Abstract
In this paper we have studied the localisation of expression of the two functional cytosolic glutamine synthetase (GS) genes, MtGSa and MtGSb, in root nodules of the model legume Medicago truncatula. We have used a combination of different techniques, including immunocytochemistry, in situ hybridisation and promoter β-glucuronidase (GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation. These studies revealed that transcriptional regulation (mRNA synthesis) plays an important part in controlling GS protein levels in nodules of M. truncatula. The major locations of cytosolic GS mRNA and protein are the central tissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GS might be involved in other physiological processes in addition to the primary assimilation of ammonia released by the bacterial nitrogenase. The two genes show different but overlapping patterns of expression with MtGSa being the major gene expressed in the infected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb of MtGSa and MtGSb, respectively, have been sequenced and primer extension revealed that the MtGSb promoter is expressed in nodules from an additional start site that is not used in roots. Generally these fragments in the homologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell types where GS proteins and transcripts are located except that the MtGSa promoter fragment did not express GUS highly in the nodule infected cells. These results indicate that the cis-acting regulatory elements responsible for infected-cell expression are missing from the MtGSa promoter fragment.
Similar content being viewed by others
References
Axelos, M., Bardet, C., Le Van Thai, A., Curie, C. and Lescure, B. 1989. The family encoding the Arabidopsis thaliana elongation factor EF-1α: molecular cloning, characterisation and expression. Mol. Gen. Genet. 219: 106–112.
Barker, D.G., Bianchi, S., Blondon, F., Dattee, Y., Duc, G., Essad, S., Flament, P., Gallusci, P., Genier, G., Guy, P., Muel, X., Tourneur, J., Denarie, J. and Huguet, T. 1990. Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol. Biol. Rep 8: 40–49.
Bennett, M.J., Lightfoot, D.A. and Cullimore, J.V. 1989. A Cdna sequence and differential expression of the gene encoding the glutamine synthetase polypeptide of Phaseolus vulgaris L. Plant Mol. Biol. 12: 553–565.
Bevan, M.W. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12: 8711–8721.
Blondon, F., Marie, D., Brown, S. and Kondorosi, A. 1993. Genome size and base composition in Medicago sativa and M. truncatula species. Genome 37: 264–270.
Brangeon, J., Hirel, B. and Forchioni, A. 1989. Immunogold localization of glutamine synthetase in soybean leaves, roots and nodules. Protoplasma 151: 88–97.
Brears, T., Walker, E.L and Coruzzi, G. 1991. A promoter sequence involved in cell-specific expression of the pea glutamine synthetase GS3A gene in organs of transgenic tobacco and alfalfa. Plant J. 1: 235–244.
Brugière, N., Dubois, F., Limami, A., Lelandais, M., Roux, Y., Sangwan, R.S. and Hirel, B. 1999. Glutamine synthetase in the phloem plays a major role in controlling proline production. Plant Cell 11: 1995–2011.
Carrayol, E., Trerce-Laforgue, T., Desbrosses, G,, Pruvot-Maschio, G., Poirier, S., Ratet, P. and Hirel, B. 1997. Ammonia regulated expression of a soybean gene encoding cytosolic glutamine synthetase is not conserved in two heterologous plant systems. Plant Sci. 125: 75–85.
Carvalho, H., Pereira, S., Sunkel, C. and Salema, R. 1992. Detection of a cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods. Plant Physiol. 100: 1591–1594.
Carvalho, H., Sunkel, C., Salema, R. and Cullimore, J. 1997. Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme. Plant Mol. Biol 35: 623–632.
Chabaud, M., Larsonneau, C., Marmouget, C. and Huguet, T. 1996. Transformation of barrel medic (Medicago truncatula Gaertn.) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep. 15: 305–310.
Chen, R., Silver, D.L. and de Bruijn, F.J. 1998. Nodule parenchymaspecific expression of the Sesbania rostrata early nodulin gene SrENOD2 is mediated by its 30 untranslated region. Plant Cell 10: 1585–1602.
Cock, J.M., Brock, I.W., Watson, A.T., Swarup, R., Morby, A.P. and Cullimore, J.V. 1991. Regulation of glutamine synthetase genes in leaves of Phaseolus vulgaris. Plant Mol. Biol. 17: 761–771.
Cullimore, J.V. and Miflin, B. 1984. Immunological studies on glutamine synthetase using antisera raised to the plant forms of the enzyme from Phaseolus root nodules. J. Exp. Bot. 153: 581–587.
