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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

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Abstract

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.

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Abbreviations

EST :

Expressed sequence tag

Fd-GOGAT :

Ferredoxin-dependent glutamate synthase

GDH :

Glutamate dehydrogenase

GS :

Glutamine synthetase

GS1a, GS1b :

Cytosolic GS

GS2 :

Chloroplastic GS

cICDH :

Cytosolic isocitrate dehydrogenase

mIDH :

Mitochondrial isocitrate dehydrogenase

MSC27 :

Medicago sativa cDNA 27

MSX :

Methionine sulfoximine

NADH-GOGAT :

NADH-dependent glutamate synthase

PEPc :

Phosphoenolpyruvate carboxylase

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Acknowledgements

The authors are grateful to Professor R. Adams for kindly correcting the English. We thank B. Jettner (Seed-Co Australia Co-Operative Ltd., Hilton, Australia) for the generous gift of M. truncatula cv. Paraggio seeds.

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Authors and Affiliations

  1. UMR INRA 1191, Physiologie Moléculaire des semences, University of Angers, 2 Bd Lavoisier, 49045, Angers cedex 01, France

    Gaëlle Glevarec, Sophie Bouton, Emmanuel Jaspard, Marie-Thérèse Riou & Anis M. Limami

  2. UMR INRA 950, Physiologie et Biochimie Végétales, University of Caen, 14032, Caen cedex, France

    Jean-Bernard Cliquet

  3. Unité de recherche Nutrition Azotée des Plantes, INRA, Route de St Cyr, 78026, Versailles cedex 01, France

    Akira Suzuki

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  1. Gaëlle Glevarec
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  2. Sophie Bouton
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  3. Emmanuel Jaspard
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  4. Marie-Thérèse Riou
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  7. Anis M. Limami
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Correspondence to Anis M. Limami.

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Glevarec, G., Bouton, S., Jaspard, E. et al. 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 . Planta 219, 286–297 (2004). https://doi.org/10.1007/s00425-004-1214-9

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