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Studies on plant secondary metabolites influencing pest behaviour.
Synthesis of both [14C-1] labelled and unlabelled non-protein amino acids has allowed investigation of the early steps of glucosinolate biosynthetic pathway in Brassica napus. Use of the [14C-1]amino acids has allowed limited characterisation of the enzymes involved in the initial oxidative decarboxylation reaction. The intermediates between the amino acid and the aldoxime have been studied by synthesis of a range of potential intermediates. Synthesis of [2H-2]homophenylalanine has allowed preliminary investigations into the intermediates by examination of the retention or loss of the label in the aldoxime product. The use of 19F NMR as a technique to examine the intermediates as they are formed in situ has been investigated with synthesis of a range of fluorine substituted amino acids and aldoximes. Synthesis of a range of unlabelled and [14C-1] methionine homologues has allowed investigation of the enzymes involved in aliphatic glucosinolate biosynthesis. The synthesis of a range of dihomomethionine analogues, and subsequent competition assays have outlined the substrate specificity of the aliphatic enzyme, and allowed the development of a crude active site model.The biosynthesis of thiohydroximates from aldoximes has been investigated, by examining the C-S bond cleavage which takes place during thiohydroximate formation. Investigation of these enzymes required the synthesis of the proposed intermediate, a cysteine thiohydroximate conjugate. This was accomplished using nitrile oxide methodology.Use of regiospecific deuteration has allowed the synthesis of deuterated homophenylalanines, which will allow further investigation of the pathway. [14C-1]Dihomomethionine has been synthesised to investigate the possibility of its incorporation into 2-propenylglucosinolate
CYP83B1 Is the Oxime-metabolizing Enzyme in the Glucosinolate Pathway in \u3ci\u3eArabidopsis\u3c/i\u3e
CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450 monooxygenase. We searched the genome of A. thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides. By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli. The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a “P450mox” that converted oximes into toxic compounds that the plant detoxified into glucosinolates
Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana : recombinant expression and characterization of methylthioalkylmalate synthase, the condensing enzyme of the chain-elongation cycle
New roles for cis-jasmone as an insect semiochemical and in plant defense
cis-Jasmone, or (Z)-jasmone, is well known as a component of plant volatiles, and its release can be induced by damage, for example during insect herbivory. Using the olfactory system of the lettuce aphid to investigate volatiles from plants avoided by this insect, (Z)-jasmone was found to be electrophysiologically active and also to be repellent in laboratory choice tests. In field studies, repellency from traps was demonstrated for the damson-hop aphid, and with cereal aphids numbers were reduced in plots of winter wheat treated with (Z)-jasmone. In contrast, attractant activity was found in laboratory and wind tunnel tests for insects acting antagonistically to aphids, namely the seven-spot ladybird and an aphid parasitoid. When applied in the vapor phase to intact bean plants, (Z)-jasmone induced the production of volatile compounds, including the monoterpene (E)-β-ocimene, which affect plant defense, for example by stimulating the activity of parasitic insects. These plants were more attractive to the aphid parasitoid in the wind tunnel when tested 48 h after exposure to (Z)-jasmone had ceased. This possible signaling role of (Z)-jasmone is qualitatively different from that of the biosynthetically related methyl jasmonate and gives a long-lasting effect after removal of the stimulus. Differential display was used to compare mRNA populations in bean leaves exposed to the vapor of (Z)-jasmone and methyl jasmonate. One differentially displayed fragment was cloned and shown by Northern blotting to be up-regulated in leaf tissue by (Z)-jasmone. This sequence was identified by homology as being derived from a gene encoding an α-tubulin isoform