Datta, D.B., Cai, X., Wong, P. and Triplett, E.W. 1991. Immunocytochemical localisation of glutamine synthetase in organs of Phaseolus vulgaris L. Plant Physiol. 96: 507–512.
de Billy, F., Barker, D.G., Gallusci, P. and Truchet, G. 1991. Leghaemoglobin gene transcription is triggered in a single cell layer in the indeterminate nitrogen-fixing root nodule of alfalfa. Plant J. 1: 27–35.
de la Pena, T.C., Frugier, F., McKhann, H.I., Bauer, P., Brown, S., Kondorosi, A. and Crespi, M. 1997. A carbonic anhydrase gene is induced in the nodule primordium and its cell specific expression is controlled by the presence of Rhizobium during development. Plant J. 11: 407–420.
Delauney, A.J. and Verma, D.P.S. 1993. Proline biosynthesis and osmoregulation in plants. Plant J. 4: 215–223.
Edwards, J.W., Walker, E.L. and Coruzzi, G.M. 1990. Cell-specific expression in trangenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthetase. Proc. Natl. Acad Sci. USA 87: 3459–3463.
Forde, B.G and Cullimore, J.V. 1989. Glutamine synthetase in higher plants. In: B.J. Miflin (Ed.), Oxford Surveys of Plant Molecular and Cell Biology, vol. 6, Oxford University Press, Oxford, pp. 247–296.
Forde, B.G., Day, H.M., Turton, J.F., Wen-Jun, S., Cullimore, J.V and Oliver, J.E. 1989. Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and patterns of expression in trangenic Lotus corniculatus plants. Plant Cell 1: 391–401.
Forde, B.G., Freeman, J., Oliver, J.E. and Pineda, M. 1990. Nuclear factors interact with conserved A/T rich elements upstream of a nodule enhanced glutamine synthetase gene from french bean. Plant Cell 2: 925–939.
Fougère, F., Rudulier, D. and Streeter, J.G. 1991. Effects of salt stress on amino acid, organic acid and carbohydrate composition of root, bacteroids, and cytosol of alfalfa (Medicago sativa L.) Plant Physiol. 96: 1228–1236.
Hunt, S. and Layzell, D. 1993. Gas exchange of legume nodules and the regulation of nitrogenase activity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 483–511.
Jefferson, R.A., Kananagh, T.A and Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in plants. EMBO J. 6: 3901–3907.
Kouchi, H., Fukai, K., Katagiri, K., Minamisawa, K. and Tajima, S. 1988. Isolation and enzymological characterisation of infected and uninfected cell protoplasts from root nodules of Glycine max. Physiol. Plant. 73: 327–334.
Lea, P.J., Robinson, S.A. and Stewart, G.R. 1990. The enzymology of and metabolism of glutamine, glutamate and asparagine. In: B.J. Miflin and P.J. Lea (Eds.), The Biochemistry of Plants, Vol. 16, Academic Press, New York, pp. 121–159.
Li, M., Villemur, R., Hussey, P., Silflow, C.D., Grantt, J.S and Snustad, P. 1993. Differential expression of six glutamine synthetase genes in Zea mays. Plant Mol. Biol. 23: 401–407.
Lullien, V., Barker, D.G., de Lajudie, P. and Huguet, T. 1987. Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol. Biol. 9: 469–478.
Marsolier, M., Carrayol, E. and Hirel, B. 1993. Multiple functions of promoter sequences involved in organ specific expression and ammonia regulation of a cytosolic soybean glutamine synthetase gene in transgenic Lotus corniculatus. Plant J. 3: 405–414.
Marsolier, M., Debrosses, G. and Hirel, B. 1995. Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus. Plant Mol. Biol. 27: 1–15.
McGrath, R.B. and Coruzzi, G.M. 1991. A gene network controlling glutamine and asparagine biosynthesis in plants. Plant J. 1: 275–280.
Miao, G., Hirel, B., Marsolier, M.C., Ridge, R.W and Verma, D.P. 1991. Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in trangenic Lotus corniculatus. Plant Cell 3: 11–22.
Miflin, B.J and Lea, P.J. 1980. Ammonia assimilation. In: B.J. Miflin (Ed.) The Biochemistry of Plants, Academic Press, New York, pp. 169–202.
Miller, S.S., Driscoll, B.T., Gregerson, R.G., Grantt, J.S. and Vance, C.P. 1998. Alfalfa malate dehydrogenase (MDH): molecular cloning and characterisation of five different forms reveals a unique nodule-enhanced MDH. Plant J. 15: 173–184.
Neo, H.H. and Layzell, D.B. 1997. Phloem glutamine and the regulation of O2 diffusion in legume nodules. Plant Physiol. 113: 259–267.
Parsons, R., Stanforth, A., Raven, J.A. and Sprent, J.I. 1993. Nodule growth and activity may be regulated by a feed-back mechanism involving phloem nitrogen. Plant Cell Environ. 16: 125–136.
Pate, J.S. and Atkins, C.A. 1983. Nitrogen uptake, transport and utilization. In: W.J. Broughton (Ed.), Nitrogen Fixation, Oxford University Press, Oxford, pp. 243–298.
