title: Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco.
abstract: Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.
authors.affiliation: Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Masclaux-Daubresse
authors.forename: Michèle
authors.initials: M
authors.lastname: Reisdorf-Cren
authors.forename: Karine
authors.initials: K
authors.lastname: Pageau
authors.forename: Maud
authors.initials: M
authors.lastname: Lelandais
authors.forename: Olivier
authors.initials: O
authors.lastname: Grandjean
authors.forename: Joceline
authors.initials: J
authors.lastname: Kronenberger
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Valadier
authors.forename: Magali
authors.initials: M
authors.lastname: Feraud
authors.forename: Tiphaine
authors.initials: T
authors.lastname: Jouglet
authors.forename: Akira
authors.initials: A
authors.lastname: Suzuki
mesh_terms: D000577:Amides / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics; D001377:Azaserine / Q000494:pharmacology; D001483:Base Sequence; D017930:Genes, Reporter; D005969:Glutamate Dehydrogenase / Q000502:physiology*; D005970:Glutamate Synthase / Q000032:analysis / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D018515:Plant Leaves / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000577:Amides; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D001377:Azaserine; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen