Skip to main content
SpringerLink
Log in

A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1, and three new site-directed mutants, omr1-5, omr1-7, and omr1-8with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7, appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5, omr1-7, and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5, omr1-7, and omr1-8, proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.

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

  • Bechtold, N., Ellis, J. and Pelletier, G. 1993. In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris Life Sci. 316: 1194–1199.

    Google Scholar 

  • Bent, A.F., Kunkel, B.N., Dahlbeck, D., Brown, K.L., Schmidt, R., Giraudat, J., Leung, J. and Staskawicz, B.J. 1994. RPS2 of Arabidopsis thaliana: A leucine-rich repeat class of plant disease resistance genes. Science 265: 1856–1860.

    Google Scholar 

  • Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12: 8711–8721.

    Google Scholar 

  • Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the method of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

  • Brears, T., Liu, C., Knight, T.J. and Coruzzi G.M. 1993. Ectopic overexpression of asparagine synthetase in transgenic tobacco. Plant Physiol. 103: 1285–1290.

    Google Scholar 

  • Bryan, J.K. (1990) Advances in the biochemistry of amino acid biosynthesis. In: B.J. Miflin, P.J. Lea (Eds.), The Biochemistry of Plants, Vol 16. Academic Press, New York, pp. 161–195.

    Google Scholar 

  • Chinchilla, D., Schwarz, F.P. and Eisenstein, E. 1998. Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. J. Biol. Chem. 273: 23219–23224.

    Google Scholar 

  • Dougall, D.K. 1970. Threonine deaminase from Paul's Scarlet Rose tissue cultures. Phytochemistry 9: 959–964.

    Google Scholar 

  • Ebmeier, A., Allison, L., Cerutti, H. and Clemente, T. 2004. Evaluation pf the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218: 751–758.

    Google Scholar 

  • Eisenstein, E. 1991. Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli. J. Biol. Chem. 266: 5801–5807.

    Google Scholar 

  • Fisher, K.E. and Eisenstein, E. 1993. An efficient approach to identify ilvA mutations reveals amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli. J. Bacteriol. 175: 6605–6613.

    Google Scholar 

  • Gallagher, D.T., Gilliland, G.L., Xiao, G., Zondlo, J., Fisher, K.E., Chinchilla, D. and Eisenstein, E. 1998. Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 6: 465–475.

    Google Scholar 

  • Guex, N. and Peitsch, M.C. 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis 18: 2714–2723.

    Google Scholar 

  • Guillouet, S., Rodal, A.A., An, G.-H., Lessard, P.A. and Sinskey, A.J. 1999. Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl. Environ. Microbiol. 65: 3100–3107.

    Google Scholar 

  • Halgand, F., Wessel, P.M., Laprevote, O. and Dumas, R. 2002. Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine. Biochemistry 41: 13767–13773.

    Google Scholar 

  • Hashiguchi, K., Kojima, H., Sato, K. and Sano, K. 1997. Effects of an Escherichia coli ilvA mutant gene encoding feedback-resistant threonine deaminase on L-isoleucine production by Brevibacterium flavum. Biosci. Biotechnol. Biochem. 61: 105–108.

    Google Scholar 

  • Haughn, G.W. and Somerville, C. 1986. Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol. Gen. Genet. 204: 430–434.

    Google Scholar 

  • Hildmann, T., Ebneth, M., Pena-Cortes, H., Sanchez-Serrano, J.J., Willmitzer, L. and Prat, S. 1992. General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding. Plant Cell 4: 1157–1170.

    Google Scholar 

  • Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.

    Google Scholar 

  • John, S.J., Srivastava, V. and Guha-Mukherjee, S. 1995. Cloning and sequencing of chickpea cDNA coding for threonine deaminase. Plant Physiol. 107: 1023–1024.

    Google Scholar 

  • Kielland-Brandt, M.C., Holmberg, S., Petersen, J.G.L. and Nilsson-Tillgren, T. 1984. Nucleotide sequence of the gene for threonine deaminase (ILV1) of Saccharomyces cerevisiae. Carlsberg Res. Commun. 49: 567–575.

    Google Scholar 

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

    Google Scholar 

  • Lawther, R.P., Wek, R.C., Lopes, J.M., Pereira, R., Taillon, B.E. and Hatfield, G.W. 1987. The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12. Nucl. Acids Res. 15: 2137–2155.

    Google Scholar 

  • Monod, J., Wyman, J. and Changeux, J.-P. 1965. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12: 88–118.

    Google Scholar 

  • Mourad, G., Emerick, R., Marion, A. and Smith, A. 1998. Cloning and sequencing of a cDNA encoding threonine dehydratase/deaminase of Arabidopsis thaliana (Accession No. AF096281) (PGR98–199). Plant Physiol. 118: 1534.

