, Volume 70, Issue 1–3, pp 149–157 | Cite as

The N-metabolites of roots and actinorhizal nodules from Alnus glutinosa and Datisca glomerata: can D. glomerata change N-transport forms when nodulated?

  • Tomas Persson
  • Thanh Van Nguyen
  • Nicole Alloisio
  • Petar Pujic
  • Alison M. Berry
  • Philippe Normand
  • Katharina Pawlowski


To gain more insight in nitrogen metabolism in actinorhizal nodules, a comparison between the N metabolite profiles in roots vs. nodules was initiated for one host plant from the best-examined order of actinorhizal plants, Fagales, A. glutinosa (Betulaceae), a temperate tree, and one host plant from the Cucurbitales order, Datisca glomerata (Datiscaceae). For both symbioses, the symbiotic transcriptomes have been published and can be used to assess the expression of genes representing specific metabolic pathways in nodules. The amino acid profiles of roots in this study suggest that A. glutinosa transported aspartate, glutamate and citrulline in the xylem, a combination of nitrogenous solutes not published previously for this species. The amino acid profiles of D. glomerata roots depended on whether the plants were nodulated or grown on nitrate; roots of nodulated plants contained increased amounts of arginine. Although bacterial transcriptome data showed no symbiotic auxotrophy for branched chain amino acids (leucine, isoleucine, valine) in either symbiosis, D. glomerata nodules contained comparatively high levels of these amino acids. This might represent a response to osmotic stress.


Actinorhiza Frankia Nitrogen-fixation Arginine Gamma-aminobutyrate (GABA) Citrulline 



We would like to thank Peter Litfors for taking care of the plants in Stockholm and Pascale Fournier for A. glutinosa growth experiments and harvesting of roots and nodules. This work was funded by grants from the Swedish Research Council Formas (229-2005-679) and the Carl Tryggers Foundation to KP, by a grant from the French ANR (Sesam ANR-10-BLAN-1708 and BugsInACell ANR-13-BSV7-0013-03), to PN, and by USDA CA-D* PLS-2173-H to AMB.

Supplementary material

13199_2016_407_MOESM1_ESM.xlsx (22 kb)
ESM 1 (XLSX 22 kb)


