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. 2003 Jul 15;373(Pt 2):357–368. doi: 10.1042/BJ20030152

Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

Barbara Montanini 1, Marco Betti 1, Antonio J Márquez 1, Raffaella Balestrini 1, Paola Bonfante 1, Simone Ottonello 1
PMCID: PMC1223491  PMID: 12683951

Abstract

The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells.

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Selected References

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  1. Balestrini R., Mainieri D., Soragni E., Garnero L., Rollino S., Viotti A., Ottonello S., Bonfante P. Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii Vittad. Fungal Genet Biol. 2000 Dec;31(3):219–232. doi: 10.1006/fgbi.2000.1242. [DOI] [PubMed] [Google Scholar]
  2. Barel I., Bignell G., Simpson A., MacDonald D. Isolation of a DNA fragment which complements glutamine synthetase deficient strains of S. pombe. Curr Genet. 1988 Jun;13(6):487–494. doi: 10.1007/BF02427754. [DOI] [PubMed] [Google Scholar]
  3. Bennett M., Cullimore J. Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant. Eur J Biochem. 1990 Oct 24;193(2):319–324. doi: 10.1111/j.1432-1033.1990.tb19340.x. [DOI] [PubMed] [Google Scholar]
  4. Brewin N. J. Development of the legume root nodule. Annu Rev Cell Biol. 1991;7:191–226. doi: 10.1146/annurev.cb.07.110191.001203. [DOI] [PubMed] [Google Scholar]
  5. Brun A., Chalot M., Botton B., Martin F. Purification and Characterization of Glutamine Synthetase and NADP-Glutamate Dehydrogenase from the Ectomycorrhizal Fungus Laccaria laccata. Plant Physiol. 1992 Jul;99(3):938–944. doi: 10.1104/pp.99.3.938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buscot F., Munch J. C., Charcosset J. Y., Gardes M., Nehls U., Hampp R. Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems. FEMS Microbiol Rev. 2000 Dec;24(5):601–614. doi: 10.1111/j.1574-6976.2000.tb00561.x. [DOI] [PubMed] [Google Scholar]
  7. Calderón J., Martínez L. M., Mora J. Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase. J Bacteriol. 1990 Sep;172(9):4996–5000. doi: 10.1128/jb.172.9.4996-5000.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clemente M. T., Márquez A. J. Functional importance of Asp56 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase. An essential residue for transferase but not for biosynthetic enzyme activity. Eur J Biochem. 1999 Sep;264(2):453–460. doi: 10.1046/j.1432-1327.1999.00636.x. [DOI] [PubMed] [Google Scholar]
  9. Eisenberg D., Gill H. S., Pfluegl G. M., Rotstein S. H. Structure-function relationships of glutamine synthetases. Biochim Biophys Acta. 2000 Mar 7;1477(1-2):122–145. doi: 10.1016/s0167-4838(99)00270-8. [DOI] [PubMed] [Google Scholar]
  10. Finnemann J., Schjoerring J. K. Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction. Plant J. 2000 Oct;24(2):171–181. doi: 10.1046/j.1365-313x.2000.00863.x. [DOI] [PubMed] [Google Scholar]
  11. Grotjohann N., Kowallik W., Huang Y., Schulte in den Bäumen A. Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus. Z Naturforsch C. 2000 Mar-Apr;55(3-4):203–212. doi: 10.1515/znc-2000-3-411. [DOI] [PubMed] [Google Scholar]
  12. Javelle A., Rodríguez-Pastrana B. R., Jacob C., Botton B., Brun A., André B., Marini A. M., Chalot M. Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum. FEBS Lett. 2001 Sep 21;505(3):393–398. doi: 10.1016/s0014-5793(01)02802-2. [DOI] [PubMed] [Google Scholar]
  13. Javelle Arnaud, Morel Mélanie, Rodríguez-Pastrana Blanca-Rosa, Botton Bernard, André Bruno, Marini Anne-Marie, Brun Annick, Chalot Michel. Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum. Mol Microbiol. 2003 Jan;47(2):411–430. doi: 10.1046/j.1365-2958.2003.03303.x. [DOI] [PubMed] [Google Scholar]