    Google Scholar 

  • Mourad, G., Emerick, R. and Smith, A. 2000. Molecular cloning and sequencing of a cDNA encoding an isoleucine feedback insensitive threonine dehydratase/deaminase of Arabidopsis thaliana line GM11b (Accession No. AF177212) (PGR00–020). Plant Physiol. 122: 619.

    Google Scholar 

  • Mourad, G. and King, J. 1995. L-O-Methylthreonine-resistant mutant of Arabidopsis defective in isoleucine feedback regulation. Plant Physiol. 107: 43–52.

    Google Scholar 

  • Mourad, G., Pandey, B. and King, J. 1993. Isolation and genetic analysis of a triazolopyrimidine-resistant mutant of Arabidopsis. J. Hered. 84: 91–96.

    Google Scholar 

  • Nicholas, K.B. and Nicholas, H.B. 1997. GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author. http:www.psc.edu/biomed/gendoc

  • Noctor, G., Arisi, A.-C.M., Jouanin, L. and Foyer, C.H. 1998. Manipulation of glutathione and amino acid biosynthesis in the chloroplast. Plant Physiol. 118: 471–482.

    Google Scholar 

  • Peitsch, M.C. 1995. Protein modeling by E-mail. Bio Technol. 13: 658–660.

    Google Scholar 

  • Peitsch, M.C. 1996. ProMod and Swiss-model: Internet-based tools for automated comparative protein modelling. Biochem. Soc. Trans. 24: 274–279.

    Google Scholar 

  • Rhodes, D. and Wood, B. 2003. Amino acid and onium compound extraction. Department of Horticulture and Landscape Architecture, Purdue University. http://www. hort.purdue.edu/rhodcr/hort64oc/method:/me00001.htm.

  • Samach, A., Hareven, D., Gutfinger, T., Ken-Dror, S. and Lifschitz, E. 1991. Biosynthetic threonine deaminase gene of tomato: Isolation, structure, and upregulation in floral organs. Proc. Natl. Acad. Sci. USA 88: 2678–2682.

    Google Scholar 

  • Schuller, D.J., Grant, G.A. and Banaszak, L.J. 1995. The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase. Nat. Struct. Biol. 2: 69–76.

    Google Scholar 

  • Sharma, R.K. and Mazumder, R. 1970. Purification, properties, and feedback control of L-threonine dehydratase from spinach. J. Biol. Chem. 245: 3008–3014.

    Google Scholar 

  • Shaul, O. and Galili, G. 1992a. Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydropicolinate synthase in their chloroplasts. Plant J. 2: 203–209.

    Google Scholar 

  • Shaul, O. and Galili, G. 1992b. Threonine overproduction in transgenic tobacco plants expressing a mutant desensitized aspartate kinase of Escherichia coli. Plant Physiol. 100: 1157–1163.

    Google Scholar 

  • Shintani, D. and DellaPenna, D. 1998. Elevating the Vitamin E content of plants through metabolic engineering. Science 282: 2098–2100.

    Google Scholar 

  • Singh, B.K. and Shaner, D.L. 1995. Biosynthesis of branched chain amino acids: From test tube to field. Plant Cell 7: 935–944.

    Google Scholar 

  • Slater, S., Mitsky, T.A., Houmiel, K.L., Hao, M., Reiser, S.E., Taylor, N.B., Tran, M., Valentin, H.E., Rodriguez, D.J., Stone, D.A., Padgette, S.R., Kishore, G. and Gruys, K.J. 1999. Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production. Nat. Biotechnol. 17: 1011–1016.

    Google Scholar 

  • Taillon, B.E., Little, R. and Lawther, R.P. 1988. Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase. Gene 63: 245–252.

    Google Scholar 

  • Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25: 4876–4882.

    Google Scholar 

  • Traber, M.G. and Sies, H. 1996. Vitamin E in humans: Demand and delivery. Annu. Rev. Nutr. 16: 321–347.

    Google Scholar 

  • Umbarger, H.E. 1956. Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine. Science 123: 848.

    Google Scholar 

  • Umbarger, H.E. 1978. Amino acid biosynthesis and its regulation. Annu. Rev. Biochem. 47: 533–606.

    Google Scholar 

  • Wessel, P.M., Graciet, E., Douce, R. and Dumas, R. 2000. Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments. Biochemistry 39: 15136–15143.

    Google Scholar 

  • Zhu, X. and Galili, G. 2003. Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds. Plant Cell 15: 845–853.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Indiana-Purdue University, 2101 East Coliseum Blvd, IN, USA

    Eric L. Garcia & George S. Mourad

Authors
  1. Eric L. Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. George S. Mourad
    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

Garcia, E.L., Mourad, G.S. A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker. Plant Mol Biol 55, 121–134 (2004). https://doi.org/10.1007/s11103-004-0500-z

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-004-0500-z

Search

Navigation