Skip to main content
SpringerLink
Log in

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher V max value and 15.9–43.1 times higher K m value for NH4 +, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with 15NH4 + showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH4 + absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CE–MS:

Capillary electrophoresis–mass spectrometry

GDH:

Glutamate dehydrogenase

GOGAT:

Glutamate synthase

GS:

Glutamine synthetase

MSX:

Methionine sulfoximine

2-OG:

2-Oxoglutarate

References

  • Abiko T, Obara M, Ushioda A, Hayakawa T, Hodges M, Yamaya T (2005) Localization of NAD-isocitrate dehydrogenase and glutamate dehydrogenase in rice roots: candidates for providing carbon skeletons to NADH-glutamate synthase. Plant Cell Physiol 46:1724–1734

    Article  CAS  PubMed  Google Scholar 

  • Ameziane R, Bernhard K, Lightfoot D (2000) Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development. Plant Soil 221:47–57

    Article  CAS  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chichkova S, Arellano J, Vance CP, Hernández G (2001) Transgenic tobacco plants that overexpress alfalfa NADH-glutamate synthase have higher carbon and nitrogen content. J Exp Bot 52:2079–2087

    CAS  PubMed  Google Scholar 

  • Fuentes SI, Allen DJ, Ortiz-Lopez A, Hernández G (2001) Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations. J Exp Bot 52:1071–1081

    Article  CAS  PubMed  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    Article  CAS  PubMed  Google Scholar 

  • Hirel B, Gadal P (1980) Glutamine synthetase in rice. Plant Physiol 66:619–623

    Article  CAS  PubMed  Google Scholar 

  • Hodges M (2002) Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation. J Exp Bot 53:905–916

    Article  CAS  PubMed  Google Scholar 

  • Ireland RJ, Lea PJ (1999) The enzymes of glutamine, glutamate, asparagine, and aspartate metabolism. In: Singh BK (ed) Plant amino acids: biochemistry and biotechnology. Marcel Dekker, New York, pp 49–109

    Google Scholar 

  • Ishiyama K, Inoue E, Watanabe-Takahashi A, Obara M, Yamaya T, Takahashi H (2004a) Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis. J Biol Chem 16(279):16598–16605

    Article  Google Scholar 

  • Ishiyama K, Inoue E, Tabuchi M, Yamaya T, Takahashi H (2004b) Biochemical background and compartmentalized functions of cytosolic glutamine synthetase for active ammonium assimilation in rice roots. Plant Cell Physiol 45:1640–1647

    Article  CAS  PubMed  Google Scholar 

  • Kamachi K, Yamaya T, Hayakawa T, Mae T, Ojima K (1992) Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol 99:1481–1486

    Article  CAS  PubMed  Google Scholar 

  • Kinghorn JR, Pateman JA (1973) NAD and NADP l-glutamate dehydrogenase activity and ammonium regulation in Aspergillus nidulans. J Gen Microbiol 78:39–46

    CAS  PubMed  Google Scholar 

  • Kisaka H, Kida T (2007) Transgenic tomato plant carrying a gene for NADP-dependent glutamate dehydrogenase (gdhA) from Aspergillus nidulans. Plant Sci 164:35–42

    Article  Google Scholar 

  • Kiyomiya S, Nakanishi H, Uchida H, Tsuji A, Nishiyama S, Futatsubashi M, Tsukada H, Ishioka NS, Watanabe S, Ito T, Mizuniwa C, Osa A, Matsuhashi S, Hashimoto S, Sekine T, Mori S (2001) Real time visualization of 13N-translocation in rice under different environmental conditions using positron emitting tracer imaging system. Plant Physiol 125:1743–1753

    Article  CAS  PubMed  Google Scholar 

  • Labboun S, Tercé-Laforgue T, Roscher A, Bedu M, Restivo FM, Velanis CN, Skopelitis DS, Moshou PN, Roubelakis-Angelakis KA, Suzuki A, Hirel B (2009) Resolving the role of plant glutamate dehydrogenase. I. In vivo real time nuclear magnetic resonance spectroscopy experiments. Plant Cell Physiol 50:1761–1773

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lea PJ, Miflin BJ (1974) Alternative route for nitrogen assimilation in higher plants. Nature 18:614–616

    Article  Google Scholar 

  • Lea PJ, Thurman DA (1972) Intracellular location and properties of plant l-glutamate dehydrogenases. J Exp Bot 23:440–449

    Article  CAS  Google Scholar 

  • Macheda ML, Hynes MJ, Davis MA (1999) The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase. Curr Genet 34:467–471

    Article  CAS  PubMed  Google Scholar 

  • Mae T, Ohira K (1981) The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant Cell Physiol 22:1067–1074

    CAS  Google Scholar 

  • Magalhães JR, Ju GC, Rich PJ, Rhodes D (1990) Kinetics of 15NH4 + assimilation in Zea mays. Plant Physiol 94:647–656

    Article  PubMed  Google Scholar 

  • Melo-Oliveira R, Oliveira IC, Coruzzi GM (1996) Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Proc Natl Acad Sci 14:4718–4723

    Article  Google Scholar 

  • Miyashita Y, Good AG (2008) NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation. J Exp Bot 59:667–680

