, Volume 234, Issue 3, pp 459–476 | Cite as

A nodulin/glutamine synthetase-like fusion protein is implicated in the regulation of root morphogenesis and in signalling triggered by flagellin

  • Anna Doskočilová
  • Ondřej Plíhal
  • Jindřich Volc
  • Jana Chumová
  • Hana Kourová
  • Petr Halada
  • Beáta Petrovská
  • Pavla BinarováEmail author
Original Article


The nodulin/glutamine synthetase-like protein (NodGS) that we identified proteomically in Arabidopsis thaliana is a fusion protein composed of an N-terminal amidohydrolase domain that shares homology with nodulins and a C-terminal domain of prokaryotic glutamine synthetase type I. The protein is homologous to the FluG protein, a morphogenetic factor in fungi. Although genes encoding NodGS homologues are present in many plant genomes, their products have not yet been characterized. The Arabidopsis NodGS was present in an oligomeric form of ~700-kDa, mainly in the cytosol, and to a lesser extent in the microsomal membrane fraction. The oligomeric NodGS was incorporated into large heterogeneous protein complexes >700 kDa and partially co-immunoprecipitated with γ-tubulin. In situ and in vivo microscopic analyses revealed a NodGS signal in the cytoplasm, with endomembranes, particularly in the perinuclear area. NodGS had no detectable glutamine synthetase activity. Downregulation of NodGS by RNAi resulted in plants with a short main root, reduced meristematic activity and disrupted development of the root cap. Y2H analysis and publicly available microarray data indicated a role for NodGS in biotic stress signalling. We found that flagellin enhanced the expression of the NodGS protein, which was then preferentially localized in the nuclear periphery. Our results point to a role for NodGS in root morphogenesis and microbial elicitation. These data might help in understanding the family of NodGS/FluG-like fusion genes that are widespread in prokaryotes, fungi and plants.


Arabidopsis Flagellin elicitation Fusion protein Glutamine synthetase-like Nodulin-like Root development 





Blue native PAGE


Dapoxyl dye




Endoplasmic reticulum


Flagellin 22


Fluorescence recovery after photobleaching


Glutamine synthetase


Murashige and Skoog


Nodulin/glutamine synthetase-like protein


Phenylmethylsulfonyl fluoride


Yeast two-hybrid analysis



We are thankful to Gabriela Kočárová, Elena Kubátová and Jan Svoboda for technical assistance. We gratefully acknowledge Dr. Csaba Koncz (Max Planck Institute, Cologne) for pAct2 cDNA library, Dr. Roger Tsien (University of California, San Diego, CA, USA) for the kind gift of the binary RFP vector pH7WGR2.0, and Dr. László Bögre (Royal Holloway University of London, UK) for GFP-MAP4 seeds. This work was supported by The Grant Agency of the ASCR grants IAA500200719 and KJB500200705 and, MSMT LC545 project, the Grant Agency of the Czech Republic grant 204/09/H084 and Institutional Research Concept AV0Z50200510.

