Skip to main content
SpringerLink
Log in

Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula–Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula–Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

ᅟ

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

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Hoffman, B.M., Lukoyanov, D., Yang, Z.Y., Dean, D.R., Seefeldt, L.C.: Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114(8), 4041–4062 (2014)

    Article  CAS  Google Scholar 

  2. Ferguson, B.J., Indrasumunar, A., Hayashi, S., Lin, M.H., Lin, Y.H., Reid, D.E., Gresshoff, P.M.: Molecular analysis of legume nodule development and autoregulation. J. Integr. Plant Biol. 52(1), 61–76 (2010)

    Article  CAS  Google Scholar 

  3. Lau, W., Fischbach, M.A., Osbourn, A., Sattely, E.S.: Key applications of plant metabolic engineering. PLoS Biol. 12(6), e1001879 (2014)

    Article  Google Scholar 

  4. Graham, P.H., Vance, C.P.: Legumes: importance and constraints to greater use. Plant Physiol. 131(3), 872–877 (2003)

    Article  CAS  Google Scholar 

  5. Dunn, M.F.: Key roles of microsymbiont amino acid metabolism in rhizobia–legume interactions. Crit. Rev. Microbiol (2014). doi:10.3109/1040841X.2013.856854

  6. Udvardi, M., Poole, P.S.: Transport and metabolism in legume-rhizobia symbioses. Annu. Rev. Plant Biol. 64, 781–805 (2013)

    Article  CAS  Google Scholar 

  7. Cook, D.: Medicago truncatula—a model in the making! Comment. Curr. Opin. Plant Biol. 2(4), 301–304 (1999)

    Article  CAS  Google Scholar 

  8. Benloch, R., Navarro, C., Beltran, J.P., Canas, L.A.: Floral development of the model legume Medicago truncatula: ontogeny studies as a tool to better characterize homeotic mutations. Sex Plant Reprod. 15(5), 231–241 (2003)

    Google Scholar 

  9. Gallardo, K., Le Signor, C., Vandekerckhove, J., Thompson, R.D., Burstin, J.: Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation. Plant Physiol. 133(2), 664–682 (2003)

    Article  CAS  Google Scholar 

  10. Wang, H.L., Chen, J.H., Wen, J.Q., Tadege, M., Li, G.M., Liu, Y., Mysore, K.S., Ratet, P., Chen, R.J.: Control of compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medicago truncatula. Plant Physiol. 146(4), 1759–1772 (2008)

    Article  CAS  Google Scholar 

  11. Branca, A., Paape, T.D., Zhou, P., Briskine, R., Farmer, A.D., Mudge, J., Bharti, A.K., Woodward, J.E., May, G.D., Gentzbittel, L., Ben, C., Denny, R., Sadowsky, M.J., Ronfort, J., Bataillon, T., Young, N.D., Tiffin, P.: Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula. Proc. Natl. Acad. Sci. U. S. A. 108(42), E864–E870 (2011)

    Article  CAS  Google Scholar 

  12. Samac, D.A., Penuela, S., Schnurr, J.A., Hunt, E.N., Foster-Hartnett, D., Vandenbosch, K.A., Gantt, J.S.: Expression of coordinately regulated defence response genes and analysis of their role in disease resistance in Medicago truncatula. Mol. Plant Pathol. 12(8), 786–798 (2011)

    Article  CAS  Google Scholar 

  13. Rasmussen, S., Parsons, A.J., Jones, C.S.: Metabolomics of forage plants: a review. Ann. Bot. 110(6), 1281–1290 (2012)

    Article  CAS  Google Scholar 

  14. Venkateshwaran, M., Volkening, J.D., Sussman, M.R., Ané, J.M.: Symbiosis and the social network of higher plants. Curr. Opin. Plant Biol. 16(1), 118–127 (2013)

    Article  CAS  Google Scholar 

  15. White, J., Prell, J., James, E.K., Poole, P.: Nutrient sharing between symbionts. Plant Physiol. 144(2), 604–614 (2007)

    Article  CAS  Google Scholar 

  16. Draper, J., Rasmussen, S., Zubair, H.: Metabolite analysis and metabolomics in the study of biotrophic interactions between plant and microbes. Annu. Plant Rev. 43, 25–59 (2011)

    CAS  Google Scholar 

  17. Desbrosses, G.G., Kopka, J., Udvardi, M.K.: Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions. Plant Physiol. 137(4), 1302–1318 (2005)

    Article  CAS  Google Scholar 

  18. Colebatch, G., Desbrosses, G., Ott, T., Krusell, L., Montanari, O., Kloska, S., Kopka, J., Udvardi, M.K.: Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus. Plant J. 39(4), 487–512 (2004)

