Abstract
We report the sequence of an essentially full-length glutamine synthetase (GS) cDNA clone (pcGS-γ1) isolated from a root nodule library ofPhaseolus vulgaris L. The polypeptide encoded by this cDNA has been producedin vitro by transcription/translation and shown to co-migrate on two-dimensional gels with the previously identified major cytosolic GS polypeptide (γ) of nodules. Two previously identified GS cDNA clones, pR-2 and pR-1 (see Gebhardtet al., EMBO J 5: 1429–1435, 1986) have similarly been shown to encode the α and β cytosolic GS polypeptides respectively. An RNase protection technique has been used to analyse specifically and quantitatively the abundance of mRNA related to these three GS cDNAs and to the cDNA (pcGS-δ1) encoding the chloroplast-located GS, during nodulation. Differences in the abundances of these mRNAs at different times suggest that they are not coordinately regulated. Moreover, using this technique mRNA specifically related to pcGS-γ1 was found at high levels in nodules but not in roots or leaves. Surprisingly the expression of this gene is not nodule-specific as previously suggested, as its mRNA was also detected, but at lower levels, in stems, petioles and in green cotyledons. By comparison, mRNA related to a leghaemoglobin gene was detected only in nodules. Comparisons of the relative abundances of the pcGS-γ1 mRNA and the γ polypeptide in different organs and at different stages during nodulation, suggest that the appearance of the γ polypeptide is largely under transcriptional control.
Similar content being viewed by others
References
Bogusz D, Appleby CA, Landsmann J, Dennis ES, Trinick MJ, Peacock WJ: Functioning haemoglobin genes in non-nodulating plants. Nature 331: 178–180 (1988).
Burnette WN: Electrophoretic transfer of proteins from sodium-dodecyl-sulphate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and iodinated protein A. Analyt Biochem 112: 195–203 (1981).
Burrell MM, Twell D, Karp A, Ooms G: Expression of shoot-inducing Ti TL-DNA in differentiated tissues of potato (Solanum tuberosum cv. Maris Bard). Plant Mol Biol 5: 213–222 (1985).
Campos F, Padilla J, Vasquez M, Ortega JL, Enriquez C, Sanchez F: Expression of nodule-specific genes inPhaseolus vulgaris L.. Plant Mol Biol 9: 521–532 (1987).
Chen F-L, Cullimore JV: Two isoenzymes of NADH-dependent glutamate synthase in root nodules ofPhaseolus vulgaris L.: purification, properties and activity changes during nodule development. Plant Physiol 88: 1411–1417 (1988).
Cullimore JV, Bennett MJ: The molecular biology and biochemistry of plant glutamine synthetase from root nodules ofPhaseolus vulgaris L. and other legumes. J Plant Physiol 132: 387–393 (1988).
Cullimore JV, Miflin BJ: Immunological studies on glutamine synthetase using antisera raised to the two plant forms of the enzyme fromPhaseolus root nodules. J Exp Bot 35: 581–587 (1984).
Cullimore JV, Sims AP: An association between photorespiration and protein catabolism: studies withChlamydomonas. Planta 150: 392–896 (1980).
Cullimore JV, Lara M, Lea PJ, Miflin BJ: Purification and properties of two forms of glutamine synthetase from the plant fraction ofPhaseolus root nodules. Planta 157: 245–253 (1983).
Cullimore JV, Gebhardt C, Saarelainen R, Miflin BJ, Idler BK, Barker RF: Glutamine synthetase ofPhaseolus vulgaris L.: organ-specific expression of a multigene family. J Mol Appl Genet 2: 589–599 (1984).
Dean C, Tamaki S, Dunsmuir P, Favreau M, Katayama C, Dooner H, Bedbrook J: mRNA transcripts of several plant genes are polyadenylated at multiple sitesin vivo. Nucl Acids Res 14: 2229–2240 (1986).
Donn G, Tischer E, Smith JA, Goodman HM: Herbicide-resistant alfalfa cells: an example of gene amplification in plants. J Mol Appl Genet 2: 621–635 (1984).
Dunn K, Dickstein R, Feinbaum R, Burnett R, Peterman K, Thoidis G, Goodman HM, Ausubel FM: Developmental regulation of nodule-specific genes in alfalfa root nodules. Mol Plant Microbe Interactions 1: 66–76 (1988).
Forde BG, Day HM, Turton JF, Shen W-J, Cullimore JV, Oliver JE: Two glutamine synthetase genes fromPhaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenicLotus corniculatus plants. The Plant Cell: in press (1989).
