Plant Molecular Biology

, Volume 23, Issue 4, pp 759–768

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase

  • Orit Shaul
  • Gad Galili
Research Articles


The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.

Key words

apical dominance aspartate kinase dihydrodipicolinate synthase flowering lysine and threonine overproduction transgenic plants 


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  1. 1.
    Bieleski, RL, Turner, NA: Separation and estimation of amino-acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal Biochem 17: 278–293 (1966).PubMedGoogle Scholar
  2. 2.
    Black, S, Wright, NG: β-aspartokinase and β-aspartyl phosphate. J Biol Chem 213: 27–38 (1955).PubMedGoogle Scholar
  3. 3.
    Boy, E, Borne, F, Patte, JC: Isolation and identification of mutants constitutive for aspartokinase III synthesis in Escherichia coli K12. Biochimie 61: 1151–1160 (1979).PubMedGoogle Scholar
  4. 4.
    Bright, SWJ, Kueh, JSH, Franklin, J, Rognes, SE, Miflin, BJ: Two genes for threonine accumulation in barley seeds. Nature 299: 278–279 (1982).Google Scholar
  5. 5.
    Bright, SWJ, Shewry, PR: Improvement of protein quality in cereals. CRC Crit Rev Plant Sci 1: 49–93 (1983).Google Scholar
  6. 6.
    Bryan, JK: Synthesis of the aspartate family and branched-chain amino acids. In: Miflin, BJ (ed) The Biochemistry of Plants, vol. 5, pp. 403–452. Academic Press, New York (1980).Google Scholar
  7. 7.
    Cassan, M, Parsot, C, Cohen, GN, Patte, JC: Nucleotide sequence of lysC gene encoding the lysine-sensitive Aspartokinase III of Escherichia coli K12. J Biol Chem 261: 1052–1057 (1986).PubMedGoogle Scholar
  8. 8.
    Cattoir-Reynaerts, A, Degryse, E, Verbruggen, I, Jacobs, M: Selection and characterization of carrot embryoid cultures resistant to inhibition by lysine and threonine. Biochem Physiol Pfl 178: 81–90 (1983).Google Scholar
  9. 9.
    Dotson, SB, Frisch, DA, Somers, DA, Gengenbach, BG: Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize. Planta 182: 546–552 (1990).Google Scholar
  10. 10.
    Duncan, DB: Multiple range and multiple F tests. Biometrics 11: 1–42 (1955).Google Scholar
  11. 11.
    Fluhr, R, Moses, P, Morelli, G, Coruzzi, G, Chua, N-H: Expression dynamics of the pea rbcS family and organ distribution of the transcripts. EMBO J 5: 2063–2071 (1986).Google Scholar
  12. 12.
    Frankard, V, Ghislain, M, Jacobs, M: Two feedback-insensitive enzymes of the spartate pathway in Nicotiana sylvestris. Plant Physiol 99: 1285–1293 (1992).Google Scholar
  13. 13.
    Frankard, V, Ghislain, M, Negrutiu, I, Jacobs, M: High threonine producer mutant of Nicotiana sylvestris (Spegg. and Comes). Theor Appl Genet 82: 273–282 (1991).Google Scholar
  14. 14.
    Fuller, MF, Mennie, I, Crofts, RMJ: The amino acid supplementation of barley for the growing pig. 2. Optimal additions of lysine and threonine for growth. Br J Nutr 41: 333–340 (1979).PubMedGoogle Scholar
  15. 15.
    Gallie, DR, Lucas, WJ, Walbot, V: Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311 (1989).CrossRefPubMedGoogle Scholar
  16. 16.
    Greve, HD, Dhaese, P, Seurinck, J, Lemmers, M, VanMontagu, M, Schell, J: Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1: 499–511 (1983).Google Scholar
  17. 17.
    Guilley, H, Dudley, RK, Jonard, G, Balazs, E, Richards, KE: Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30: 763–773 (1982).CrossRefPubMedGoogle Scholar
  18. 18.
    Hibberd, KA, Walter, T, Green, CE, Gengenbach, BG: Selection and characterization of feedback-insensitive tissue culture of maize. Planta 148: 183–187 (1980).Google Scholar
  19. 19.
    Hurst, WJ: o-phthaldehyde derivative of amino acids in cocoa beans. In: Hancock, WS (ed) CRC Handbook of HPLC for the Separation of Amino Acids, Peptides and Proteins, vol. 1, pp. 325–330. CRC Press, Boca Raton, FL (1984).Google Scholar
  20. 20.
    Matthews, PF, Farrar, MJ, Gray, AC: Purification and interconversion of homoserine dehydrogenase from Daucus carota cell suspension cultures. Plant Physiol 91: 1569–1574 (1989).Google Scholar
  21. 21.
    Matthews, BF, Widholm, JM: Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots. Planta 141: 315–321 (1978).Google Scholar
  22. 22.
    Mills, WR, Lea, PJ, Miflin, BJ: Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts. Plant Physiol 65: 1166–1172 (1980).Google Scholar
  23. 23.
    Negrutiu, I, Cattoir-Reynearts, A, Verbruggen, I, Jacobs, M: Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes). Theor Appl Genet 68: 11–20 (1984).CrossRefGoogle Scholar
  24. 24.
    Nitsch, JP: Experimental androgenesis in Nicotiana. Phytomorphology 19: 389–404 (1969).Google Scholar
  25. 25.
    Odell, JT, Nagy, F, Chua, N-H: Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).PubMedGoogle Scholar
  26. 26.
    Richaud, F, Richaud, C, Rafet, P, Patte, JC: Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene. J Bact 166: 297–300 (1986).PubMedGoogle Scholar
  27. 27.
    Shaul, O, Galili, G: Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts. Plant J 2: 203–209 (1992).Google Scholar
  28. 28.
    Shaul, O, Galili, G: Threonine overproduction in transgenic tobacco plants expressing a mutant desensitized aspartate kinase of Escherichia coli. Plant Physiol 100: 1157–1163 (1992).Google Scholar
  29. 29.
    Umbarger, HE: Amino acid biosynthesis and its regulation. Annu Rev Biochem 47: 533–606 (1978).CrossRefGoogle Scholar
  30. 30.
    Yugari, Y, Gilvarg, C: The condensation step in diaminopimelate synthesis. J Biol Chem 240: 4710–4716 (1965).PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Orit Shaul
    • 1
  • Gad Galili
    • 1
  1. 1.Department of Plant GeneticsThe Weizmann Institute of ScienceRehovotIsrael

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