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Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

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Abstract

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, 15NH +4 was incorporated into [5−15N]glutamine and [2−15N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−15N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−15N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation.

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Abbreviations

AAT2:

Cytosolic aspartate aminotransferase

ASN :

Asparagine synthetase gene

Fd:

Ferredoxin

FNR:

Ferredoxin:NADP+ oxidoreductase

FR:

Far-red

FW:

Fresh weight

GDH :

NADH-glutamate dehydrogenase gene

GLN1(2) :

Cytosolic (chloroplastic) glutamine synthetase gene

GLT :

NADH-glutamate synthase gene

GLU1 (GLU2):

Ferredoxin-glutamate synthase gene 1 (2)

GOGAT:

Glutamate synthase

Lhcb :

Light-harvesting chlorophyll a/b-protein gene of photosystem II

NH +4 :

Ammonium

NII :

Nitrite reductase gene

NIR :

Nitrate reductase gene

NO −3 :

Nitrate

PPFD:

Photosynthetic photon flux density

PSI:

Photosystem I

R:

Red

uidA :

ß-Glucuronidase uidA (gusA) gene (GUS)

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Acknowledgements

We would like to thank Dr. Steven Rothstein (Department of Molecular Biology and Genetics, University of Guelph, Canada) for his helpful comments in preparing the manuscript, and Dr. JP Boutin (Laboratoire de Biologie des Semences, INRA, Versailles, France) and Dr. M Reisdorf-Cren (Unité de Nutrition Azotée des Plantes, INRA, Versailles, France) for their help in amino acid analysis and 15N labeling experiments.

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Authors and Affiliations

  1. Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique (INRA), Route de St-Cyr, 78026, Versailles cedex, France

    Magali Feraud, Céline Masclaux-Daubresse, Sylvie Ferrario-Méry, Karine Pageau, Maud Lelandais, Christine Ziegler, Edouard Leboeuf, Tiphaine Jouglet, Lauriane Viret, Axelle Spampinato, Vanina Paganelli, Mounir Ben Hammouda & Akira Suzuki

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  1. Magali Feraud
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  2. Céline Masclaux-Daubresse
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  3. Sylvie Ferrario-Méry
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  6. Christine Ziegler
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  9. Lauriane Viret
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  10. Axelle Spampinato
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  11. Vanina Paganelli
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  12. Mounir Ben Hammouda
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  13. Akira Suzuki
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Corresponding author

Correspondence to Akira Suzuki.

Additional information

The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525

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Feraud, M., Masclaux-Daubresse, C., Ferrario-Méry, S. et al. Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.). Planta 222, 667–677 (2005). https://doi.org/10.1007/s00425-005-0013-2

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