    Article  Google Scholar 

  19. Suzuki, H., Reddy, M.S.S., Naoumkina, M., Aziz, N., May, G.D., Huhman, D.V., Sumner, L.W., Blount, J.W., Mendes, P., Dixon, R.A.: Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic reprogramming in cell suspension cultures of the model legume Medicago truncatula. Planta 220(5), 696–707 (2005)

    Article  CAS  Google Scholar 

  20. Farag, M.A., Huhman, D.V., Lei, Z.T., Sumner, L.W.: Metabolic profiling and systematic identification of flavonoids and isoflavonoids in roots and cell suspension cultures of Medicago truncatula using HPLC-UV-ESI-MS and GC-MS. Phytochemistry 68(3), 342–354 (2007)

    Article  CAS  Google Scholar 

  21. Farag, M.A., Huhman, D.V., Dixon, R.A., Sumner, L.W.: Metabolomics reveals novel pathways and differential mechanistic and elicitor-specific responses in phenylpropanoid and isoflavonoid biosynthesis in Medicago truncatula cell cultures. Plant Physiol. 146(2), 387–402 (2008)

    Article  CAS  Google Scholar 

  22. Harada, K., Fukusaki, E.: Profiling of primary metabolite by means of capillary electrophoresis-mass spectrometry and its application for plant science. Plant Biotech. 26(1), 47–52 (2009)

    Article  CAS  Google Scholar 

  23. Kueger, S., Steinhauser, D., Willmitzer, L., Giavalisco, P.: High-resolution plant metabolomics: from mass spectral features to metabolites and from whole-cell analysis to subcellular metabolite distributions. Plant J. 70(1), 39–50 (2012)

    Article  CAS  Google Scholar 

  24. Lee, Y.J., Perdian, D.C., Song, Z.H., Yeung, E.S., Nikolau, B.J.: Use of mass spectrometry for imaging metabolites in plants. Plant J. 70(1), 81–95 (2012)

    Article  CAS  Google Scholar 

  25. Kaspar, S., Peukert, M., Svatoš, A., Matros, A., Mock, H.P.: MALDI-imaging mass spectrometry—an emerging technique in plant biology. Proteomics 11(9), 1840–1850 (2011)

    Article  CAS  Google Scholar 

  26. Gemperline, E., Li, L.: MALDI-mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules. J. Vis. Exp. 85, (2014). doi:10.3791/51434

  27. Ye, H., Gemperline, E., Venkateshwaran, M., Chen, R., Delaux, P.M., Howes-Podoll, M., Ane, J.M., Li, L.: MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis. Plant J. 75(1), 130–145 (2013)

    Article  CAS  Google Scholar 

  28. Bjarnholt, N., Li, B., D'Alvise, J., Janfelt, C.: Mass spectrometry imaging of plant metabolites—principles and possibilities. Nat. Prod. Rep. 31(6), 818–837 (2014)

    Article  CAS  Google Scholar 

  29. Catoira, R., Galera, C., de Billy, F., Penmetsa, R., Journet, E., Maillet, F., Rosenberg, C., Cook, D., Gough, C., Denarie, J.: Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell 12(9), 1647–1665 (2000)

    Article  CAS  Google Scholar 

  30. Oke, V., Long, S.R.: Bacteroid formation in the rhizobium-legume symbiosis. Curr. Opin. Microbiol. 2(6), 641–646 (1999)

    Article  CAS  Google Scholar 

  31. Robichaud, G., Garrard, K.P., Barry, J.A., Muddiman, D.C.: MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform. J. Am. Soc. Mass Spectrom. 24(5), 718–721 (2013)

    Article  CAS  Google Scholar 

  32. Wolf, S., Schmidt, S., Muller-Hannemann, M., Neumann, S.: In silico fragmentation for computer assisted identification of metabolite mass spectra. BMC Bioinformatics 11, (2010). doi:10.1186/1471-2105-11-148

  33. Mitra, R.M., Long, S.R.: Plant and bacterial symbiotic mutants define three transcriptionally distinct stages in the development of the Medicago truncatula–Sinorhizobium meliloti symbiosis. Plant Physiol. 134(2), 595–604 (2004)

    Article  CAS  Google Scholar 

  34. Wang, D., Griffitts, J., Starker, C., Fedorova, E., Limpens, E., Ivanov, S., Bisseling, T., Long, S.R.: A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science 327(5969), 1126–1129 (2010)

