Skip to main content
SpringerLink
Log in

Isolation and characterization of a cDNA encoding NADP+-specific isocitrate dehydrogenase from soybean (Glycine max)

  • Research Articles
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A cDNA that encodes an NADP-specific isocitrate dehydrogenase (IDH) was cloned from a soybean nodule cDNA library by complementation of an Escherichia coli mutant that lacked IDH. DNA sequence analysis showed that the 1583 bp soybean cDNA could encode a protein that shares 63.9% amino acid sequence identity with the Saccharomyces cerevisiae NADP-IDH and long sequences of identity to an IDH from pig. Southern blot analysis suggests that this gene corresponds to a gene family made up of no more than two loci. The IDH cDNA hybridized to a 1.7 kb soybean mRNA and the relative amount of this transcript in soybean leaves, nodules and roots was 1:3.4:7.7. In alfalfa, a 1.7 kb mRNA was also found but the ratios for the corresponding tissues were 1:7.4:7.7. IDH activity was detected in the complemented E. coli strain and the electrophoretic mobility of this activity in nondenaturing polyacrylamide gels was identical to that of an IDH in extracts from soybean cotyledons or nodule cytosol. NADP-IDH specific activity in the E. coli host strain varied with growth phase; the highest rates (ca. 180 nmol/min per mg protein) were observed in late-stationary-phase cells. The enzyme had a broad pH optimum of 8.0 to 9.5 and had an absolute metal cofactor requirement, preferring Mn2+ below pH 8.0 and Mg2+ above pH 8.0. The K m for isocitrate and NADP was 21 μM and 11 μM respectively with Mn2+ as cofactor and 13 μM and 12 μM with Mg2+ as cofactor.

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. Aerbersold PB, Winans GA, Teel DJ, Milner GB, Utter FM: Manual for starch gel electrophoresis: a method for detection of genetic variation, pp. 12–19. National Oceanic and Atmospheric Administration National Marine Fisheries Service Technical Report NMFS 61 (1987).

    Google Scholar 

  2. Bilofsky HS, Burks C, Fickett JW, Goad WB, Lewitter FI, Rindone WP, Swindell CD, Tung CS: The Gen-BankTM genetic sequence data bank. Nucl Acids Res 14: 1–4 (1986).

    Google Scholar 

  3. Bowman EJ, Ikuma H, Stein HJ: Citric acid cycle activity in mitochondria isolated form mung bean hypocotyls. Plant Physiol 58: 426–432 (1976).

    Google Scholar 

  4. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Article  PubMed  Google Scholar 

  5. Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD: A method for isolation of intact, translationally active ribonucleic acid. DNA 2: 239–335 (1983).

    Google Scholar 

  6. Chen R, LeMarechal P, Vidal J, Jacquot J-P, Gadal P: Purification and comparative properties of the cytosolic isocitrate dehydrogenase (NADP) from pea (Pisum sativum) root and green leaves. Eur J Biochem 175: 565–572 (1988).

    PubMed  Google Scholar 

  7. Cox GF: Isocitrate dehydrogenase (NAD+ specific) from pea mitochondria. Meth Enzymol 13: 47–51 (1969).

    Google Scholar 

  8. Cupp JR, McAlister-Henn L: NAD+-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the idh2 gene from Saccharomyces cerevisiae. J Biol Chem 266: 22199–22205 (1991).

    PubMed  Google Scholar 

  9. Curry RA, Ting IP: Purification, properties and kinetic observations on the isozymes of NADP-isocitrate dehydrogenase of maize. Arch Biochem Biophys 176: 501–509 (1976).

    PubMed  Google Scholar 

  10. Day DA, Price GD, Gresshoff PM: Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules. Protoplasma 134: 121–129 (1986).

    Google Scholar 

  11. Delauney AJ, Verma DPS. A soybean gene encoding Δ-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet 221: 299–305 (1990).

    Article  PubMed  Google Scholar 

  12. Devereaux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).

    PubMed  Google Scholar 

  13. Elias BA, Givan CV: Alpha-ketoglutarate supply for amino acid synthesis in higher plant chloroplasts. Plant Physiol 59: 738–740 (1977).

