Skip to main content
SpringerLink
Log in

Structure and expression of a nitrite reductase gene from bean (Phaseolus vulgaris) and promoter analysis in transgenic tobacco

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

Abstract

A structural gene encoding nitrite reductase (NiR) in bean (Phaseolus vulgaris) has been cloned and sequenced. The NiR gene is present as a single copy encoding a protein of 582 amino acids. The bean NiR protein is synthezised as a precursor with an amino-terminal transit peptide (TP) consisting of 18 amino acid residues. The bean NiR transit peptide shows similarity to the TPs of other known plant NiRs.

The NiR gene is expressed in trifoliate leaves and in roots of 20-day old bean plants where transcript accumulation is nitrate-inducible. Gene expression occurs in a circadian rhythm and induced by light in leaves of dark-adapted plants.

A particular 100 bp sequence is present in the promoter and in the first intron of the NiR gene. Several copies of this 100 bp sequence are present in the bean genome. Comparisons between the promoter of the bean NiR gene and of two bean nitrate reductase genes (NR1 and NR2) show a limited number of conserved motifs, although the genes are presumed to be co-regulated. Comparisons are also made between the bean NiR promoter and the spinach NiR promoter.

Transformation of tobacco plants with the bean NiR promoter fused to the GUS reporter gene (β-glucuronidase) shows that the bean NiR promoter is nitrate-regulated and that the presence of the 100 bp sequence influences the level of GUS activity. NiR-coding sequences are not required for nitrate regulation but have a quantitative effect on the measured GUS activity.

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. Aslam M, Huffaker RC: Role of nitrate and nitrite in the induction of nitrite reductase in leaves of barley seedlings. Plant Physiol 91: 1152–1156 (1989).

    Google Scholar 

  2. Augier V, Guigliarelli B, Asso M, Bertrand P, Froxon C, Giordano G, Chippaux M, Blasco F: Site-directed mutagenesis of conserved cysteine residues within the β-subunit of Escherichia coli nitrate reductase. Physiological, biochemical and EPR characterization of the mutated enzymes. Biochemistry 32: 2013–2023 (1993).

    Google Scholar 

  3. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Strul K: Current Protocols in Molecular Biology. Greene Wiley-Interscience, New York (1987).

    Google Scholar 

  4. Back E, Burkhart W, Moyer M, Privalle L, Rothstein S: Isolation of cDNA clones coding for spinach nitrite reductase: complete sequence and nitrate induction. Mol Gen Genet 212: 20–26 (1988).

    Google Scholar 

  5. Back E, Dunne W, Schneiderbauer A, de Framond A, Rastogi R, Rothstein SJ: Isolation of the spinach nitrite reductase gene promoter which confers nitrate inducibility on GUS gene expression in transgenic tobacco. Plant Mol Biol 17: 9–18 (1991).

    Google Scholar 

  6. Becker TW, Foyer C, Caboche M: Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytocrome-deficient aurea mutant of tomato. Planta 188: 39–47 (1992).

    Google Scholar 

  7. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).

    Google Scholar 

  8. Boutry M, Chua N-H: A nuclear gene encoding the β-subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia. EMBO J 4: 2159–2165 (1985).

    Google Scholar 

  9. Bradford M: 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).

    Google Scholar 

  10. Brown JWS: A catalogue of splice junction and putative branch poin sequences from plant introns. Nucl Acids Res 14: 9549–9559 (1986).

    Google Scholar 

  11. Cheng C-L, Acedo GN, Christinsin M, Conkling MA: Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription. Proc Natl Acad Sci USA 89: 1861–1864 (1992).

    Google Scholar 

  12. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: Version II. Plant Mol Biol 1: 19–21 (1983).

    Google Scholar 

  13. Duncanson E, Gilkes AF, Kirk DW, Sherman A, Wray JL: Nirl, a conditional-lethal mutation in barley causing a defect in nitrite reduction. Mol Gen Genet 236: 275–282 (1993).

    Google Scholar 

  14. Faure J-D, Vincentz M, Kronenberger J, Caboche M: Co-regulated expression of nitrate and nitrite reductases. Plant J 1: 107–113 (1991).

    Google Scholar 

  15. Friemann A, Brinkmann K, Hachtel W: Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions. Mol Gen Genet 231: 411–416 (1992).

    Google Scholar 

  16. Friemann A, Lange M, Hachtel W, Brinkmann K: Induction of nitrate assimilatory enzymes in the tree Betula pendula. Plant Physiol 99: 837–842 (1992).

    Google Scholar 

  17. Hasumi H, Nagata E, Nakamura S: Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves. Biochem Biophys Res Commun 110: 280–286 (1983).