    Article  CAS  Google Scholar 

  35. Sprent, J.I., James, E.K.: Legume evolution: where do nodules and mycorrhizas fit in? Plant Physiol. 144(2), 575–581 (2007)

    Article  CAS  Google Scholar 

  36. Sprent, J. I.: Legume nodulation: a global perspective. pp. 79–94, Wiley-Blackwell, Chichester, U.K. (2009)

  37. Sulieman, S., Tran, L.S.P.: Asparagine: an amide of particular distinction in the regulation of symbiotic nitrogen fixation of legumes. Crit. Rev. Biotechnol. 33(3), 309–327 (2013)

    Article  CAS  Google Scholar 

  38. Schulze, J.: Source-sink manipulations suggest an N-feedback mechanism for the drop in N-2 fixation during pod-filling in pea and broad bean. J. Plant Physiol. 160(5), 531–537 (2003)

    Article  CAS  Google Scholar 

  39. Fischinger, S.A., Drevon, J.J., Claassen, N., Schulze, J.: Nitrogen from senescing lower leaves of common bean is re-translocated to nodules and might be involved in a N-feedback regulation of nitrogen fixation. J. Plant Physiol. 163(10), 987–995 (2006)

    Article  CAS  Google Scholar 

  40. Sulieman, S., Fischinger, S.A., Gresshoff, P.M., Schulze, J.: Asparagine as a major factor in the N-feedback regulation of N-2 fixation in Medicago truncatula. Physiol. Plant. 140(1), 21–31 (2010)

    Article  CAS  Google Scholar 

  41. Parsons, R., Stanforth, A., Raven, J.A., Sprent, J.I.: Nodule growth and activity may be regulated by a feedback mechanism involving phloem nitrogen. Plant, Cell Environ. 16(2), 125–136 (1993)

    Article  CAS  Google Scholar 

  42. Touraine, B.: Nitrate uptake by roots-transporters and root development. In: Amancio, S., Stulen, I. (eds.) Nitrogen Acquisition and Assimilation in Higher Plants, p. 1–34. Springer Netherlands, Dordrecht (2004)

  43. Schubert, S.: The apoplast of indeterminate legume nodules: compartment for transport of amino acids, amides and sugars. In: Sattelmacher, B., Horst, W.J. (eds.) The Apoplast of Higher Plants: Compartment of Storage, Transport and Reactions, pp. 445–454. Springer Netherlands, Dordrecht (2007)

  44. Boscari, A., Van de Sype, G., Le Rudulier, D., Mandon, K.: Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation. Mol. Plant-Microbe Interact. 19(8), 896–903 (2006)

    Article  CAS  Google Scholar 

  45. Alloing, G., Travers, I., Sagot, B., Le Rudulier, D., Dupont, L.: Proline betaine uptake in Sinorhizobium meliloti: characterization of Prb, an Opp-like ABC transporter regulated by both proline betaine and salinity stress. J. Bacteriol. 188(17), 6308–6317 (2006)

    Article  CAS  Google Scholar 

  46. Luyten, E., Vanderleyden, J.: Survey of genes identified in Sinorhizobium meliloti spp., necessary for the development of an efficient symbiosis. Eur. J. Soil Biol 36(1), 1–26 (2000)

    Article  CAS  Google Scholar 

  47. Appleby, C.A.: Leghemoglobin and rhizobium respiration. Ann. Rev. Plant Physiol. Plant Mol. Biol. 35, 443–478 (1984)

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by funding from the University of Wisconsin Graduate School and the Wisconsin Alumni Research Foundation (WARF), a Romnes Faculty Research Fellowship program to L.L. and a National Science Foundation (NSF) grant to J.M.A. (NSF#0701846). E.G. acknowledges an NSF Graduate Research Fellowship (DGE-1256259). The MALDI-Orbitrap and Q-Exactive instruments were purchased through an NIH shared instrument grant (NCRR S10RR029531).

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Erin Gemperline & Lingjun Li

  2. Department of Agronomy, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Dhileepkumar Jayaraman, Junko Maeda & Jean-Michel Ané

  3. School of Pharmacy, University of Wisconsin-Madison, Madison, WI, 53705, USA

    Lingjun Li

Authors
  1. Erin Gemperline
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dhileepkumar Jayaraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junko Maeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean-Michel Ané
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lingjun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lingjun Li.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 186 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gemperline, E., Jayaraman, D., Maeda, J. et al. Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS. J. Am. Soc. Mass Spectrom. 26, 149–158 (2015). https://doi.org/10.1007/s13361-014-1010-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-014-1010-0

Keywords

Search

Navigation