Pate, J.S., Gunning, B.E.S. and Briarty, L.G. 1969. Ultrastructure and functioning of the transport system of the leguminous root nodule. Planta 85: 11–34.
Pathirana, M.S., Samac, D.A., Roeven, R., Yoshioka, H., Vance, C.P. and Grantt, J.S. 1997. Analysis of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules. Plant J. 12: 293–304.
Pereira, S., Carvalho, H., Sunkel, C. and Salema, R. 1992. Immunocytolocalisation of glutamine synthetase in mesophyl and phloem leaves of Solanum tuberosum L. Protoplasma 167: 66–73.
Peterman, T.K. and Goodman, H.M. 1991. The glutamine synthetase gene family of Arabidopsis thaliana: light regulation and differential expression in leaves, roots and seeds. Mol. Gen. Genet. 230: 145–154.
Robinson, D.L., Kahn, M.L. and Vance, C.P. 1994. Cellular localisation of nodule enhanced aspartate aminotransferase in Medicago sativa. Planta 92: 202–210.
Robinson, D.L., Pathirana, S.M., Grantt, J.S. and Vance, C.P. 1996. Immunogold localisation of nodule enhanced phosphoenolpyruvate carboxylase in alfalfa. Plant Cell Environ. 19: 602–608.
Sandal, N.N., Bojsen, K. and Marcker, K.A. 1987. A family of nodule specific genes from soybean. Nucl. Acids Res. 15: 1507–1519.
Shi, L., Twary, S.N., Yoshioka, H., Gregerson, R.G., Miller, S.S., Samac, D.A., Grantt, J.S., Unkefer, P.J and Vance, C.P. 1997. Nitrogen assimilation in alfalfa: isolation and characterisation of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves. Plant Cell 9: 1339–1356.
Stanford, C., Larsen, K., Barker, D.G. and Cullimore, J.V. 1993.Differential expression within the glutamine synthetase gene family of the model legume, Medicago truncatula. Plant Physiol. 103: 73–81.
Taylor, C.B. 1996. Proline and water deficit: ups, downs, ins and outs. Plant Cell 8: 1221–1224.
Taylor, C.B. 1997. Promoter fusion analysis: an insufficient measure of gene expression. Plant Cell 9: 273–274.
Temple, S.J., Heard, J., Ganter, G., Dunn, K. and 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. 2: 218–227.
Tercé-Laforgue, T., Carrayol, E., Cren, M., Desbrosses, G., Hecht, V. and Hirel, B. 1999. A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding glutamine synthetase. Plant Mol. Biol. 39: 551–564.
Thykjaer, T., Danielsen, D., She, Q. and Stougaard, J. 1997. Organisation and expression of genes in the genomic region surrounding the glutamine synthetase gene Gln1 from Lotus japonicus. Mol. Gen. Genet. 255: 628–636.
Tischer, E., Dassarma, S. and Goodman, H.M. 1986. Nucleotide sequence of an alfalfa glutamine synthetase gene. Mol. Gen. Genet. 203: 221–229.
Tjaden, G., Edwards, J.V and Coruzzi, G.M. 1995. cis elements and trans-acting factors affecting regulation of a non-photosynthetic light-regulated gene for chloroplast glutamine synthetase. Plant Physiol. 108: 1109–1117.
Trepp, G.B., Temple, S.J., Bucciarelli, B., Shi, L.F and Vance, C.P. 1999a. Expression map for genes involved in nitrogen and carbon metabolism in alfalfa root nodules. Mol. Plant-Microbe Interact. 12: 526–535.
Trepp, G.B., Plank, D.W., Grantt, J.S. and Vance, C.P. 1999b. NADH-glutamate synthase in alfalfa root nodules. Immunocytochemical localization. Plant Physiol. 119: 829–837.
Vance, C.P., Miller, S.S., Gregerson, R.G., Samac, D.A., Robinson, D.L and Gantt, J.S. 1995. Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation. Plant J. 8: 345–358.
Vasse, J., de Billy, F., Camut, S. and Truchet, G. 1990. Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J. Bact. 172: 4295–4306.
Verma, D.P., Fortin, M.G., Stanley, J., Mauro, V.P., Purohits, S. and Morrison, N. 1986. Nodulins and nodulin genes of Glycine max. Plant Mol. Biol. 7: 51–61.
Walker, E.L. and Coruzzi, G.M. 1989. Developmental regulated expression of the gene family for the cytosolic glutamine synthetase in Pisum sativum. Plant Physiol. 91: 702–708.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Carvalho, H., Lescure, N., de Billy, F. et al. Cellular expression and regulation of the Medicago truncatula cytosolic glutamine synthetase genes in root nodules. Plant Mol Biol 42, 741–756 (2000). https://doi.org/10.1023/A:1006304003770
Issue Date:
DOI: https://doi.org/10.1023/A:1006304003770