, Volume 156, Issue 1–2, pp 103–116 | Cite as

Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E. coli

  • David A. LightfootEmail author
  • Rajsree Mungur
  • Rafiqa Ameziane
  • Scott Nolte
  • Lynn Long
  • Karen Bernhard
  • Andrew Colter
  • Karen Jones
  • M. J. Iqbal
  • Edward Varsa
  • Brian Young


Genetic modification of nitrogen metabolism via bacterial NADPH- dependent glutamate dehydrogenase (GDH; E.C. favorably alters growth and metabolism of C3 plants. The aim of this study was to examine the effect of expression of GDH in the cytoplasmic compartment of Zea mays cells. The gdhA gene from Escherichia coli , that encoded a NADPH-GDH, was ligated to the ubiquitin promoter that incorporated the first intron enhancer and used to transform Z. mays cv. ‘H99’ embryo cultures by biolistics. R0–R3 generations included selfed inbreds, back-crossed inbreds, and hybrids with B73 derivatives. The lines with the highest GDH specific activity produced infertile R0 plants. The highest specific activity of GDH from the fertile Z. mays plants was sufficient to alter phenotypes. Plant damage caused by the phosphinothricin in gluphosinate-type herbicides, glutamine synthetase (GS; EC inhibitors, was less pronounced in Z. mays plants with gdhA pat than in gusA pat plants. Germination and grain biomass production were increased in gdhA transgenic plants in the field during seasons with significant water deficits but not over all locations. Water deficit tolerance under controlled conditions was increased. Crops modified with gdhA may have value in semi-arid locations.


Ammonia assimilation Glutamate dehydrogenase Transgenic plants Phosphinothricin Gluphosinate 



Plant materials were developed with a grant from the Herman Frasch foundation and the Illinois Maize Marketing Board. Analyses were supported by grants from the Illinois Missouri Biotechnology Alliance and the Illinois Council for Food and Agricultural Research. We thank P. Bullock at Garst Inc., Highway 210 W, PO Box 500, Slater, Iowa 50244.; and D. Hondred, now at Pioneer Hi-Bred International Inc., Trait and Technology Development, 7300 NW 62 nd Ave., PO Box 1004, Johnson, Iowa 50131-1004 for transformation an analysis advice. We thank Dr. J. Preece and Dr. G. Kapusta for advice, E. Cerny and R. Lang for technical assistance.


