, 223:558 | Cite as

Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula

  • Lígia Lima
  • Ana Seabra
  • Paula Melo
  • Julie Cullimore
  • Helena CarvalhoEmail author
Original Article


In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)6-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.


14-3-3 proteins Glutamine Synthetase Medicago Phosphorylation Proteolysis 



glutamine synthetase


plastid GS


nitrate reductase


nickel-nitrilotriacetic acid

6x his-tag

six histidine tag



We gratefully acknowledge Dr. Carol Mackintosh (MRC unit, University of Dundee, UK) for providing anti-14-3-3 antibodies. We are also extremely grateful to Michel Rossignol and Giselle Borderie (IFR40, Toulouse, France) for expert assistance in 2D electrophoresis. We are also grateful to Jorge Azevedo and Pedro Pereira (IBMC, Porto, Portugal) for helpful discussions. This work was supported by the Fundação para a Ciência e Tecnologia (Project no. POC/PI/41433/2001)


  1. Adam Z (1996) Protein stability and degradation in chloroplasts. Plant Mol Biol. 32: 773–83CrossRefPubMedGoogle Scholar
  2. Callis J (1995) Regulation of protein degradation. Plant Cell 7: 845–857CrossRefPubMedGoogle Scholar
  3. Carvalho H, Cullimore JV (2003) Regulation of glutamine synthetase isoenzymes and genes in the model legume Medicago truncatula. In: Pandalai SG (ed) Recent research developments in plant molecular biology, vol 1, part 1. Research Signpost Publishers.Trivandrum, pp. 157–175Google Scholar
  4. Cock JM, Brock IW, Watson AT, Swarup R, Morby AP, Cullimore JV (1991) Regulation of glutamine synthetase genes in leaves of Phaseolus vulgaris. Plant Mol Biol 17:761–771CrossRefPubMedGoogle Scholar
  5. Cock JM, Hermon P, Cullimore JV (1992) Characterization of a gene encoding the plastid-located glutamine synthetase of Phaseolus vulgaris: regulation of β–glucuronidase gene fusions in transgenic tobacco. Plant Mol Biol 18: 1141–1149CrossRefPubMedGoogle Scholar
  6. Cotelle V, Meek SEM, Provan F, Milne FC, Morrice N, Mackintosh C (2000) 14–3-3s regulate cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. EMBO J 19:2869–2876CrossRefPubMedGoogle Scholar
  7. Cullimore JV, Miflin BJ (1984) Immunological studies on glutamine synthetase using antisera raised to the two plant forms of the enzyme from Phaseolus root nodules. J Exp Bot 35:581–587CrossRefGoogle Scholar
  8. Cullimore JV, Sims AP (1980) An association between photorespiration and protein catabolism: studies in Chlamydomonas. Planta 150:392–396CrossRefGoogle Scholar
  9. Dubois F, Brugiere N, Sangwan RS, Hirel B (1996) Localization of tobacco citosolic glutamine synthetase enzymes and corresponding transcripts shows organ and cell-specific patterns of protein synthesis and gene expression. Plant Mol Biol 31:803–817CrossRefPubMedGoogle Scholar
  10. Finnemann J, Schjoerring JK (2000) Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14–3-3 protein interaction. Plant J 24:171–181CrossRefPubMedGoogle Scholar
  11. Forde BG, Cullimore JV (1989) The molecular biology of glutamine synthetase in higher plants. In: Miflin B (ed) Oxford Survey Plant Mol Cell Biol, vol 6. Oxford University Press, pp 247–296Google Scholar
  12. Forde BG, Day HM, Turton JF, Shen W-J, Cullimore JV, Oliver JE (1989) Two glutamine synthetases genes from Phaseolus vulgaris L. display contrasting developmental and special patterns of expression in transgenic Lotus corniculatus plants. Plant Cell 1:391–401CrossRefPubMedGoogle Scholar
  13. Frohlich V, Fischer A, Ochs G, Wild A, Feller U (1994) Proteolytic inactivation of glutamine synthetase in extracts from wheat leaves: effects of pH, inorganic ions and metabolites. Aust J Plant Physiol 21:303–310Google Scholar
  14. Hardin SC, Huber SC (2004) Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves. Plant Physiol Biochem 42:197–208CrossRefPubMedGoogle Scholar
