PLANT PHYSIOLOGY , Vol 111, Issue 3 831-837, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L
L. Du and B. A. Halkier
Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark
An in vitro system that converts phenylalanine to phenylacetaldoxime in the
biosynthesis of the glucosinolate glucotropaeolin has been established in
seedlings of Tropaeolum majus L. exposed to the combined treatment of
jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold
induction, compared with untreated, dark-grown seedlings, of de novo
biosynthesis measured as incorporation of radioactively labeled
phenylalanine into glucotropaeolin. Formation of the inhibitory degradation
product benzylisothiocyanate during tissue homogenization was prevented by
inactivation of the thioglucosidase myrosinase by addition of 100 mM
ascorbic acid to the isolation buffer. This allowed the isolation of a
biosynthetically active microsomal preparation from the induced T. majus
plant material. The enzyme, which catalyzes the conversion of phenylalanine
to the corresponding oxime, was sensitive to cytochrome P450 inhibitors,
indicating the involvement of a cytochrome P450 in the biosynthetic
pathway. It has previously been shown that the oxime-producing enzyme in
the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is
dependent on cytochrome P450, whereas the oxime-producing enzymes in
Brassica species have been suggested to be flavin monooxygenases or
peroxidase-type enzymes. The result with T. majus provides additional
experimental documentation for a similarity between the enzymes converting
amino acids into the corresponding oximes in the biosynthesis of
glucosinolates and cyanogenic glucosides.