Fortin MG, Verma DPS: Peribacteroid membrane nodulins of soybean. In: Verma DPS, Brisson N (eds) Molecular Genetics of Plant-Microbe Interactions, pp. 102–107. Martinus Nijhoff Publishers, Dordrecht (1987).
Gebhardt C, Oliver JE, Forde BG, Saarelainen R, Miflin BJ: Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves ofPhaseolus vulgaris. EMBO J 5: 1429–1435 (1986).
Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).
Hirel B, Bouet C, King B, Layzell D, Jacobs F, Verma DPS: Glutamine synthetase genes are regulated by ammonium provided externally or by symbiotic nitrogen fixation. EMBO J 6: 1167–1171 (1987).
Kreig PA, Melton DA:In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol 155: 397–415 (1987).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lara M, Cullimore JV, Lea PJ, Miflin BJ, Johnston AWB, Lamb JW: Appearance of a novel form of plant glutamine synthetase during nodule development inPhaseolus vulgaris L. Planta 157: 254–258 (1983).
Lara M, Porta H, Padilla J, Folch J, Sanchez F: Heterogeneity of glutamine synthetase polypeptides inPhaseolus vulgaris L. Plant Physiol 76: 1019–1023 (1984).
Lara M, Ortega JL, Olguin BE, Sanchez F: Nodulin expression and nitrogen metabolism inPhaseolus vulgaris root nodules. In: Bothe H, deBruijn FJ, Newton WE (eds) Nitrogen Fixation: Hundred Years After, pp. 617–622. Gustav-Fischer Verlag, Stuttgart (1988).
Larsen K, Jochimsen BU: Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen. EMBO J 5: 25–29 (1986).
Lightfoot DA, Green NK, Cullimore JV: The chloroplast-located glutamine synthetase ofPhaseolus vulgaris L.: nucleotide sequence, expression in different organs and uptake into isolated chloroplasts. Plant Mol Biol 11: 191–202 (1988).
Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Analyt Biochem 163: 16–20 (1987).
Maniatis T, Fritsch E, Sambrook J: Molecular Cloning: Laboratory Manual. Cold Spring Harbor Laboratory Cold Spring Harbor, NY (1982).
Maxam AM, Gilbert W: Sequencing end-labelled DNA with base-specific chemical changes. Methods Enzymol 65: 499–560 (1980).
Miflin BJ, Cullimore JV: Nitrogen assimilation in the legume-Rhizobium symbiosis: a joint endeavour. In: Verma DPS, Hohn Th (eds) Genes Involved in Microbe-Plant Interactions, pp. 129–196. Springer-Verlag, Vienna (1984).
Miflin BJ, Lea PJ: Ammonia assimilation. In: Miflin BJ (ed) The Biochemistry of Plants vol 5, pp. 169–202. Academic Press, New York (1980).
Padilla JE, Campos F, Conde V, Lara M, Sanchez F: Nodule-specific glutamine synthetase is expressed before the onset of nitrogen fixation inPhaseolus vulgaris L. Plant Mol Biol 9: 65–74 (1987).
Robertson JG, Farnden KJF: Ultrastructure and metabolism of the developing legume root nodule. In: Miflin BJ (ed) The Biochemistry of Plants vol 5, pp. 66–113. Academic Press, New York (1980).
Roscoe TJ, Ellis RJ: Two-dimensional gel electrophoresis of chloroplast proteins. In: Edelman M, Hallik R, Chua N-H (eds) Methods in Chloroplast Biology, pp. 1015–1028. Elsevier-North Holland Biomedical Press, Amsterdam (1982).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Sengupta-Gopalan C, Pitas JW: Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans. Plant Mol Biol 7: 189–199 (1986).
Tingey SV, Walker EL, Coruzzi GM: Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. EMBO J 6: 1–9 (1987).
Tingey SV, Tsai F-Y, Edwards JW, Walker EL, Coruzzi GM: Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressedin vivo. J Biol Chem 263: 9651–9657 (1988).
Tischer E, DasSarma S, Goodman HM: Nucleotide sequence of an alfalfa glutamine synthetase gene. Mol Gen Genet 203: 221–229 (1986).
Verma DPS, Fortin MG, Stanley J, Mauro VP, Purohit S, Morrison N: Nodulins and nodulin genes ofGlycine max. Plant Mol Biol 7: 51–61 (1986).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bennett, M.J., Lightfoot, D.A. & Cullimore, J.V. cDNA sequence and differential expression of the gene encoding the glutamine synthetase γ polypeptide ofPhaseolus vulgaris L.. Plant Mol Biol 12, 553–565 (1989). https://doi.org/10.1007/BF00036969
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00036969