  15. Hardin SC, Tang G-Q, Scholz A, Holtgraewe D, Winter H, Huber SC (2003) Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis. Plant J 35:588–603CrossRefPubMedGoogle Scholar
  16. Hoelzle I, Finner JJ, McMullen MD, Streeter JG (1992) Induction of glutamine synthetase activity in nonnodulated roots of Glicine max, Phaseolus vulgaris and Pisum sativum. Plant Physiol 100:525–528PubMedCrossRefGoogle Scholar
  17. Huber SC, Bachmann M, Huber JL (1996) Post-translational regulation of nitrate reductase activity: a role for Ca2+ and 14–3-3 proteins. Trends Plant Sci 1:432–438CrossRefGoogle Scholar
  18. Journet EP, van Tuinen D, Gouzy J, Carreau V, Farmer MJ, Niebel A, Schiex T, Crespeau H, Jaillon O, Chatagnier O, Godiard L, Gianinazzi-Pearson V, Kahn D, Gamas P (2002) Exploring the root symbiotic programs of the model legume Medicago truncatula using EST analysis. Nucleic Acids Res 30: 5579–5592CrossRefPubMedGoogle Scholar
  19. Kaiser WM, Weiner H, Kandlbinder A, Tsai C-B, Rockel P, Sonoda M, Planchet E (2002) Modulation of nitrate reductase: some new insights, an unusual case and potentially important side reaction. J Exp Bot vol 53:875–882CrossRefGoogle Scholar
  20. Lea PJ, Robinson SA, Stewart GR (1990) The enzymology and metabolism of glutamine, glutamate and asparagine. In: Miflin BJ, Lea PJ (eds) The Biochemistry of Plants, vol 16. Academic Press, New York, pp 121–160Google Scholar
  21. Li MG, Villemur R, Hussey PJ, Silflow CD, Gantt JS, Snustad DP (1993) Differential expression of six glutamine synthetase genes in Zea mays. Plant Mol Biol 23:401–407CrossRefPubMedGoogle Scholar
  22. Lullien V, Barker DG, da Lajudie P, Huguet T (1987) Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol Biol 9:469–478CrossRefGoogle Scholar
  23. Mackintosh C, Meek SE (2001) Regulation of plant NR activity by reversible phosphorylation, 14–3-3 proteins and proteolysis. Cell Mol Life Sci 58:205–214PubMedCrossRefGoogle Scholar
  24. Man H-M, Kaiser WM (2001) Increased glutamine synthetase activity and changes in amino acid pools in leaves treated with 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR). Physiol Plant 111:291–296CrossRefPubMedGoogle Scholar
  25. Melo PM, Lima LM, Santos IM, Carvalho HG, Cullimore JV (2003) Expression of the plastid-located glutamine synthetase of Medicago truncatula. Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids. Plant Physiol 132:390–399CrossRefPubMedGoogle Scholar
  26. Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, Mackintosh C (1999) Phosphorylation-dependent interactions between enzymes of plant metabolism and 14–3-3 proteins. Plant J 18:1–12CrossRefPubMedGoogle Scholar
  27. Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14–3-3 with signalling proteins is mediated by the recognition of phosphoserine. Cell 84:889–897CrossRefPubMedGoogle Scholar
  28. Oliveira IC, Lam H-M, Coschigano K, Melo-Oliveira R, Coruzzi G (1997) Molecular-genetic dissection of ammonium assimilation in Arabidopsis thaliana. Plant Physiol Biochem 35:185–198Google Scholar
  29. Ortega JL, Roche D, Sengupta-Gopalan C (1999) Oxidative turnover of soybean root glutamine synthetase. In vitro and in vivo studies. Plant Physiol 119:1483–1495CrossRefPubMedGoogle Scholar
  30. Ortega JL, Temple SJ, Sengupta-Gopolan C (2001) Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 may be regulated at the level of RNA stability and protein turnover. Plant Physiol 126:109–121CrossRefPubMedGoogle Scholar
  31. Ortega JL, Temple SJ Bagga S, Ghoshroy S, Sengupta-Gopolan C (2004) Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively overexpressing a cytosolic glutamine synthetase gene. Planta 219:807–818CrossRefPubMedGoogle Scholar
  32. Palatnik JF, Carrillo N, Valle EM (1999) The role of photosynthetic electron transport in the oxidative degradation of chloroplastic glutamine synthetase. Plant Physiol. 121: 471–478CrossRefPubMedGoogle Scholar