    Google Scholar 

  18. Hirasawa M, de Best JH, Knaff DB: The effect of lysine- and arginine-modifying reagents on spinach ferredoxin: nitrite oxidoreductase. Biochim Biophys Acta 1140: 304–312 (1993).

    Google Scholar 

  19. Hoff T, Stummann BM, Henningsen KW: Cloning and expression of a gene encoding a root specific nitrate reductase in bean (Phaseolus vulgaris). Physiol Plant 82: 197–204 (1991).

    Google Scholar 

  20. Horsch RB, Fry JW, Hoffman NL, Eicholtz D, Rogers SG, Fraley RJ: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  21. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    Google Scholar 

  22. Jensen PE, Hoff T, Møller MG, Stummann BM, Henningsen KW: Identification and characterization of a nitrate reductase gene from bean (Phaseolus vulgaris) containing four introns. Submitted.

  23. Kavanagh TA, Jefferson RA, Bevan MW: Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein. Mol Gen Genet 215: 38–45 (1988).

    Google Scholar 

  24. Keegstra K, Olsen LJ, Theg SM: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Physiol Plant Mol Biol 40: 471–501 (1989).

    Google Scholar 

  25. Kronenberger J, Lepingle A, Caboche M, Vaucheret H: Cloning and expression of distinct nitrite reductases in tobacco leaves and roots. Mol Gen Genet 236: 203–208 (1993).

    Google Scholar 

  26. Lahners K, Kramer V, Back E, Privalle L, Rothstein S: Molecular cloning of complementary DNA encoding maize nitrite reductase. Plant Physiol 88: 741–746 (1988).

    Google Scholar 

  27. Mohr H, Neininger A, Seith B: Control of nitrate reductase and nitrite reductase gene expression by light, nitrate and a plastidic factor. Bot Acta 105: 81–89 (1992).

    Google Scholar 

  28. Murashige T, Skoog F: A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).

    Google Scholar 

  29. Neininger A, Bichler J, Schneiderbauer A, Mohr H: Response of a nitrite-reductase 3.1-kilobase upstream regulatory sequence fro spinach to nitrate and light in transgenic tobacco. Planta 189: 440–442 (1993).

    Google Scholar 

  30. Neininger A, Kronenberger J, Mohr H: Coaction of light and a plastidic factor in controlling nitrite-reductase gene expression in tobacco. Planta 187: 381–387 (1992).

    Google Scholar 

  31. Oelmuller R, Schuster C, Mohr H: Physiological characterization of a plastidic signal required for nitrate induced appearance of nitrate and nitrite reductases. Planta 174: 75–83 (1988).

    Google Scholar 

  32. Rastogi R, Back E, Schneiderbauer A, Bowsher CG, Moffat B, Rothstein SJ: A 330 bp region of the spinach nitrite reductase gene promoter directs nitrate-inducible tissue-specific expression in transgenic tobacco. Plant J 4: 317–326 (1993).

    Google Scholar 

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

    Google Scholar 

  34. Schuster C, Mohr H: Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by phytocrome. Planta 181: 327–334 (1990).

    Google Scholar 

  35. Seith B, Schuster C, Mohr H: Coaction of light, nitrate and a plastidic factor in controlling nitrite-reductase gene expression in spinach. Planta 184: 74–80 (1991).

    Google Scholar 

  36. Tsay Y-F, Schroeder JI, Feldmann KA, Crawford NM: The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72: 705–713 (1993).

    Google Scholar 

  37. Vaucheret H, Kronenberger J, Lepingle A, Vilaine F, Boutin J-P, Caboche M: Inhibition of tobacco nitrite reductase activity by expression of antisense RNA. Plant J 2: 559–569 (1992).

    Google Scholar 

  38. Vincentz M, Moureaux T, Leydecker M-T, Vaucheret H, Caboche M: Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant J 3: 315–324 (1993).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genetics Section, Institute of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Bülowsvej 13, DK-1870, Frederiksberg C, Denmark

    Lilli Sander, Poul E. Jensen, Louise F. Back, Bjarne M. Stummann & Knud W. Henningsen

Authors
  1. Lilli Sander
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Poul E. Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Louise F. Back
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bjarne M. Stummann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Knud W. Henningsen
    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

Sander, L., Jensen, P.E., Back, L.F. et al. Structure and expression of a nitrite reductase gene from bean (Phaseolus vulgaris) and promoter analysis in transgenic tobacco. Plant Mol Biol 27, 165–177 (1995). https://doi.org/10.1007/BF00019188

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00019188

Key words

Search

Navigation