    Google Scholar 

  14. Gubler V, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).

    Article  PubMed  Google Scholar 

  15. Gupta VK, Singh R: Partial purification and characterization of NADP+-isocitrate dehydrogenase from immature pod walls of chickpea (Cicer arietinum L.). Plant Physiol 87: 741–744 (1986).

    Google Scholar 

  16. Hamm GH, Cameron GN: The EMBL data library. Nucl Acids Res 14: 5–10 (1988).

    Google Scholar 

  17. Hartl F-U, Pfanner N, Nicholson DW, Neupert W: Mitochondrial protein import. Biochim Biophys Acta 988: 1–45 (1989).

    PubMed  Google Scholar 

  18. Haselbeck RJ, McAlister-Henn L: Isolation, nucleotide sequence, and distribution of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J Biol Chem 266: 2339–2345 (1991).

    PubMed  Google Scholar 

  19. Haselbeck RJ, Colman RF, McAlister-Henn L: Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase. Biochemistry 31: 6219–6223 (1992).

    PubMed  Google Scholar 

  20. Hedges BR, Sellner JM, Devine TE, Palmer RG: Assigning isocitrate dehydrogenase to linkage group 11 in soybean. Crop Sci 30: 940–942 (1990).

    Google Scholar 

  21. Henson CA, Schrader LE, Duke SH: Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max (L) Merr.) cultivars. Physiol Plant 48: 168–174 (1980).

    Google Scholar 

  22. Henson CA, Collins M, Duke SH: Subcellular localization of enzymes of carbon and nitrogen metabolism in nodules of Medicago sativa. Plant Cell Physiol 23: 227–235 (1982).

    Google Scholar 

  23. Henson CA, Duke SH, Collins M: Characterization of NADP+ isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules. Physiol Plant 67: 538–544 (1986).

    Google Scholar 

  24. Hurley JH, Thorsness PE, Ramalingam V, Helmers NH, Koshland DEJr, Stroud RM: Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci USA 86: 8635–8639 (1989).

    PubMed  Google Scholar 

  25. Hurley JH, Dean AM, Koshland DEJr, Stroud RM: Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: Implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry 30: 8671–8678 (1991).

    PubMed  Google Scholar 

  26. Kiang YT, Gorman MB: Soybean. In: Tanksley SD, Orton TJ (eds) Isozymes in Plant Genetics and Breeding, Part B, pp. 295–328. Elsevier Science Publishers, Amsterdam (1983).

    Google Scholar 

  27. Kiang YT, Gorman MB: Inheritance of NADP-active isocitrate dehydrogenase isozymes in soybeans. J Hered 76: 279–284 (1985).

    Google Scholar 

  28. Kittell BL, Helinski DR, Ditta GS: Aromatic aminotrans-ferase activity and indoleacetic acid production in Rhizobium meliloti. J Bact 171: 5458–5466 (1989).

    PubMed  Google Scholar 

  29. Leech RM: Comparative biochemistry and comparative morphology of chloroplasts isolated by different methods. In: Goodwin TW (ed) Biochemistry of Chloroplasts, vol. 1, pp. 65–74. Academic Press, New York (1966).

    Google Scholar 

  30. Maloney RJ, Dennis DT: The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L. Can J Biochem 55: 928–934 (1977).

    PubMed  Google Scholar 

  31. McDermott TR, Kahn ML: Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene. J Bact 174: 4790–4797 (1992).

    PubMed  Google Scholar 

  32. Miao G-H, Hirel B, Marsolier MC, Ridge R, Verma DPS: Ammonia-regulated expression of a soybean gene encoding glutamine synthetase in transgenic Lotus corniculatus. Plant Cell 3: 11–22 (1991).

    Article  PubMed  Google Scholar 

  33. Miller JH: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972).

    Google Scholar 

  34. Ni W, Robertson EF, Reeves HC: Purification and characterization of cytosolic NADP specific isocitrate dehydrogenase from Pisum sativum. Plant Physiol 83: 785–788 (1987).

