Abstract
Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer. Glucosinolates are derived from amino acids and have aldoximes as intermediates. We report that cytochrome P450 CYP79F1 catalyzes aldoxime formation in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A. thaliana, i.e. 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp. Transgenic A. thaliana with cosuppression of CYP79F1 have a reduced content of aliphatic glucosinolates and a highly increased level of dihomomethionine and trihomomethionine. The transgenic plants have a morphological phenotype showing loss of apical dominance and formation of multiple axillary shoots. Our data provide the first evidence that a cytochrome P450 catalyzes the N-hydroxylation of chain-elongated methionine homologues to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates.
Highlights
Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer
Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A. thaliana, i.e. 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp
We report the identification of a cytochrome P450 of the CYP79 family, CYP79F1, which catalyzes the conversion of dihomomethionine and trihomomethionine to 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively
Summary
Plant Material—A. thaliana ecotype Columbia was used for all experiments. The plants were grown in a controlled-environment Arabidopsis Chamber (Ar-60 I, Percival, Boone, IA) at a photosynthetic flux of 100 –200 mol photons mϪ2 sϪ1, 20 °C, and 70% humidity. For GC-MS analysis, 450 l of reaction mixture containing 3.3 mM L-methionine (Sigma), 3.3 mM DL-dihomomethionine or 3.3 mM DL-trihomomethionine, respectively, were incubated for 4 h at 25 °C and extracted with a total volume of 600 l CHCl3. The cDNA was synthesized in First Strand buffer (Life Technologies, Inc.) supplemented with 0.5 mM dNTPs, 10 mM dithiothreitol, 200 ng of random hexamers (Amersham Pharmacia Biotech), 3 pg of control RNA (internal standard), and 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.) in a total volume of 20 l. PCR analysis of the internal standard showed that the RT reactions ran with the same efficiency in samples prepared with different amounts of RNA isolated from different plant tissues
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