  14. Kersten M. A., Müller Y., Op den Camp H. J., Vogels G. D., Van Griensven L. J., Visser J., Schaap P. J. Molecular characterization of the glnA gene encoding glutamine synthetase from the edible mushroom Agaricus bisporus. Mol Gen Genet. 1997 Sep;256(2):179–186. doi: 10.1007/pl00008612. [DOI] [PubMed] [Google Scholar]
  15. Lacourt Isabelle, Duplessis Sébastien, Abbà Simona, Bonfante Paola, Martin Francis. Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii. Appl Environ Microbiol. 2002 Sep;68(9):4574–4582. doi: 10.1128/AEM.68.9.4574-4582.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  18. Legrain C., Vissers S., Dubois E., Legrain M., Wiame J. M. Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis. Eur J Biochem. 1982 Apr;123(3):611–616. doi: 10.1111/j.1432-1033.1982.tb06576.x. [DOI] [PubMed] [Google Scholar]
  19. Listrom C. D., Morizono H., Rajagopal B. S., McCann M. T., Tuchman M., Allewell N. M. Expression, purification, and characterization of recombinant human glutamine synthetase. Biochem J. 1997 Nov 15;328(Pt 1):159–163. doi: 10.1042/bj3280159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Margelis S., D'Souza C., Small A. J., Hynes M. J., Adams T. H., Davis M. A. Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans. J Bacteriol. 2001 Oct;183(20):5826–5833. doi: 10.1128/JB.183.20.5826-5833.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marzluf G. A. Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev. 1997 Mar;61(1):17–32. doi: 10.1128/mmbr.61.1.17-32.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchell A. P., Magasanik B. Purification and properties of glutamine synthetase from Saccharomyces cerevisiae. J Biol Chem. 1983 Jan 10;258(1):119–124. [PubMed] [Google Scholar]
  23. Montanini Barbara, Moretto Nadia, Soragni Elisabetta, Percudani Riccardo, Ottonello Simone. A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii. Fungal Genet Biol. 2002 Jun;36(1):22–34. doi: 10.1016/S1087-1845(02)00001-4. [DOI] [PubMed] [Google Scholar]
  24. Muhitch M. J. Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels. Plant Physiol. 1989 Nov;91(3):868–875. doi: 10.1104/pp.91.3.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pesole G., Bozzetti M. P., Lanave C., Preparata G., Saccone C. Glutamine synthetase gene evolution: a good molecular clock. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):522–526. doi: 10.1073/pnas.88.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Riedel J., Tischner R., Mäck G. The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast. Planta. 2001 Jul;213(3):396–401. doi: 10.1007/s004250000509. [DOI] [PubMed] [Google Scholar]
  27. Sakakibara H., Shimizu H., Hase T., Yamazaki Y., Takao T., Shimonishi Y., Sugiyama T. Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots. J Biol Chem. 1996 Nov 22;271(47):29561–29568. doi: 10.1074/jbc.271.47.29561. [DOI] [PubMed] [Google Scholar]
  28. Sims A. P., Toone J., Box V. The regulation of glutamine synthesis in the food yeast Candida utilis: the purification and subunit structure of glutamine synthetase and aspects of enzyme deactivation. J Gen Microbiol. 1974 Feb;80(2):485–499. doi: 10.1099/00221287-80-2-485. [DOI] [PubMed] [Google Scholar]
  29. Soragni E., Bolchi A., Balestrini R., Gambaretto C., Percudani R., Bonfante P., Ottonello S. A nutrient-regulated, dual localization phospholipase A(2) in the symbiotic fungus Tuber borchii. EMBO J. 2001 Sep 17;20(18):5079–5090. doi: 10.1093/emboj/20.18.5079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de la Torre Fernando, García-Gutiérrez Angel, Crespillo Remedios, Cantón Francisco R., Avila Concepción, Cánovas Francisco M. Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties. Plant Cell Physiol. 2002 Jul;43(7):802–809. doi: 10.1093/pcp/pcf094. [DOI] [PubMed] [Google Scholar]

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