  1. Alloisio N, Queiroux C, Fournier P, Pujic P, Normand P, Vallenet D, Médigue C, Yamaura M, Kakoi K, Kucho K (2010) The Frankia alni symbiotic transcriptome. Mol Plant-Microbe Interact 23:593–607CrossRefPubMedGoogle Scholar
  2. Bai C, Reilly CC, Wood BW (2007) Identification and quantitation of asparagine and citrulline using high-performance liquid chromatography (HPLC). Anal Chem Insights 2:31–36PubMedPubMedCentralGoogle Scholar
  3. Barbosa JM, Singh NK, Cherry JH, Locy RD (2010) Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings. Plant Physiol Biochem 48:443–450CrossRefPubMedGoogle Scholar
  4. Berry AM, Murphy TM, Okubara PA, Jacobsen KR, Swensen SM, Pawlowski K (2004) Novel expression pattern of cytosolic glutamine synthetase in nitrogen-fixing root nodules of the actinorhizal host, Datisca glomerata. Plant Physiol 135:1849–1862CrossRefPubMedPubMedCentralGoogle Scholar
  5. Berry AM, Mendoza-Herrera A, Guo Y-Y, Hayashi J, Persson T, Barabote R, Demchenko K, Zhang S, Pawlowski K (2011) New perspectives on nodule nitrogen assimilation in actinorhizal symbioses. Funct Plant Biol 38:645–652CrossRefGoogle Scholar
  6. Bouché N, Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 9:110–115CrossRefPubMedGoogle Scholar
  7. Bown AW, Shelp BJ (1997) The metabolism and functions of γ-aminobutyric acid. Plant Physiol 115:1–5CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brooks JM, Benson DR (2016) Comparative metabolomics of root nodules infected with Frankia sp. strains and uninfected roots from Alnus glutinosa and Casuarina cunninghamiana reflects physiological integration. Symbiosis. doi: 10.1007/s13199-016-0379-x Google Scholar
  9. Carro L, Pujic P, Alloisio N, Fournier P, Boubakri H, Hay AE, Poly F, François P, Hocher V, Mergaert P, Balmand S, Rey M, Heddi A, Normand P (2015) Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia. ISME J 9:1723–1733CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chen TH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20CrossRefPubMedGoogle Scholar
  11. Couturier J, Doidy J, Guinet F, Wipf D, Blaudez D, Chalot M (2010) Glutamine, arginine and the amino acid transporter Pt-CAT11 play important roles during senescence in poplar. Ann Bot 105:1159–1169CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cramer VA, Schmidt S, Stewart GR, Thorburn PJ (2002) Can the nitrogenous composition of xylem sap be used to assess salinity stress in Casuarina glauca? Tree Physiol 22:1019–1026CrossRefPubMedGoogle Scholar
  13. Dawson JO (2008) Ecology of actinorhizal plants. In: Pawlowski K, W.E. N (eds) Nitrogen-fixing actinorhizal symbioses. Springer, Netherlands, pp. 199–237CrossRefGoogle Scholar
  14. Demina IV, Persson T, Santos P, Plaszczyca M, Pawlowski K (2013) Comparison of the nodule vs. root transcriptome of the actinorhizal plant Datisca glomerata: actinorhizal nodules contain a specific class of defensins. PLoS One 8:e72442CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fåhraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–3781PubMedGoogle Scholar
  16. Gardner IC, Leaf G (1960) Translocation of citrulline in Alnus glutinosa. Plant Physiol 35:948–950CrossRefPubMedPubMedCentralGoogle Scholar
  17. Geurts R, Xiao TT, Reinhold-Hurek B (2016) What does it take to evolve a nitrogen-fixing endosymbiosis? Trends Plant Sci 21:199–208CrossRefPubMedGoogle Scholar
  18. Gtari M, Ghodhbane-Gtari F, Nouioui I, Ktari A, Hezbri K, Mimouni W, Sbissi I, Ayari A, Yamanaka T, Normand P, Tisa LS, Boudabous A (2015) Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains. Sci Rep 5:13112CrossRefPubMedPubMedCentralGoogle Scholar
  19. Guan C, Ribeiro A, Akkermans AD, Jing Y, van Kammen A, Bisseling T, Pawlowski K (1996) Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase. Plant Mol Biol 32:1177–1184CrossRefPubMedGoogle Scholar
  20. Guérin V, Huché-Thélier L, Charpentier S (2007) Mobilisation of nutrients and transport via the xylem sap in a shrub (Ligustrum ovalifolium) during spring growth: N and C compounds and interactions. J Plant Physiol 164:562–573CrossRefPubMedGoogle Scholar
  21. Hacham Y, Avraham T, Amir R (2002) The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism. Plant Physiol 128:454–462CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hoagland DR, Arnon DT (1938) The water-culture method for growing plants without soil, California Agriculture Experiment Station Circular 347. University of CA, BerkeleyGoogle Scholar
