Skip to main content
SpringerLink
Log in

Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5′-deletion fragments in front of the uidA gene encoding β-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.

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. Ainley WM, Walker JC, Nagao RT, Key JL: Sequence and characterization of two auxin-regulated genes from soybean. J Biol Chem 263: 10658–10666 (1988).

    PubMed  Google Scholar 

  2. An G, Costa MA, Ha SV: Nopaline synthase promoter iswound inducible and auxin inducible. Plant Cell 2: 225–233 (1990).

    Article  PubMed  Google Scholar 

  3. Bartel B, Fink GR: Differential regulation of an auxin producing gene family in Arabidopsis thaliana. Proc Natl Acad Sci USA 91: 6649–6653 (1994).

    PubMed  Google Scholar 

  4. Bartling D, Seedorf M, Mithöfer A, Weiler EW: Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone indole-3-acetic acid. Eur J Biochem 205: 417–424 (1992).

    Google Scholar 

  5. Bartling D, Seedorf M, Schmidt RC, Weiler EW: Molecular characterization of two cloned nitrilases from Arabidopsis thaliana, key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid. Proc Natl Acad Sci USA 91: 6021–6025 (1994).

    PubMed  Google Scholar 

  6. Block A, Dangl JL, Hahlbrock K, Schulze-Lefert P: Functional borders, genetic fine structure and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proc Natl Acad Sci USA 87: 5387–5391 (1990).

    PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  8. Brown JWS: Acatalogue of splice junction and putative branch point sequences from plant introns. Nucl Acids Res 14: 9549–9559 (1986).

    PubMed  Google Scholar 

  9. Bustos MM, Guiltinan MJ, Jordano J, Begum D, Kalkan FA, Hall TC: Regulation of β-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a french bean β-phaseolin gene. Plant Cell 1: 839–853 (1989).

    Article  PubMed  Google Scholar 

  10. Datta N, Cashmore AR: Binding of a pea nuclear protein to promotors of certain photoregulated genes is modulated by phosphorylation. Plant Cell 1: 1069–1077 (1989).

    Article  PubMed  Google Scholar 

  11. Donald RGK, Schindler U, Batschauer A, Cashmore AR: The plant G-box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G-box binding factor. EMBO J 9: 1727–1735 (1990).

    PubMed  Google Scholar 

  12. Ellis JG, Llewellyn DJ, Walker JC, Dennis ES, Peacock WJ: The ocs element: a 16 base pair palindrome essential for activity of the octopine synthase enhancer. EMBO J 11: 3203–3208 (1987).

    Google Scholar 

  13. Fenwick GR, Heaney RK, Mullin WJ: Glucosinolates and their breakdown products in food and food plants. Crit Rev Food Sci Nutr 18: 123–301 (1983).

    PubMed  Google Scholar 

  14. Foster R, Izawa T, Chua NH: Plant bZIP proteins gather at ACGT elements. FASEB J 8: 192–200 (1994).

    PubMed  Google Scholar 

  15. Garbers C, Simmons C: Approaches to understandig auxin action. Trends Cell Biol 4: 242–249 (1994).

    Google Scholar 

  16. Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR: An evolutionary conserved protein binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci USA 85: 7089–7093 (1988).

    PubMed  Google Scholar 

  17. Goldberg RB: Regulation of plant gene expression. Phil Trans R Soc Lond B 314: 343–353 (1986).

    Google Scholar 

  18. Goldberg RB, Barker SJ, Perez-Grau L: Regulation of gene expression during plant embryogenesis. Cell 56: 149–160 (1989).

    Article  PubMed  Google Scholar 

  19. Goldsmith MHM: The transport of auxin. Annu Rev Plant Physiol 19: 347–360 (1968).

    Google Scholar 

  20. Goodall GJ, Filipowicz W: The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58: 473–483 (1989).

    Article  PubMed  Google Scholar 

  21. Green PJ, Kay SA, Chua NH: Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene. EMBO J 6: 2543–2549 (1987).

    PubMed  Google Scholar 

  22. Guiltinan MJ, Marcotte WRJ, Quatrano RS: A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250: 267–271 (1990).

    PubMed  Google Scholar 

  23. Hillebrand H, Tiemann B, Hell R, Bartling D, Weiler EW: Structure of the gene encoding nitrilase 1 from Arabidopsis thaliana. Gene 170: 197–200 (1996).