  1. Ameziane R, Bernhard K, Lightfoot DA (2000a) Expression of the Escherichia coli glutamate dehydrogenase gene in tobacco affects plant growth and development. Plant and Soil 221:45–57CrossRefGoogle Scholar
  2. Ameziane R, Bernhardt K, Lightfoot DA (2000b) Expression of the bacterial gdhA gene encoding a glutamate dehydrogenase in tobacco and maize increased tolerance to the phosphinothricin herbicide. In: Martins-Loucao MA, Lips SH (eds) “Nitrogen in a sustainable ecosystem, from the cell to the plant”. Backhuys Publishers, Leiden, The NetherlandsGoogle Scholar
  3. Andrews M, Lea PJ, Raven JA, Lindsey K (2004) Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and greater N-use efficiency? An assessment. Ann Appl Biol 145:25–35CrossRefGoogle Scholar
  4. Aubert S, Bligny R, Douce R, Gout E, Ratcliffe RG, Roberts JKM (2001) Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by C-13 and P-31 nuclear magnetic resonance. J Exp Bot 52:37–45PubMedCrossRefGoogle Scholar
  5. Beagle JM, Apgar GA, Jones KL, Griswold KE, Radcliffe JS, Qiu X, Lightfoot DA, Iqbal MJ. (2006) The digestive fate of Escherichia coli glutamate dehydrogenase deoxyribonucleic acid from transgenic corn in diets fed to weanling pigs. J Anim Sci 84:597–607PubMedGoogle Scholar
  6. Becker TW, Carrayol E, Hirel B (2000) Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves, localization, relative proportion and their role in ammonium assimilation or nitrogen transport. Planta 211:800–806PubMedCrossRefGoogle Scholar
  7. Blum A, Ebercon A (1976) Genotypic responses in sorghum to drought stress. III. Free proline accumulation and drought resistance. Crop Sci 16:428–431CrossRefGoogle Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  9. Chen TH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol. 5:250–257PubMedCrossRefGoogle Scholar
  10. Colter RA (2001) Nitrogen response in glutamate dehydrogenase transgenic corn. MS Thesis, SIUC, Carbondale IL USAGoogle Scholar
  11. Coruzzi GM, Zhou L (2001) Carbon and nitrogen sensing and signaling in plants, emerging ‘matrix effects’. Curr Opp Plant Biol 4:247–253CrossRefGoogle Scholar
  12. David MB, Gentry LE, Kovacic DA, Smith KM (1997) Nitrogen balance in and export from an agricultural watershed. J Env Qual 26:1038–1048CrossRefGoogle Scholar
  13. Doehlert D, Lambert RJ (1991) Metabolic Characteristics associated with starch, protein and oil deposition in developing maize kernels. Crop Sci 31:151–157CrossRefGoogle Scholar
  14. Dubois V, Botton E, Meyer C, Rieu A, Bedu M, Maisonneuve B, Mazier M (2005) Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.). J Exp Bot 56:2379–2388PubMedCrossRefGoogle Scholar
  15. Duncan D, Dykhuisen R, Frazer A, MacKenzie H, Golden M, Bejamin N, Leifert C, (1998) Human health effects of nitrate. Gut 42:334–340CrossRefGoogle Scholar
  16. Duvick DN (1991) Progress in conventional plant breeding. Springer Verlag, AmsterdamGoogle Scholar
  17. Fei H, Chaillou S, Hirel B, Mahon JD, Vessey JK (2003) Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate. Planta 216:467–474PubMedGoogle Scholar
  18. Gallais A, Hirel B (2004) An approach to the genetics of nitrogen use efficiency in maize. J Exp Bot 55:295–306PubMedCrossRefGoogle Scholar
  19. Guthrie TA, Apgar GA, Griswold KE, Lindemann MD, Radcliffe JS, Jacobson BN (2004) Nutritional value of a corn containing a glutamate dehydrogenase gene for growing pigs. J Anim Sci 82:1693–1698PubMedGoogle Scholar
  20. Hirel B, Terce-LeForge T, Gonzalo-Moro MB, Estavillo JM (2005a) Physiology of maize I: a comprehensive and integrated view of nitrogen metabolism in a C4 plant. Physiol Plantar 124:167–177CrossRefGoogle Scholar
  21. Hirel B, Andrieu B, Valadier MH, Renard S, Quilleré I, Chelle M, Pommel B, Fournier C, Drouet JL (2005b) Physiology of maize II: identification of physiological markers representative of the nitrogen status of maize (Zea mays) leaves during grain filling. Physiol Plantar 124:178–188CrossRefGoogle Scholar
  22. Kichey T, Heumez E, Pocholle D, Pageau K, Vanacker H, Dubois F, Le Gouis J, Hirel B (2006) Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. New Phytol 169:265–278PubMedCrossRefGoogle Scholar
  23. Kichey T, Le Gouis J, Sangwan B, Hirel B, Dubois F (2005) Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.). Plant Cell Physiol 46:964–974PubMedCrossRefGoogle Scholar
  24. Lightfoot DA, Baron AJ, Wootton JC (1988) Expression of Escherichia coli glutamate dehydrogenase in the cyanobacterium Synechococcus PCC6301 causes ammonia tolerance. Plant Molec Biol 11:335–344CrossRefGoogle Scholar
  25. Lightfoot DA, Long LM, Vidal ME (1999) Plants containing the gdhA gene and methods of use thereof. US Patent # 5,998,700Google Scholar
