AUTHOR]

Published
2010
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12. Phosphate (Pi) Starvation Effect on the Cytosolic Pi Concentration and Pi Exchanges across the Tonoplast in Plant Cells: An in Vivo 31P-Nuclear Magnetic Resonance Study Using Methylphosphonate as a Pi Analog.

Author

Pratt, James, Boisson, Anne-Marie, Gout, Elisabeth, Bligny, Richard, Douce, Roland, and Aubert, Serge

Subjects
CYTOSOL, SYCAMORES, ARABIDOPSIS thaliana, TONOPLASTS, CELL membranes, PLANT cells & tissues, BIOCHEMISTRY, NUCLEAR magnetic resonance
Abstract

In vivo 31P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplafanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 μM instead of >1 mM) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects. [ABSTRACT FROM AUTHOR]

Published
2009
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13. Accumulation of 2- C-methyl-d-erythritol 2,4-cyclodiphosphate in illuminated plant leaves at supraoptimal temperatures reveals a bottleneck of the prokaryotic methylerythritol 4-phosphate pathway of isoprenoid biosynthesis.

Author

RIVASSEAU, CORINNE, SEEMANN, MYRIAM, BOISSON, ANNE-MARIE, STREB, PETER, GOUT, ELISABETH, DOUCE, ROLAND, ROHMER, MICHEL, and BLIGNY, RICHARD

Subjects
METABOLIC profile tests, METABOLISM testing, NUCLEAR magnetic resonance, TEMPERATURE, ISOPENTENOIDS, BIOCHEMISTRY
Abstract

Metabolic profiling using phosphorus nuclear magnetic resonance (31P-NMR) revealed that the leaves of different herbs and trees accumulate 2- C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach ( Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only 31P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by ( E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O2 and ROS produced by photosynthesis damage this O2-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd2+ does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature. [ABSTRACT FROM AUTHOR]

Published
2009
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14. Early response of plant cell to carbon deprivation: in vivo.

Author

Gout, Elisabeth, Bligny, Richard, Douce, Roland, Boisson, Anne-Marie, and Rivasseau, Corinne

Subjects
*PLANT cells & tissues, *CELL metabolism, *ACER pseudoplatanus, *CYTOSOL, *CYTOPLASM, *NUCLEAR magnetic resonance, *PLANT physiology
Abstract

In plant cells, sugar starvation triggers a cascade of effects at the scale of 1-2 days. However, very early metabolic response has not yet been investigated. Soluble phosphorus (P) compounds and intracellular pHs were analysed each 2.5 min intervals in heterotrophic sycamore (Acer pseudoplatanus) cells using in vivo phosphorus nuclear magnetic resonance (31P-NMR). Upon external-sugar withdrawal, the glucose 6-P concentration dropped in the cytosol, but not in plastids. The released inorganic phosphate (Pi) accumulated transiently in the cytosol before influx into the vacuole; nucleotide triphosphate concentration doubled, intracellular pH increased and cell respiration decreased. It was deduced that the cytosolic free-sugar concentration was low, corresponding to only 0.5 mM sucrose in sugar-supplied cells. The release of sugar from the vacuole and from plastids is insufficient to fully sustain the cell metabolism during starvation, particularly in the very short term. Similarly to Pi-starvation, the cell's first response to sugar starvation occurs in the cytosol and is of a metabolic nature. Unlike the cytoplasm, cytosolic homeostasis is not maintained during starvation. The important metabolic changes following cytosolic sugar exhaustion deliver early endogenous signals that may contribute to trigger rescue metabolism. [ABSTRACT FROM AUTHOR]

Published
2011
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