    PubMed  Google Scholar 

  24. Hobbie L, Estelle M: Genetic approaches to auxin action. Plant Cell Environ 17: 525–540 (1994).

    PubMed  Google Scholar 

  25. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transfering genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

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

    PubMed  Google Scholar 

  27. Katagiri F, Lam E, Chua NH: Two tobacco DNA-binding proteins with homology to the nuclear factor CREB. Nature 340: 727–730 (1989).

    Article  PubMed  Google Scholar 

  28. Kim S-R, Kim Y, An G: Identification ofmethyl jasmonate and salicylic acid response elements from the nopaline synthase (nos) promoter. Plant Physiol 103: 97–103 (1993).

    Article  PubMed  Google Scholar 

  29. Koncz C, Schell J: The promotor of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383–396 (1986).

    Article  Google Scholar 

  30. Lohmer S, Maddaloni M, Motto M, Di Fonzo N, Hartings H, Salamini F, Thompson RD: The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J 10: 617–624 (1991).

    PubMed  Google Scholar 

  31. Lois R, Dietrich A, Hahlbrock K, Schulz W: A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J 8: 1641–1648 (1989).

    PubMed  Google Scholar 

  32. Manzara T, Gruissem W: Organization and expression of the genes encoding ribulose-1,5-bisphosphate carboxylase in higher plants. Photosynth Res 16: 117–130 (1988).

    Google Scholar 

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

    Google Scholar 

  34. Normanly J, Cohen JD, Fink GR: Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid. Proc Natl Acad Sci USA 90: 10355–10359 (1993).

    PubMed  Google Scholar 

  35. Normanly J, Slovin JP, Cohen JD: Rethinking auxin biosynthesis and metabolism. Plant Physiol 107: 323–329 (1995).

    PubMed  Google Scholar 

  36. Oeda K, Salinas J, Chua NH: A tobacco bZIP transcription activator (TAF-1) binds to a G-box-like motif conserved in plant genes. EMBO J 10: 1793–1802 (1991).

    PubMed  Google Scholar 

  37. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, (2nd ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  38. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA74: 5463–5467 (1977).

    PubMed  Google Scholar 

  39. Schindler U, Beckmann H, Cashmore AR: TGA1 and G-box binding factors: Two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG. Plant Cell 4: 1309–1319 (1992).

    Article  PubMed  Google Scholar 

  40. Schmidt RJ, Burr FA, Aukermann MJ, Burr B: Maize regulatory gene opaque-2 encodes a protein with a ‘leucine-zipper’ motif that binds to zein DNA. Proc Natl Acad Sci USA 87: 46–50 (1990).

    PubMed  Google Scholar 

  41. Schmidt RC, Müller A, Hain R, Bartling D, Weiler EW: Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme. Plant J 9: 683–691 (1996).

    PubMed  Google Scholar 

  42. Sembdner G, Gross D, Liebisch HW, Schneider G: Molecular aspects of plant hormones. In: McMillan (ed) Hormonal Regulation of Development I, pp. 281–444. Springer-Verlag, Berlin (1981).

    Google Scholar 

  43. Thimann KV, Skoog F: On the inhibition of bud development and other functions of growth substances in Vicia faba. Proc R Soc B 114: 317–339 (1934).

    Google Scholar 

  44. Thimann KV: Hormone action in the whole life of plants. University of Massachusettes Press (1977).

  45. Tsunoda H, Yamaguchi K: The cDNA sequence of an auxinproducing nitrilase homologue in tobacco. Plant Physiol 109: 339 (1995).

    Google Scholar 

  46. Weisshaar B, Armstrong GA, Block A, da Costa e Silva O, Hahlbrock K: Light-inducible and constitutively expressed DNA-binding proteins recognizing a plant promoter element with functional relevance in light responsiveness. EMBO J 10: 1777–1786 (1991).

    PubMed  Google Scholar 

  47. Went FW, Thimann KW: Phytohormones. McMillan, New York (1937).

    Google Scholar 

  48. Wetmore RH, Jacobs WP: Studies on abscission; the inhibiting effect of auxin. Amer J Bot 40: 272–276 (1953).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Ruhr-Universität Bochum, 44780, Bochum, Germany

    Helke Hillebrand, Dieter Bartling & Elmar W. Weiler

Authors
  1. Helke Hillebrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dieter Bartling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elmar W. Weiler
    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

Hillebrand, H., Bartling, D. & Weiler, E.W. Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana. Plant Mol Biol 36, 89–99 (1998). https://doi.org/10.1023/A:1005998918418

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1005998918418

Search

Navigation