  23. Hocher V, Alloisio N, Auguy F, Fournier P, Doumas P, Pujic P, Gherbi H, Queiroux C, Da Silva C, Wincker P, Normand P, Bogusz D (2011) Transcriptomics of actinorhizal symbioses reveals homologs of the whole common symbiotic signaling cascade. Plant Physiol 156:700–711CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hosie AHF, Allaway D, Galloway CS, Dunsby HA, Poole PS (2002) Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family. J Bacteriol 184:4071–4080CrossRefPubMedPubMedCentralGoogle Scholar
  25. Huguet V, Mergeay M, Cervantes E, Fernandez MP (2004) Diversity of Frankia strains associated to Myrica gale in Western Europe: impact of host plant (Myrica vs. Alnus) and of edaphic factors. Environ Microbiol 6:1032–1041CrossRefPubMedGoogle Scholar
  26. Jeong J, Suh S, Guan C, Tsay YF, Moran N, Oh CJ, An CS, Demchenko KN, Pawlowski K, Lee Y (2004) A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family. Plant Physiol 134:969–978CrossRefPubMedPubMedCentralGoogle Scholar
  27. Joshi V, Joung JG, Fei Z, Jander G (2010) Interdependence of threonine, methionine and isoleucine metabolism in plants: accumulation and transcriptional regulation under abiotic stress. Amino Acids 39:933–947CrossRefPubMedGoogle Scholar
  28. Karp PD, Paley SM, Krummenacker M, Latendresse M, Dale JM, Lee TJ, Kaipa P, Gilham F, Spaulding A, Popescu L, Altman T, Paulsen I, Keseler IM, Caspi R (2010) Pathway tools version 13.0: integrated software for pathway/genome informatics and systems biology. Brief Bioinform 11:40–79CrossRefPubMedGoogle Scholar
  29. Kinnersley AM, Torano FJ (2000) Gamma aminobutyric acid (GABA) and plant responses to stress. Crit Rev Plant Sci 19:479–509CrossRefGoogle Scholar
  30. Mirza MS, Hameed S, Akkermans ADL (1994) Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene. Appl Environ Microbiol 60:2371–2376PubMedPubMedCentralGoogle Scholar
  31. Nabais C, Hagemeyer J, Freitas H (2005) Nitrogen transport in the xylem sap of Quercus ilex: the role of ornithine. J Plant Physiol 162:603–606CrossRefPubMedGoogle Scholar
  32. Normand P, Lalonde M (1982) Evaluation of Frankia strains isolated from provenances of two Alnus species. Can J Microbiol 28:1133–1142CrossRefGoogle Scholar
  33. Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J, Evtushenko L, Misra AK (1996) Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae. Int J Syst Bacteriol 46:1–9CrossRefPubMedGoogle Scholar
  34. Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, Bagnarol E, Bassi CA, Berry AM, Bickhart DM, Choisne N, Couloux A, Cournoyer B, Cruveiller S, Daubin V, Demange N, Francino MP, Goltsman E, Huang Y, Kopp OR, Labarre L, Lapidus A, Lavire C, Marechal J, Martinez M, Mastronunzio JE, Mullin BC, Niemann J, Pujic P, Rawnsley T, Rouy Z, Schenowitz C, Sellstedt A, Tavares F, Tomkins JP, Vallenet D, Valverde C, Wall LG, Wang Y, Medigue C, Benson DR (2007) Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genome Res 17:7–15CrossRefPubMedPubMedCentralGoogle Scholar
  35. Oakley B, North M, Franklin JF, Hedlund BP, Staley JT (2004) Diversity and distribution of Frankia strains symbiotic with Ceanothus in California. Appl Environ Microbiol 70:6444–6452CrossRefPubMedPubMedCentralGoogle Scholar
  36. Oldroyd GE, Murray JD, Poole PS, Downie JA (2011) The rules of engagement in the legume-rhizobial Symbiosis. Annu Rev Genet 45:119–144CrossRefPubMedGoogle Scholar
  37. Orgován G, Noszál B (2011) The complete microspeciation of arginine and citrulline. J Pharm Biomed Anal 54:965–971CrossRefPubMedGoogle Scholar
  38. Pate JS (1980) Transport and partioning of nitrogenous solutes. Annu Rev Plant Physiol 31:313–340CrossRefGoogle Scholar
  39. Pawlowski K, Demchenko KN (2012) The diversity of actinorhizal symbiosis. Protoplasma 249:967–979CrossRefPubMedGoogle Scholar
  40. Persson T, Benson DR, Normand P, Vanden Heuvel B, Pujic P, Chertkov O, Teshima H, Bruce BC, Detter C, Tapia R, Han S, Han J, Woyke T, Pitluck S, Pennacchio L, Nolan M, Ivanova N, Pati A, Land ML, Pawlowski K, Berry AM (2011) The genome of Candidatus Frankia datiscae Dg1, the uncultured microsymbiont from nitrogen-fixing root nodules of the dicot Datisca glomerata. J Bacteriol 193:7017–7018CrossRefPubMedPubMedCentralGoogle Scholar
  41. Persson T, Battenberg K, Demina IV, Vigil-Stenman T, Vanden Heuvel B, Pujic P, Facciotti MT, Wilbanks EG, O’Brien A, Fournier P, Cruz Hernandez MA, Mendoz Herrera A, Médigue C, Normand P, Pawlowski K, Berry AM (2015) Candidatus Frankia datiscae Dg1, the actinobacterial microsymbiont of Datisca glomerata, expresses the canonical nod genes nodABC in symbiosis with its host plant. PLoS One 10:e0127630CrossRefPubMedPubMedCentralGoogle Scholar
  42. Prell J, Bourdès A, Karunakaran R, Lopez-Gomez M, Poole P (2009a) Pathway of γ-aminobutyrate metabolism in Rhizobium leguminosarum 3841 and its role in symbiosis. J Bacteriol 191:2177–2186CrossRefPubMedPubMedCentralGoogle Scholar
