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. 1993 Feb;5(2):215–226. doi: 10.1105/tpc.5.2.215

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

R G Gregerson 1, S S Miller 1, S N Twary 1, J S Gantt 1, C P Vance 1
PMCID: PMC160264  PMID: 8453303

Abstract

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.

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Selected References

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  1. Anderson M. P., Vance C. P., Heichel G. H., Miller S. S. Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules. Plant Physiol. 1989 May;90(1):351–358. doi: 10.1104/pp.90.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brears T., Walker E. L., Coruzzi G. M. A promoter sequence involved in cell-specific expression of the pea glutamine synthetase GS3A gene in organs of transgenic tobacco and alfalfa. Plant J. 1991 Sep;1(2):235–244. doi: 10.1111/j.1365-313x.1991.00235.x. [DOI] [PubMed] [Google Scholar]
  3. Brutlag D. L., Dautricourt J. P., Maulik S., Relph J. Improved sensitivity of biological sequence database searches. Comput Appl Biosci. 1990 Jul;6(3):237–245. doi: 10.1093/bioinformatics/6.3.237. [DOI] [PubMed] [Google Scholar]
  4. Chen F. L., Cullimore J. V. Two Isoenzymes of NADH-dependent Glutamate Synthase in Root Nodules of Phaseolus vulgaris L: Purification, Properties and Activity Changes during Nodule Development. Plant Physiol. 1988 Dec;88(4):1411–1417. doi: 10.1104/pp.88.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cullimore J. V., Gebhardt C., Saarelainen R., Miflin B. J., Idler K. B., Barker R. F. Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family. J Mol Appl Genet. 1984;2(6):589–599. [PubMed] [Google Scholar]
  6. Fedoroff N., Wessler S., Shure M. Isolation of the transposable maize controlling elements Ac and Ds. Cell. 1983 Nov;35(1):235–242. doi: 10.1016/0092-8674(83)90226-x. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Gantt J. S., Larson R. J., Farnham M. W., Pathirana S. M., Miller S. S., Vance C. P. Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels. Plant Physiol. 1992 Mar;98(3):868–878. doi: 10.1104/pp.98.3.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gebhardt C., Oliver J. E., Forde B. G., Saarelainen R., Miflin B. J. Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. EMBO J. 1986 Jul;5(7):1429–1435. doi: 10.1002/j.1460-2075.1986.tb04379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Griffith S. M., Vance C. P. Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization. Plant Physiol. 1989 Aug;90(4):1622–1629. doi: 10.1104/pp.90.4.1622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Groat R. G., Schrader L. E. Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules. Plant Physiol. 1982 Dec;70(6):1759–1761. doi: 10.1104/pp.70.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Groat R. G., Vance C. P. Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.). Plant Physiol. 1982 Mar;69(3):614–618. doi: 10.1104/pp.69.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grossberger D. Minipreps of DNA from bacteriophage lambda. Nucleic Acids Res. 1987 Aug 25;15(16):6737–6737. doi: 10.1093/nar/15.16.6737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hemmilä I. A., Mäntsälä P. I. Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium. Biochem J. 1978 Jul 1;173(1):45–52. doi: 10.1042/bj1730045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  16. Hirsch A. M., Bang M., Ausubel F. M. Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):367–380. doi: 10.1128/jb.155.1.367-380.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hummelt G., Mora J. Regulation and function of glutamate synthase in Neurospora crassa. Biochem Biophys Res Commun. 1980 Oct 31;96(4):1688–1694. doi: 10.1016/0006-291x(80)91368-6. [DOI] [PubMed] [Google Scholar]
  18. Knaff D. B., Hirasawa M., Ameyibor E., Fu W., Johnson M. K. Spectroscopic evidence for a [3Fe-4S] cluster in spinach glutamate synthase. J Biol Chem. 1991 Aug 15;266(23):15080–15084. [PubMed] [Google Scholar]
  19. Lederer F., Cortial S., Becam A. M., Haumont P. Y., Perez L. Complete amino acid sequence of flavocytochrome b2 from baker's yeast. Eur J Biochem. 1985 Oct 15;152(2):419–428. doi: 10.1111/j.1432-1033.1985.tb09213.x. [DOI] [PubMed] [Google Scholar]
  20. Leigh J. A., Reed J. W., Hanks J. F., Hirsch A. M., Walker G. C. Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion. Cell. 1987 Nov 20;51(4):579–587. doi: 10.1016/0092-8674(87)90127-9. [DOI] [PubMed] [Google Scholar]
  21. Masters D. S., Jr, Meister A. Inhibition of homocysteine sulfonamide of glutamate synthase purified from Saccharomyces cerevisiae. J Biol Chem. 1982 Aug 10;257(15):8711–8715. [PubMed] [Google Scholar]
  22. Oliver G., Gosset G., Sanchez-Pescador R., Lozoya E., Ku L. M., Flores N., Becerril B., Valle F., Bolivar F. Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12. Gene. 1987;60(1):1–11. doi: 10.1016/0378-1119(87)90207-1. [DOI] [PubMed] [Google Scholar]
  23. Pathirana S. M., Vance C. P., Miller S. S., Gantt J. S. Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules. Plant Mol Biol. 1992 Nov;20(3):437–450. doi: 10.1007/BF00040603. [DOI] [PubMed] [Google Scholar]
  24. Sakakibara H., Watanabe M., Hase T., Sugiyama T. Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf. J Biol Chem. 1991 Feb 5;266(4):2028–2035. [PubMed] [Google Scholar]
  25. Scrutton N. S., Berry A., Perham R. N. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature. 1990 Jan 4;343(6253):38–43. doi: 10.1038/343038a0. [DOI] [PubMed] [Google Scholar]
  26. Suzuki A., Vidal J., Gadal P. Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues. Plant Physiol. 1982 Sep;70(3):827–832. doi: 10.1104/pp.70.3.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trotta P. P., Platzer K. E., Haschemeyer R. H., Meister A. Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4607–4611. doi: 10.1073/pnas.71.11.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vance C. P., Stade S. Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase. Plant Physiol. 1984 May;75(1):261–264. doi: 10.1104/pp.75.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Virts E. L., Stanfield S. W., Helinski D. R., Ditta G. S. Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti. Proc Natl Acad Sci U S A. 1988 May;85(9):3062–3065. doi: 10.1073/pnas.85.9.3062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Walker E. L., Coruzzi G. M. Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum. Plant Physiol. 1989 Oct;91(2):702–708. doi: 10.1104/pp.91.2.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yarosh O. K., Charles T. C., Finan T. M. Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti. Mol Microbiol. 1989 Jun;3(6):813–823. doi: 10.1111/j.1365-2958.1989.tb00230.x. [DOI] [PubMed] [Google Scholar]
  32. von Heijne G., Steppuhn J., Herrmann R. G. Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem. 1989 Apr 1;180(3):535–545. doi: 10.1111/j.1432-1033.1989.tb14679.x. [DOI] [PubMed] [Google Scholar]

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