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  1. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983PubMedCrossRefGoogle Scholar
  2. Bhalerao RP, Salchert K, Bako L, Okresz L, Szabados L, Muranaka T, Machida Y, Schell J, Koncz C (1999) Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases. Proc Natl Acad Sci USA 96:5322–5327PubMedCrossRefGoogle Scholar
  3. Binarova P, Cihalikova J, Dolezel J (1993) Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells. Cell Biol Int 17:847–856PubMedCrossRefGoogle Scholar
  4. Binarova P, Cenklova V, Sulimenko V, Drykova D, Volc J, Draber P (2003) Distribution of gamma-tubulin in cellular compartments of higher plant cells. Cell Biol Int 27:167–169PubMedCrossRefGoogle Scholar
  5. Binarova P, Cenklova V, Prochazkova J, Doskocilova A, Volc J, Vrlik M, Bogre L (2006) Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis. Plant Cell 18:1199–1212PubMedCrossRefGoogle Scholar
  6. Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, He P, Bush J, Cheng SH, Sheen J (2010) Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464:418–422PubMedCrossRefGoogle Scholar
  7. Bulow L, Schindler M, Hehl R (2007) PathoPlant: a platform for microarray expression data to analyze co-regulated genes involved in plant defense responses. Nucleic Acids Res 35:D841–D845PubMedCrossRefGoogle Scholar
  8. Chabaud M, Genre A, Sieberer BJ, Faccio A, Fournier J, Novero M, Barker DG, Bonfante P (2011) Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis. New Phytol 189:347–355PubMedCrossRefGoogle Scholar
  9. Charpentier M, Bredemeier R, Wanner G, Takeda N, Schleiff E, Parniske M (2008) Lotus japonicus CASTOR and POLLUX are ion channels essential for perinuclear calcium spiking in legume root endosymbiosis. Plant Cell 20:3467–3479PubMedCrossRefGoogle Scholar
  10. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  11. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469PubMedCrossRefGoogle Scholar
  12. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP:cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97:3718–3723PubMedCrossRefGoogle Scholar
  13. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17PubMedCrossRefGoogle Scholar
  14. D’Souza CA, Lee BN, Adams TH (2001) Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans. Genetics 158:1027–1036PubMedGoogle Scholar
  15. Davis AM, Hall A, Millar AJ, Darrah C, Davis SJ (2009) Protocol: streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods 5:3PubMedCrossRefGoogle Scholar
  16. Drykova D, Cenklova V, Sulimenko V, Volc J, Draber P, Binarova P (2003) Plant gamma-tubulin interacts with alphabeta-tubulin dimers and forms membrane-associated complexes. Plant Cell 15:465–480PubMedCrossRefGoogle Scholar
  17. Enright AJ, Iliopoulos I, Kyrpides NC, Ouzounis CA (1999) Protein interaction maps for complete genomes based on gene fusion events. Nature 402:86–90PubMedCrossRefGoogle Scholar
  18. Gamas P, Niebel Fde C, Lescure N, Cullimore J (1996) Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development. Mol Plant Microbe Interact 9:233–242PubMedCrossRefGoogle Scholar
  19. Gao M, Liu J, Bi D, Zhang Z, Cheng F, Chen S, Zhang Y (2008) MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants. Cell Res 18:1190–1198PubMedCrossRefGoogle Scholar
  20. Gawronski JD, Benson DR (2004) Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection. Anal Biochem 327:114–118PubMedCrossRefGoogle Scholar
  21. Genre A, Chabaud M, Timmers T, Bonfante P, Barker DG (2005) Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. Plant Cell 17:3489–3499PubMedCrossRefGoogle Scholar
  22. Gifford ML, Dean A, Gutierrez RA, Coruzzi GM, Birnbaum KD (2008) Cell-specific nitrogen responses mediate developmental plasticity. Proc Natl Acad Sci USA 105:803–808PubMedCrossRefGoogle Scholar
  23. Halada P, Man P, Grebenova D, Hrkal Z, Havlicek V (2001) Identification of HL60 proteins affected by 5-aminolevulinic acid-based photodynamic therapy using mass spectrometric approach. Collect Czech Chem Commun 66:1720–1728CrossRefGoogle Scholar
