Mutants in the Biosynthesis of Amino Acids

  • M. Jacobs
  • V. Frankard
  • M. Ghislain
Part of the Current Plant Science and Biotechnology in Agriculture book series (PSBA, volume 8)

Abstract

Mutagenesis-selection procedures used in our laboratory resulted in the obtention of plants with modified regulatory mechanisms leading to a higher production of specific free amino acids. In particular, two mutants of the aspartate pathway in Nicotiana sylvestris were isolated following selection on growth inhibitory concentrations of lysine plus threonine, and on a lysine analog, S-(2-ammoethyl)-L- cysteine (AEC), respectively named RLT 70 and RAEC-l.The resistance property in bath cases was inherited as a monogenic dominant nuclear character. In RLT 70, up to 70% of the pool of free amino acids was made up of threonine, against 6% in the wild type plant. A completely desensitized aspartate kinase (AK) isoenzyme normally feedback inhibited by lysine was found to be the molecular basis of this overproduction. In RAEC-1, up to 25% of lysine could be found in the pool of free amino acids, compared to 1.5% in the wild type plant. The enzyme implicated in this mutant was dihydrodipicolinate synthase (DHDPS) totally desensitized to normal feedback inhibition by lysine. Evolution of the expression of both mutations during plant development revealed a peak of accumulation of the concerned amino acid, just before the elongation phase. In calli, overproduction was elevated and constant throughout the subcultures. Purification to homogeneity of the DHDPS subunit was achieved through a 2D polyacrylamide gel electrophoresis with a partially purified enzyme fraction. It was found to be a tetramer of four identical subunits, each of 39 kDa, and to be localized in the chloroplast. The purified protein was then microsequenced and the first 11 amino acids of the NH2-end were determined (collaboration with Dr.J.C. Guillemot, ELF Sanofi Bioresearch). Moreover the eluted protein was injected into rabbits to induce the production of anti-DHDPS antibodies. These polyclonal antibodies were used as probe to screen a cDNA library built in lambda gt11 from total poly A+ RNA fractions of N. Sylvestris. Several positive clones were isolated and are now being characterized.

Keywords

Free Amino Acid Total Free Amino Acid Lysine Content Aspartate Kinase Growth Inhibitory Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. (1).
    Bryan J K (1980), “Synthesis of the aspartate family and branched-chain amino acids”, in Miflin B J (ed.), The Biochemistry of Plants, Vol 5, Amino acids and derivatives, Academic Press, New York, pp 404–452.Google Scholar
  2. (2).
    Yamada Y, Kumpaisal R, Hashimoto T, Sugimoto Y, Suzuka Y (1986), “Growth and aspartate kinase activity in wheat cell suspension culture: effects of lysine analogs and aspartate-derived amino acids”, Plant Cell Physiol. 27: pp 607–617.Google Scholar
  3. (3).
    Giovanelli J, Mudd S H, Datko A (1989), “Aspartokinase of Lemna paucicostataHegelm. 6746”, Plant Physiol. 90: pp 1577–1583.PubMedCrossRefGoogle Scholar
  4. (4).
    Dotson S B, Somers D A, Gengenbach B G (1990),“Purification and characterization of lysine-sensitive aspartate kinase from maize cell cultures”, Planta (submitted).Google Scholar
  5. (5).
    Ghislain M, Frankard V, Jacobs M (1990),“Purification and characterization of dihydrodipicolinate synthase of Nicotiana sylvestris (Spegg. and Comes)”, Planta (in press).Google Scholar
  6. (6).
    Sainis, Mayne, Wallsgrove R, Lea P, Miflin B (1981), “Localisation and characterisation of homoserine deshydrogenase isolated from barley and pea leaves”, Planta 152: pp 491–496.CrossRefGoogle Scholar
  7. (7).
    Green C E, Phillips R L (1974), “Potential selection system for mutants with increased lysine, threonine, and methionine in cereal crops”, Crop Science 14: pp 827–830.CrossRefGoogle Scholar
  8. Cattoir-Reynaerts A, Degryse E, Jacobs M (1981), “Selection and analysis of mutants overproducing amino acids of the aspartate family in barley, Arabidopsis, and carrot”, in “Induced mutations as a tool for crop plant improvement”, IAEA-SM-251/28, Vienna, pp 58–63.Google Scholar
  9. (9).
    Negrutiu I, Jacobs M, Cattoir-Reynaerts A (1984), “Progress in cellular engineering of plants: biochemical and genetic assessment of selectable markers from cultured cells”, Plant Molec. Biol. 3: pp 289–302.CrossRefGoogle Scholar
  10. Jacobs M, Negrutiu I, Dirks R, Cammaerts D (1987), “Selection programmes for isolation and analysis of mutants in plant celle cultures”, in: Green C E, Somers D A, Hackett W P, Biesboer D D, (eds), Plant Biology, vol 3, Plant tissue and cell culture, Alan R Liss, New York, pp. 243–264.Google Scholar
  11. (11).
    Negrutiu I., Cattoir-Reynaerts A., Verbruggen I., Jacobs M. (1984), “Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris(Spegazzini and Comes)”, Theor. Appl. Genet 68, 11–20CrossRefGoogle Scholar
  12. (12).
    Kumpaisal R., Hashimoto T., Yamada Y. (1987), “Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures”, Plant Physiol. 85, 145–151PubMedCrossRefGoogle Scholar
  13. (13).
    Glassman, Barnes, and Ernst (1989), “Elevation of free lysine in plants by the introduction of a bacterial gene”, Dechema European Workshop, Bad Soden, Germany (Poster).Google Scholar
  14. (14).
    Hallings S.M., Stahly D.P. (1976) “Dihydrodipicolinic acid synthase of Bacillus licheniformis, quaternary structure, kinetics,’ and stability in the presence of sodium chloride and substrates”, B. B. A. 452, 580–596Google Scholar

Copyright information

© Kluwer Academic Publishers 1990

Authors and Affiliations

  • M. Jacobs
    • 1
  • V. Frankard
    • 1
  • M. Ghislain
    • 1
  1. 1.Laboratory of Plant GeneticsVrije Universiteit BrusselSint-Genesius RodeBelgium

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