Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula.
Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis.
UMR INRA 1191, Physiologie Moléculaire des semences, University of Angers, 2 Bd Lavoisier, 49045 Angers cedex 01, France.
Gaëlle
G
Glevarec
Sophie
S
Bouton
Emmanuel
E
Jaspard
Marie-Thérèse
MT
Riou
Jean-Bernard
JB
Cliquet
Akira
A
Suzuki
Anis M
AM
Limami
D002244:Carbon / Q000378:metabolism; D018548:Cotyledon / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D003470:Culture Media; D015870:Gene Expression; D018525:Germination; D005969:Glutamate Dehydrogenase / Q000502:physiology*; D005970:Glutamate Synthase / Q000502:physiology*; D005974:Glutamate-Ammonia Ligase / Q000502:physiology*; D029902:Medicago / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000201:enzymology / Q000254:growth & development; D013997:Time Factors
D003470:Culture Media; D000644:Quaternary Ammonium Compounds; D002244:Carbon; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen