Skip to main content
SpringerLink
Log in

Synchronous expression of leghaemoglobin genes inMedicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Two leghaemoglobin genes from the diploid, autogamousMedicago truncatula (Mtlb1 andMtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups ofMedicago leghaemoglobin genes [2]. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule ofM. truncatula. WhilstMtlb1 mRNA is present at approximatively 3-fold higher steady-state levels thanMtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation withRhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulatedM. truncatula is supplemented with 5 mM KNO3 over a period of 2–3 days there is a progressive drop in specific nitrogen fixation activity to only 20–25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to bothMtlb1 andMtlb2. By contrast, the expression of the nodule parenchyma-specific geneENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

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

Similar content being viewed by others

References

  1. Appleby CA: Leghemoglobin andRhizobium respiration. Ann Rev Plant Physiol 35: 443–478 (1984).

    Google Scholar 

  2. Barker DG, Gallusci P, Lullien V, Khan H, Ghérardi M, Huguet T: Identification of two groups of leghemoglobin genes in alfalfa (Medicago sativa) and a study of their expression during root nodule development. Plant Mol Biol 11: 761–772 (1988).

    Google Scholar 

  3. Barker DG, Bianchi S, Blondon F, Dattée Y, Duc G, Flament P, Gallusci P, Génier G, Guy P, Muel X, Tourneur J, Dénarié J, Huguet T:M. truncatula, a model plant for studying the molecular genetics of theRhizobium-legume symbiosis. Plant Mol Biol Rep 8: 40–49 (1990).

    Google Scholar 

  4. Becana M, Sprent JI: Effect of nitrate on components of nodule leghemoglobins. J Exp Bot 40: 725–731 (1989).

    Google Scholar 

  5. de Billy F, Barker DG, Gallusci P, Truchet G: Leghemoglobin gene transcription is triggered in a single cell layer in the indeterminate nitrogen-fixing nodule of alfalfa. Plant J, in press (1991).

  6. Bojsen K, Abildsten D, Jensen EO, Paludan K, Marcker KA: The chromosomal arrangement of six soybean leghemoglobin genes. EMBO J 2: 1165–1168 (1983).

    Google Scholar 

  7. Brown GG, Lee JS, Brisson N, Verma DPS: The evolution of a plant globin gene family. J Mol Evol 21: 19–32 (1984).

    Google Scholar 

  8. Essad S: Sur la relation possible d'une unité de variation saltatoire de l'ADN nucléaire, le nucléon, et des chromosomes B dans le genreMedicago L. C R Acad Sci Paris série III 305: 307–310 (1987).

    Google Scholar 

  9. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).

    Google Scholar 

  10. Franssen HJ, Nap JP, Gloudemans T, Stiekema W, van Dam H, Govers F, Louwerse J, van Kammen A, Bisseling T: Characterisation of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development. Proc Natl Acad Sci USA 84: 4495–4499 (1987).

    Google Scholar 

  11. Govers F, Moerman M, Downie JA, Hooykaas P, Franssen HJ, Louwerse J, van Kammen A, Bisseling T:Rhizobium nod genes are involved in inducing an early nodulin gene. Nature 323: 564–566 (1986).

    Google Scholar 

  12. Harper JE, Gibson AH: Differential nodulation tolerance to nitrate among legume species. Crop Sci 24: 797–801 (1984).

    Google Scholar 

  13. Heckmann MO, Drevon JJ, Saglio P, Salsac L: Effect of oxygen and malate on NO3-inhibition of nitrogenase in soybean nodules. Plant Physiol 90: 224–229 (1989).

    Google Scholar 

  14. Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).

    Google Scholar 

  15. Jensen EO, Marcker KA, Schell J, de Bruijn FJ: Interaction of a nodule specifictrans-acting factor with distinct DNA elements in the soybean leghaemoglobinlbc 3 5′ upstream region. EMBO J 7: 1265–1271 (1988).

    Google Scholar 

  16. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: A compilation and analysis. Nucl Acids Res 15: 9627–9640 (1987).

    Google Scholar 

  17. Kanayama K, Watanabe I, Yamamoto Y: Inhibition of nitrogen fixation in soybean plants supplied with nitrate. I. Nitrite accumulation and formation of nitrosylleghe-moglobin in nodules. Plant Cell Physiol 31: 341–346 (1990).

    Google Scholar 

  18. Kiss GB, Vincze E, Vegh Z, Toth G, Soos J: Identification and cDNA cloning of a new nodule specific gene, Nms-25 (nodulin 25) ofMedicago sativa. Plant Mol Biol 14: 467–475 (1990).

    Google Scholar 

  19. Kuhse J, Pühler A: Conserved sequence motifs in the untranslated 3′ end of leghemoglobin transcripts isolated from broadbean nodules. Plant Sci 49: 137–143 (1987).

    Google Scholar 

  20. Landsmann J, Dennis ES, Higgins TJV, Appleby CA, Kortt AA, Peacock WJ: Common evolutionary origin of legume and non-legume plant haemoglobins. Nature 324: 166–168 (1986).

    Google Scholar 

  21. Lee JS, Verma DPS: Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization. EMBO J 3: 2745–2752 (1984).

    Google Scholar 

  22. Lehtovaara P, Lappalainen A, Ellfolk N: The amino acid sequence of pea (Pisum sativum) leghemoglobin. Biochim Biophys Acta 623: 98–106 (1980).

    Google Scholar 

  23. Long SR:Rhizobium-legume nodulation: Life together in the underground. Cell 56: 203–214 (1989).

    Google Scholar 

  24. Lullien V, Barker DG, de Lajudie P, Huguet T: Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol Biol 9: 469–478 (1987).

    Google Scholar 

  25. Marcker A, Lund M, Jensen EO, Marcker KA: Transcription of the soybean leghemoglobin genes during nodule development. EMBO J 3: 1691–1695 (1984).

    Google Scholar 

  26. Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ: Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legumeSesbania rostrata: Conserved coding sequences,cis-elements andtrans-acting factors. Mol Gen Genet 214: 181–191 (1988).

    Google Scholar 

  27. Minchen FR, Minguez MI, Sheehy JE, Witty JF, Skot L: Relationship between nitrate and oxygen supply in symbiotic nitrogen fixation by white clover. J Exp Bot 37: 1103–1113 (1986).

    Google Scholar 

  28. Murphy G, Kavanagh T: Speeding-up the sequencing of double-stranded DNA. Nucl Acids Res 16: 5198 (1988).

    Google Scholar 

  29. Rigaud J, Puppo A: Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation. Biochim Biophys Acta 497: 702–706 (1977).

    Google Scholar 

  30. Rosenberg C, Boistard P, Dénarié J, Casse-Delbart F: Genes controlling early and late functions in symbiosis are located on a megaplasmid inRhizobium meliloti. Mol Gen Genet 184: 326–333 (1981).

    Google Scholar 

  31. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  32. Sandal NN, Bojsen K, Marcker KA: A small family of nodule specific genes from soybean. Nucl Acids Res 15: 1507–1519 (1987).

    Google Scholar 

  33. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  34. Scheres B, van Engelen F, van der Knaap E, van de Wiel C, van Kammen A, Bisseling T: Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell 2: 687–700 (1990).

    Google Scholar 

  35. Scheres B, van de Wiel C, Zalensky A, Horvath B, Spaink H, van Eck H, Zwartkruis F, Wolters A-M, Gloudemans T, van Kammen A, Bisseling T: TheENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction. Cell 60: 281–294 (1990).

    Google Scholar 

  36. Stougaard J, Sandal NN, Gron A, Kühle A, Marcker KA: 5′ analysis of the soybean leghemoglobinlbc 3 gene: Regulatory elements required for promoter activity and organ specificity. EMBO J 6: 3565–3569 (1987).

    Google Scholar 

  37. Stougaard J, Jorgensen J-E, Christensen T, Kühle A, Marcker KA: Inter-dependence and nodule specificity ofcis-acting regulatory elements in the soybean leghemoglobinlbc 3 and N23 gene promoters. Mol Gen Genet 220: 353–360 (1990).

    Google Scholar 

  38. Streeter J: Inhibition of legume nodule formation and N2 fixation by nitrate. CRC Crit Rev Plant Sci 7: 1–23 (1988).

    Google Scholar 

  39. Trinchant JC, Rigaud J: Nitrite inhibition of nitrogenase from soybean bacteroids. Arch Microbiol 124: 49–54 (1981).

    Google Scholar 

  40. Truchet G, Barker DG, Camut S, de Billy F, Vasse J, Huguet T: Alfalfa nodulation in the absence ofRhizobium. Mol Gen Genet 219: 65–68 (1989).

    Google Scholar 

  41. Turner GL, Gibson AH: Measurement of nitrogen fixation by indirect means. In: Bergersen FJ (ed) Methods for Evaluating Biological Nitrogen Fixation, pp. 111–138. Wiley, Chichester (1980).

    Google Scholar 

  42. van de Wiel C, Scheres B, Franssen HJ, Van Lierop MJ, Van Lammeren A, Van Kammen A, Bisseling T: The early nodulin transcriptENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybean root nodules. EMBO J 9: 1–7 (1990).

    Google Scholar 

  43. van Kammen A: Suggested nomenclature for plant genes involved in nodulation and symbiosis. Plant Mol Biol Rep 2: 43–45 (1984).

    Google Scholar 

  44. Verma DPS, Delauney AJ: Root nodule symbiosis: nodulins and nodulin genes. In: Verma DPS, Goldberg RB (eds) Temporal and Spatial Regulation of Plant Genes, pp. 169–199. Springer-Verlag, Wien/New York (1988).

    Google Scholar 

  45. Vincze E, Kiss GB: A phosphate group at thecos ends of phage lambda is not a prerequisite forin vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, pGY97. Gene 96: 17–22 (1990).

    Google Scholar 

  46. Wiborg O, Hyldig-Nielsen JJ, Jensen EO, Paludan K, Marcker KA: The nucleotide sequences of two leghemoglobin genes from soybean. Nucl Acids Res 10: 3487–3494 (1982).

    Google Scholar 

  47. Wiborg O, Pederson MS, Wind A, Berglund LE, Marcker KA, Vuust J: The human ubiquitin multigene family: Some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J 4: 755–759 (1985).

    Google Scholar 

  48. Witty JF, Minchen FR, Skot L, Sheehy JE: Nitrogen fixation and oxygen in legume root nodules. Oxford Surv Plant Mol Cell Biol 3: 275–314 (1986).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire des Relations Plantes Microorganismes, INRA-CNRS, BP27, 31326, Castanet-Tolosan Cédex, France

    Philippe Gallusci, Annie Dedieu, Etienne P. Journet, Thierry Huguet & David G. Barker

Authors
  1. Philippe Gallusci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Annie Dedieu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Etienne P. Journet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thierry Huguet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David G. Barker
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gallusci, P., Dedieu, A., Journet, E.P. et al. Synchronous expression of leghaemoglobin genes inMedicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate. Plant Mol Biol 17, 335–349 (1991). https://doi.org/10.1007/BF00040629

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00040629

Key words

Search

Navigation