  26. Lightfoot DA, Long LM, Vidal ME (2001) Plants containing the gdhA gene and methods of use thereof. US Patent # 6,329,573Google Scholar
  27. Limami A, Phillipson B, Ameziane R, Pernollet N, Jiang Q, Roy R, Deleens E, Chaumont-Bonnet M, Gresshoff PM, Hirel B (1999) Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta. 209:495–502PubMedCrossRefGoogle Scholar
  28. Loulakakis KA, Primikirios NI, Nikolantonakis MA, Roubelakis-Angelakis KA (2002) Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development. Planta 215:630–638PubMedCrossRefGoogle Scholar
  29. Magalhaes JR, Ju GC, Rich PJ, Rhodes D (1990) Kinetics of 15NH4+ assimilation in Zea mays. Plant Physiol 94:647–656PubMedCrossRefGoogle Scholar
  30. Melo-Oliveira R, Oliveira IC, Coruzzi GM (1996) Arabidopsis mutant analysis and gene regulation define a non-redundant role for glutamate dehydrogenase in nitrogen assimilation. Proc Natl Acad Sci USA 93:4718–4723PubMedCrossRefGoogle Scholar
  31. Mungur R (2002) Metabolic profiles of GDH transgenic crops. MS thesis, SIUC CarbondaleGoogle Scholar
  32. Mungur R, Glass AD, Goodenow D, Lightfoot DA (2005) Metabolite fingerprint changes in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. J Biomed Biotech 2:198–214CrossRefGoogle Scholar
  33. Mungur R, Wood AJ, Lightfoot DA (2006) Water potential is maintained during water deficit in Nicotiana tabacum expressing the Escherichia coli glutamate dehydrogenase gene. Plant Growth Regul 50:231–238CrossRefGoogle Scholar
  34. Noctor G, Novitskaya L, Lea PJ, Foyer CH (2002) Co-ordination of leaf minor amino acid contents in crop species, significance and interpretation. J Exp Bot 53:939–945PubMedCrossRefGoogle Scholar
  35. Nolte SA, Young BG, Mungur R, Lightfoot DA (2004) The glutamate dehydrogenase gene gdhA increased the resistance of tobacco to glufosinate. Weed Res 44:335–339CrossRefGoogle Scholar
  36. Oliveira IC, Brears T, Knight TJ, Clark A, Coruzzi GM (2002) Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration. Plant Physiol 129:1170–1180PubMedCrossRefGoogle Scholar
  37. Ortega JL, Moguel-Esponda S, Potenza C, Conklin CF, Quintana A, Sengupta-Gopalan C (2006) The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS) affects transcript stability and protein accumulation in transgenic alfalfa. Plant J 45:832–846PubMedCrossRefGoogle Scholar
  38. Sakakibara H, Fujii K, Sugiyama T (1995) Isolation and characterization of a cDNA that encodes maize glutamate dehydrogenase. Plant Cell Physiol 36:789–797PubMedGoogle Scholar
  39. Schmidt RR, Miller P (1999) Polypeptides and polynucleotides relating to alpha and beta subunits of glutamate dehydrogenase and methods of use. US patent # 5879941Google Scholar
  40. Schuster S, Dandekar T, Fell DA (1999) Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering. Trends Biotech 17:53–60CrossRefGoogle Scholar
  41. Stitt M, Muller C, Matt P, Gibon Y, Carillo P, Morcuende R, Scheible WR, Krapp A (2002) Steps towards an integrated view of nitrogen metabolism. J Exp Bot 53:959–970PubMedCrossRefGoogle Scholar
  42. Sun H, Huang QM, Su J (2005) Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance. J Plant Physiol Molec Biol 31:492–498Google Scholar
  43. Terce-Laforgue T, Mack G, Hirel B (2004a) New insights towards the function of glutamate dehydrogenase revealed during source-sink transition of tobacco (N. tabacum) plants grown under different nitrogen regimes. Physiol Plantar 120:220–228CrossRefGoogle Scholar
  44. Terce-Laforgue T, Dubois F, Ferrario-Mery S, de Crecenzo MA, Sangwan R, Hirel B (2004b) Glutamate dehydrogenase of tobacco is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration. Plant Physiol 136:4308–4317CrossRefGoogle Scholar
  45. Wang R, Guegler K, LaBrie ST, Crawford NM (2000) Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. Plant Cell 128:1491–1510CrossRefGoogle Scholar
  46. Wang X, Larkins B (2001) Genetic analysis of amino acid accumulation in opaque-2 maize endosperm. Plant Physiol 125:1766–1777PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • David A. Lightfoot
    • 1
    Email author
  • Rajsree Mungur
    • 1
    • 2
  • Rafiqa Ameziane
    • 1
    • 3
  • Scott Nolte
    • 1
  • Lynn Long
    • 1
  • Karen Bernhard
    • 1
  • Andrew Colter
    • 1
  • Karen Jones
    • 4
  • M. J. Iqbal
    • 1
  • Edward Varsa
    • 1
  • Brian Young
    • 1
  1. 1.The Department of Plant, Soil and Agricultural SystemsSouthern Illinois UniversityCarbondaleUSA
  2. 2.University of BerkleySan FranciscoUSA
  3. 3.Molecular, Cellular, and Developmental Biology (MCDB)University of MichiganAnn ArborUSA
  4. 4.The Department of Animal Science, Food and NutritionSouthern Illinois UniversityCarbondaleUSA

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