Planta

, Volume 212, Issue 4, pp 612–618 | Cite as

Characterization of transgenic Arabidopsis thaliana with metabolically engineered high levels of p-hydroxybenzylglucosinolate

  • Bent Larsen Petersen
  • Erik Andréasson
  • Søren Bak
  • N. Agerbirk
  • Barbara Ann Halkier

Abstract.

The cytochrome P450 CYP79A1 catalyzes the conversion of l-tyrosine to p-hydroxyphenylacetaldoxime, the first step in the biosynthetic pathway of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. We have demonstrated that introduction of CYP79A1 into Arabidopsis thaliana (L.) Heynh. results in the production of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate (p-OHBG), not found in wild-type A. thaliana (Bak et al., 1999, Plant J. 20: 663–671). In the present study, glucosinolate profiles and contents in various tissues (roots, leaves, stems, closed flower buds and green siliques) of A. thaliana plants expressing CYP79A1 were analyzed by high-performance liquid chromatography. The total glucosinolate content in these tissues was increased 3.5- to 4.5-fold in comparison with the level of the control plants. The increase was due solely to the production of p-OHBG, as the composition of the major endogenous aliphatic and indole glucosinolates was not affected. Conversely, in mature seeds the total glucosinolate content of CYP79A1 and control plants was similar, with p-OHBG accounting for ca. 30%. The transcript level of the post-oxime enzyme UDP-glucose:thiohydroximate glucosyltransferase in leaves of CYP79A1 plants was increased ca. 50% compared with control plants, indicating that the post-oxime enzymes in the biosynthetic pathway are up-regulated. Western blot analysis and activity measurements showed similar amounts and activities of myrosinase in CYP79A1 and control plants. Thus, the increase in glucosinolate content in CYP79A1 plants was not accompanied by an increase in content or activity of degradation enzyme. The present data demonstrate that the high biosynthetic capacity of the post-oxime enzymes combined with a low substrate-specificity of the post-oxime enzymes in A. thaliana provide a highly flexible system for metabolic engineering of glucosinolate profiles, including new (non-endogenous) glucosinolates derived from oximes introduced into the plant, e.g. by transformation with CYP79 homologues.

Key words:Arabidopsis (glucosinolate) Cytochrome P450 Glucosinolate Metabolic engineering Myrosinase Transgenic Arabidopsis 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  • Bent Larsen Petersen
    • 1
  • Erik Andréasson
    • 2
  • Søren Bak
    • 1
  • N. Agerbirk
    • 3
  • Barbara Ann Halkier
    • 1
  1. 1.Department of Plant Biology, Center of Molecular Plant Physiology (PlaCe), Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, 1871 Frederiksberg C, Copenhagen, DenmarkDK
  2. 2.Department of Plant Biology, Box 7080 Swedish University of Agricultural Sciences, 5007 Uppsala, SwedenSE
  3. 3.Department of Chemistry, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, 1871 Frederiksberg C, Copenhagen, DenmarkDK

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