  33. Pozuelo M, Mackintosh C, Galvan A, Fernandez E (2001) Cytosolic glutamine synthetase and not nitrate reductase from the green alga Chlamydomonas reinhardtii is phosphorylated and binds 14–3-3 proteins. Planta 212:264–269CrossRefPubMedGoogle Scholar
  34. Riedel J, Tischner R, Mäck G (2001) The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14–3-3 proteins inside the chloroplast. Planta 213:396–401CrossRefPubMedGoogle Scholar
  35. Roche D, Temple SJ, Sengupta-Gopalan C (1993) Two classes of differentially regulated glutamine synthetase genes are expressed in the soybean nodule: a nodule-specific class and a constitutively expressed class. Plant Mol Biol 22:971–983CrossRefPubMedGoogle Scholar
  36. Roulin S, Feller U (1997) Light-induced proteolysis of stromal proteins in pea (Pisum sativum L.) chloroplasts: requirement for intact organelles. Plant Sci 128:31–41CrossRefGoogle Scholar
  37. Sakakibara H, Kawabata S, Takahashi H, Hase T, Sugiyama T (1992) Molecular cloning of the family of glutamine synthetase genes from maize: expression of genes for glutamine synthetase and ferredoxin-dependent glutamate synthase in photosynthetic and non-photosynthetic tissues. Plant Cell Physiol 33:49–58Google Scholar
  38. Streit L, Feller U (1983) Chandging activities and different resistence to proteolytic activity of two forms of glutamine synthetase in wheat leaves during senescence. Physiol Vég 21:103–108Google Scholar
  39. Sukanya R, Li M-G, Snustad DP (1994) Root- and shoot-specific responses of individual glutamine synthetase genes of maize to nitrate and ammonium. Plant Mol Biol 26:1935–1946CrossRefPubMedGoogle Scholar
  40. Tang G-Q, Hardin SC, Dewey R, Huber SC (2003) A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds. Plant J 34:77–93CrossRefPubMedGoogle Scholar
  41. Temple SJ, Heard J, Ganter G, Dunn K, Sengupta-Gopalan C (1995) Characterization of a nodule-enhanced glutamine synthetase from alfalfa: nucleotide sequence, in situ localization and transcript analysis. Mol Plant-Microbe Interact 8:218–227PubMedGoogle Scholar
  42. Temple SJ, Heard J, Kunjibettu S, Roche D, Sengupta-Gopalan C (1996) Total glutamine synthetase activity during soybean nodule development is controlled at the level of transcription and holoprotein turnover. Plant Physiol 112:1723–1733PubMedGoogle Scholar
  43. Temple SJ, Bagga S, Sengupta-Gopolan C (1998) Down-regulation of specific members of the glutamine synthetase gene family by antisense RNA technology. Plant Mol Biol 37:535–547CrossRefPubMedGoogle Scholar
  44. Thoenen M, Feller U (1998) Degradation of glutamine synthetase in intact chloroplasts isolated from pea (Pisum sativum) leaves. Aust J Plant Physiol 25:279–286CrossRefGoogle Scholar
  45. Toroser D, Athwal GS, Huber SC (1998) Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14–3-3 proteins. FEBS Lett 435:110–114CrossRefPubMedGoogle Scholar
  46. Walker EL, Coruzzi GM (1989) Developmental regulated expression of the gene family for cytosolic glutamine synthetase in Pisum sativum. Plant Physiol 91:702–708PubMedGoogle Scholar
  47. Weiner H, Kaiser WM (1999) 14–3-3 proteins control proteolysis of nitrate reductase in spinach leaves. FEBS Lett 455:75–78CrossRefPubMedGoogle Scholar
  48. Yamamoto Y, Inagaki N, Satoh K (2001) Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein: regulation of enzyme-substrate interaction by molecular environments. J Biol Chem 276(10): 7518–25CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Lígia Lima
    • 1
  • Ana Seabra
    • 1
  • Paula Melo
    • 1
  • Julie Cullimore
    • 2
  • Helena Carvalho
    • 1
    Email author
  1. 1.Instituto de Biologia Molecular e Celular Rua do Campo AlegrePortoPortugal
  2. 2.Laboratoire des Interactions Plantes-Microorganismes INRA-CNRSCastanet-Tolosan CedexFrance

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