    Google Scholar 

  35. Omran RG, Dennis DT: Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from a higher plant. Isolation and characterization. Plant Physiol 47: 43–47 (1971).

    PubMed  Google Scholar 

  36. Randall DD, Givan CV: Subcellular location of NADP+-isocitrate dehydrogenase in Pisum sativum leaves. Plant Physiol 68: 70–73 (1981).

    Google Scholar 

  37. Rasmusson AG, Moller IM: NADP-utilizing enzymes in the matrix of plant mitochondria. Plant Physiol 94: 1012–1018 (1990).

    Google Scholar 

  38. Rawsthorne S, Minchin FR, Summerfield RJ, Cookson C, Coombs J: Carbon and nitrogen metabolism in legume root nodules. Phytochemistry 19: 341–355 (1980).

    Article  Google Scholar 

  39. Robertson JG, Farnden KJF: Ultrastructure and metabolism of the developing legume root nodule. In: Stumpf PK, Conn EE (eds) The Biochemistry of Plants, vol 5, pp. 65–113. Academic Press, New York (1980).

    Google Scholar 

  40. Rogers SO, Bendich AJ: Extraction of DNA from milligram amounts of fresh, herbarium and mummufied plant tissues. Plant Mol Biol 5: 69–76 (1985).

    Google Scholar 

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

    Google Scholar 

  42. Satoh Y: Changes in the stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds. Biochim Biophys Acta 630: 352–360 (1980).

    PubMed  Google Scholar 

  43. Schubert KR: Products of biological nitrogen fixation in higher plants: synthesis, transport and metabolism. Annu Rev Plant Physiol 37: 539–574 (1986).

    Google Scholar 

  44. Snustad DP, Hunsperger JP, Chereskin BM, Messing J: Maize glutamine synthetase cDNAs: Isolation by direct selection in Escherichia coli. Genetics 120: 1111–1124 (1988).

    PubMed  Google Scholar 

  45. Sprent JI: Root nodule anatomy, type of export product and evolutionary origin in some leguminosae. Plant Cell Environ 3: 35–43 (1980).

    Google Scholar 

  46. Surguchov AP: Common genes for mitochondrial and cytoplasmic proteins. Trends Biochem Sci 12: 335–338 (1987).

    Article  Google Scholar 

  47. Thorsness PE, Koshland DE: Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem 262: 10422–10425 (1987).

    PubMed  Google Scholar 

  48. Udvardi MK, Kahn ML: Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase. Mol Gen Genet 231: 97–105 (1991).

    Article  PubMed  Google Scholar 

  49. Wych RD, Rains DW: Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans. Plant Physiol 62: 443–448 (1978).

    Google Scholar 

  50. Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13-mp 18 and pUC19 vectors. Gene 33: 103–119 (1985).

    Article  PubMed  Google Scholar 

  51. Zemlyanukhin AA, Popova TN, Eprintsev AT, Zemlyanukhin LA: Isoforms of NADP+-dependent pea leaf isocitrate dehydrogenase and their properties. Fiziologiya Rastenii 34: 499–506 (1987).

    Google Scholar 

Download references

Author information

Author notes

  1. Michael K. Udvardi

    Present address: CSIRO Division of Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia

Authors and Affiliations

  1. Institute of Biological Chemistry, Washington State University, 99164-6340, Pullman, WA, USA

    Michael K. Udvardi, Timothy R. McDermott & Michael L. Kahn

  2. Department of Microbiology, Washington State University, 99164-6340, Pullman, WA, USA

    Michael L. Kahn

Authors
  1. Michael K. Udvardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Timothy R. McDermott
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael L. Kahn
    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

Udvardi, M.K., McDermott, T.R. & Kahn, M.L. Isolation and characterization of a cDNA encoding NADP+-specific isocitrate dehydrogenase from soybean (Glycine max). Plant Mol Biol 21, 739–752 (1993). https://doi.org/10.1007/BF00027108

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00027108

Key words

Search

Navigation