  43. Prell J, White JP, Bourdes A, Bunnewell S, Bongaerts RJ, Poole PS (2009b) Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids. Proc Natl Acad Sci U S A 106:12477–12482CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher-plants. Annu Rev Plant Physiol Plant Mol Biol 44:357–384CrossRefGoogle Scholar
  45. Scholz SS, Reichelt M, Mekonnen DW, Ludewig F, Mithöfer A (2015) Insect herbivory-elicited GABA accumulation in plants is a wound-induced, direct, systemic, and jasmonate-independent defense response. Front Plant Sci 6:1128CrossRefPubMedPubMedCentralGoogle Scholar
  46. Schubert KR (1986) Products of biological nitrogen fixation in higher plants: synthesis, transport, and metabolism. Annu Rev Plant Physiol 37:539–574CrossRefGoogle Scholar
  47. Schwencke J (1991) Rapid, exponential growth and increased biomass yield of some Frankia strains in buffered and stirred mineral medium (BAP) with phosphatidyl choline. Plant Soil 137:37–41CrossRefGoogle Scholar
  48. Sellstedt A, Atkins CA (1991) Composition of amino compounds transported in xylem of Casuarina sp. J Exp Bot 42:1493–1497CrossRefGoogle Scholar
  49. Sen A, Daubin V, Abrouk D, Gifford I, Berry AM, Normand P (2014) Phylogeny of the class Actinobacteria revisited in the light of complete genomes. The orders ‘Frankiales’ and Micrococcales should be split into coherent entities: proposal of Frankiales ord. nov., Geodermatophilales ord. nov., Acidothermales ord. nov. and Nakamurellales ord. nov. Int J Syst Evol Microbiol 64:3821–3832CrossRefPubMedGoogle Scholar
  50. Shelp BJ, Bown AW, McLean MD (1999) Metabolism and functions of γ-aminobutyric acid. Trends Plant Sci 4:446–452CrossRefPubMedGoogle Scholar
  51. Shelp BJ, Bown AW, Faure D (2006) Extracellular γ-aminobutyrate mediates communication between plants and other organisms. Plant Physiol 142:1350–1352CrossRefPubMedPubMedCentralGoogle Scholar
  52. Simonet P, Navarro E, Rouvier C, Reddell P, Zimpfer J, Dawson J, Dommergues Y, Bardin R, Combarro P, Hamelin J, Domenach A-M, Gourbière F, Prin Y, Normand P (1999) Co-evolution between Frankia populations and host plants in the family Casuarinaceae and consequent patterns of global dispersal. Environ Microbiol 1:525–535CrossRefPubMedGoogle Scholar
  53. Snedden WA, Fromm H (1999) Regulation of the γ-aminobutyrate-synthesizing enzyme, glutamate decarboxylase, by calcium–calmodulin: a mechanism for rapid activation in response to stress. In: Lerner HR (ed) Plant responses to environmental stresses: from phytohormones to genome reorganization. Marcel Dekker, New York, pp. 549–574Google Scholar
  54. Sulieman S, Schulze J (2010) Phloem derived γ-aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula. Plant Cell Environ 33:2162–2172CrossRefPubMedGoogle Scholar
  55. Tajima S, Nomura M, Kouchi H (2004) Ureide biosynthesis in legume nodules. Front Biosci 9:1374–1381CrossRefPubMedGoogle Scholar
  56. Tisa L, McBride M, Ensign JC (1983) Studies of growth and morphology of Frankia strains EAN1pec, EUI1c, CpI1 and ACN1AG. Can J Bot 61:2768–2773CrossRefGoogle Scholar
  57. Valverde C, Huss-Danell K (2008) C and N metabolism in actinorhizal nodules. In: Pawlowski K, Newton WE (eds) Nitrogen-fixing actinorhizal symbioses. Springer, Dordrecht, pp. 167–198CrossRefGoogle Scholar
  58. Vercruysse M, Fauvart M, Beullens S, Braeken K, Cloots L, Engelen K, Marchal K, Michiels J (2011) A comparative transcriptome analysis of Rhizobium etli bacteroids: specific gene expression during symbiotic nongrowth. Mol Plant-Microbe Interact 24:1553–1561CrossRefPubMedGoogle Scholar
  59. Wheeler CT, Bond G (1970) The amino acids of non-legume root nodules. Phytochemistry 9:705–708CrossRefGoogle Scholar
  60. Winter G, Todd CD, Trovato M, Forlani G, Funck D (2015) Physiological implications of arginine metabolism in plants. Front Plant Sci 6:534CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zhang X, Benson DR (1992) Utilization of amino acids by Frankia sp. strain CpI1. Arch Microbiol 158:256–261CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Tomas Persson
    • 1
  • Thanh Van Nguyen
    • 1
  • Nicole Alloisio
    • 2
  • Petar Pujic
    • 2
  • Alison M. Berry
    • 3
  • Philippe Normand
    • 2
  • Katharina Pawlowski
    • 1
  1. 1.Department of Ecology, Environment and Plant SciencesStockholmSweden
  2. 2.Université Lyon 1, Université de Lyon, CNRS, Ecologie Microbienne UMR5557VilleurbanneFrance
  3. 3.Department of Plant SciencesUniversity of CaliforniaDavisUSA

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