    Article  CAS  PubMed  Google Scholar 

  • Mungur R, Glass AD, Goodenow DB, Lightfoot DA (2005) Metabolite fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. J Biomed Biotechnol 30:198–214

    Article  Google Scholar 

  • Noor S, Punekar NS (2005) Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface. Microbiology 151:1409–1419

    Article  CAS  PubMed  Google Scholar 

  • Obara M, Sato T, Yamaya T (2000) High contents of glutamine synthetase does not accompany a high activity of the enzyme in rice (Oryza sativa) leaves of indica cultivars. Physiol Plant 108:11–18

    Article  CAS  Google Scholar 

  • Obara M, Tamura W, Ebitani T, Yano M, Sato T, Yamaya T (2010) Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions. Theor Appl Genet. doi:10.1007/s00122-010-1328-3

  • Oliveira IC, Brears T, Knight TJ, Clark A, Coruzzi GM (2002) Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration. Plant Physiol 129:170–180

    Article  Google Scholar 

  • Paczek V, Dubois F, Sangwan R, Morot-Gaudry JF, Roubelakis-Angelakis KA, Hirel B (2003) Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism. Planta 216:245–254

    Article  Google Scholar 

  • Purnell MP, Botella JR (2007) Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo. Plant Physiol 143:530–539

    Article  CAS  PubMed  Google Scholar 

  • Purnell MP, Skopelitis DS, Roubelakis-Angelakis KA, Botella JR (2005) Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels. Planta 222:167–180

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sato S, Soga T, Nishioka T, Tomita M (2004) Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection. Plant J 40:151–163

    Article  CAS  PubMed  Google Scholar 

  • Skopelitis DS, Paranychianakis NV, Kouvarakis A, Spyros A, Stephanou EG, Roubelakis-Angelakis KA (2007) The isoenzyme 7 of tobacco NAD(H)-dependent glutamate dehydrogenase exhibits high deaminating and low aminating activities in vivo. Plant Physiol 145:1726–1734

    Article  CAS  PubMed  Google Scholar 

  • Tabuchi M, Sugiyama K, Ishiyama K, Inoue E, Sato T, Takahashi H, Yamaya T (2005) Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1. Plant J 42:641–651

    Article  CAS  PubMed  Google Scholar 

  • Temple SJ, Knight TJ, Unkefer PJ, Sengupta-Gopalan C (1993) Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis. Mol Gen Genet 236:315–325

    Article  CAS  PubMed  Google Scholar 

  • Tercé-Laforgue T, Dubois F, Ferrario-Méry S, de Crecenzo MA, Sangwan R, Hirel B (2004) Glutamate dehydrogenase of tobacco is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration. Plant Physiol 136:4308–4317

    Article  PubMed  Google Scholar 

  • Wakayama M, Abiko T, Asano T, Kawakami A, Egami T, Aoki N, Sasaki H, Ohsugi R (2008) Analysis of carboxylic acids and amino acids in plant extracts by using the capillary zone electrophoresis/tandem mass spectroscopy. In: Conference abstract book, 5th international conference on plant metabolomics, 15–18 July 2008, Yokohama, Japan, pp 162–163

  • Wootton JC (1983) Re-assessment of ammonium-ion affinities of NADP-specific glutamate dehydrogenases. Biochem J 209:527–531

    CAS  PubMed  Google Scholar 

  • Yamaya T, Oaks A, Matsumoto H (1984) Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots. Plant Physiol 76:1009–1013

    Article  CAS  PubMed  Google Scholar 

  • Yamaya T, Hayakawa T, Tanasawa K, Kamachi K, Mae T, Ojima K (1992) Tissue distribution of glutamate synthase and glutamine synthetase in rice leaves. Plant Physiol 100:1427–1432

    Article  CAS  PubMed  Google Scholar 

  • Yamaya T, Obara M, Nakajima H, Sasaki S, Hayakawa T, Sato T (2002) Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. J Exp Bot 53:917–925

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. Tatsuro Hirose for provision of the antibody of susy2, Dr. Nakagawa for provision of the pGWB5 vector, and Dr. Masaru Fujimoto for assistance with confocal laser microscopy. The field trial in South Korea was practiced in cooperation with Inplanta Innovations Inc. (Yokohama, Japan). This work was supported by a grant from the PROBRAIN (Program for promotion of Basic Research Activities for Innovative Biosciences) project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Author information

Authors and Affiliations

  1. Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Tomomi Abiko, Masataka Wakayama, Akira Kawakami, Naohiro Aoki & Ryu Ohsugi

  2. Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ohwashi 1-1, Tsukuba, Ibaraki, 305-8686, Japan

    Mitsuhiro Obara

  3. Production and Technology Administration Center, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa, 210-8681, Japan

    Hiroaki Kisaka

  4. Research Institute for Health Fundamentals, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa, 210-8681, Japan

    Tetsuya Miwa

Authors
  1. Tomomi Abiko
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masataka Wakayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akira Kawakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mitsuhiro Obara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroaki Kisaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tetsuya Miwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Naohiro Aoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ryu Ohsugi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ryu Ohsugi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Abiko, T., Wakayama, M., Kawakami, A. et al. Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA). Planta 232, 299–311 (2010). https://doi.org/10.1007/s00425-010-1172-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1172-3

Keywords

Search

Navigation