  24. Lee BN, Adams TH (1994) The Aspergillus nidulans fluG gene is required for production of an extracellular developmental signal and is related to prokaryotic glutamine synthetase I. Genes Dev 8:641–651PubMedCrossRefGoogle Scholar
  25. Li J, Zhu S, Song X, Shen Y, Chen H, Yu J, Yi K, Liu Y, Karplus VJ, Wu P, Deng XW (2006) A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem. Plant Cell 18:340–349PubMedCrossRefGoogle Scholar
  26. Llorca O, Betti M, Gonzalez JM, Valencia A, Marquez AJ, Valpuesta JM (2006) The three-dimensional structure of an eukaryotic glutamine synthetase: functional implications of its oligomeric structure. J Struct Biol 156:469–479PubMedCrossRefGoogle Scholar
  27. Marc J, Granger CL, Brincat J, Fisher DD, Kao T, McCubbin AG, Cyr RJ (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927–1940PubMedCrossRefGoogle Scholar
  28. Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D (1999) Detecting protein function and protein-protein interactions from genome sequences. Science 285:751–753PubMedCrossRefGoogle Scholar
  29. Margelis S, D’Souza C, Small AJ, Hynes MJ, Adams TH, Davis MA (2001) Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans. J Bacteriol 183:5826–5833PubMedCrossRefGoogle Scholar
  30. Mashiguchi K, Asami T, Suzuki Y (2009) Genome-wide identification, structure and expression studies, and mutant collection of 22 early nodulin-like protein genes in Arabidopsis. Biosci Biotechnol Biochem 73:2452–2459PubMedCrossRefGoogle Scholar
  31. Mathis R, Grosjean C, de Billy F, Huguet T, Gamas P (1999) The early nodulin gene MtN6 is a novel marker for events preceding infection of Medicago truncatula roots by Sinorhizobium meliloti. Mol Plant Microbe Interact 12:544–555PubMedCrossRefGoogle Scholar
  32. Mathis R, Gamas P, Meyer Y, Cullimore JV (2000) The presence of GSI-like genes in higher plants: support for the paralogous evolution of GSI and GSII genes. J Mol Evol 50:116–122PubMedGoogle Scholar
  33. Menges M, Doczi R, Okresz L, Morandini P, Mizzi L, Soloviev M, Murray JA, Bogre L (2008) Comprehensive gene expression atlas for the Arabidopsis MAP kinase signalling pathways. New Phytol 179:643–662PubMedCrossRefGoogle Scholar
  34. Nap JP, Bisseling T (1990) Developmental biology of a plant–prokaryote symbiosis: the legume root nodule. Science 250:948–954PubMedCrossRefGoogle Scholar
  35. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45PubMedCrossRefGoogle Scholar
  36. Rexer HU, Schaberle T, Wohlleben W, Engels A (2006) Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2). Arch Microbiol 186:447–458PubMedCrossRefGoogle Scholar
  37. Toufighi K, Brady SM, Austin R, Ly E, Provart NJ (2005) The botany array resource: e-Northerns, expression angling, and promoter analyses. Plant J 43:153–163PubMedCrossRefGoogle Scholar
  38. Trevaskis B, Wandrey M, Colebatch G, Udvardi MK (2002) The soybean GmN6L gene encodes a late nodulin expressed in the infected zone of nitrogen-fixing nodules. Mol Plant Microbe Interact 15:630–636PubMedCrossRefGoogle Scholar
  39. Walch-Liu P, Ivanov II, Filleur S, Gan Y, Remans T, Forde BG (2006) Nitrogen regulation of root branching. Ann Bot 97:875–881PubMedCrossRefGoogle Scholar
  40. Weingartner M, Binarova P, Drykova D, Schweighofer A, David JP, Heberle-Bors E, Doonan J, Bogre L (2001) Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis. Plant Cell 13:1929–1943PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Anna Doskočilová
    • 1
  • Ondřej Plíhal
    • 1
    • 3
  • Jindřich Volc
    • 1
  • Jana Chumová
    • 1
  • Hana Kourová
    • 1
  • Petr Halada
    • 1
  • Beáta Petrovská
    • 2
    • 4
  • Pavla Binarová
    • 1
    Email author
  1. 1.Institute of Microbiology, v.v.i.Academy of Sciences of the Czech RepublicPrague 4Czech Republic
  2. 2.Institute of Experimental Botany, v.v.i.Academy of Sciences of the Czech RepublicOlomoucCzech Republic
  3. 3.Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Biochemistry, Faculty of SciencePalacký University in OlomoucOlomoucCzech Republic
  4. 4.Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental Botany AS CROlomoucCzech Republic

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