Identification of a CYP84 family of cytochrome P450-dependent mono-oxygenase genes in Brassica napus and perturbation of their expression for engineering sinapine reduction in the seeds

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxy genases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis . Until recently F5H has been thought to catalyze the hydroxylation of feru late to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derive d ester in the seeds, is antinutritional and a target for elimination in ca nola meal. We have isolated three FSH-like genes (BNFSH1-3) from a cultivat ed Brassica napus, whose amphidiploid progenitor is considered to have aris en from a fusion of the diploids Brassica rapa and Brassica oleracea. Two c ultivated varieties of the diploids were also found to contain BNF5H3 and a dditionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >9 0% identical in their coding sequence, BNF5H1 and BNF5H2 are closer to each other than to BNF5H3. This and additional data suggest that the two groups of genes have diverged in an ancestor of the diploids. B. napus showed max imal F5H expression in the stems, least in the seeds, and subtle difference s among the expression profiles of the three genes elsewhere. Transgenic B. napus with cauliflower mosaic virus 35S-antisense BNF5H contained up to 40 % less sinapine, from 9.0 +/- 0.3 mg in the controls to 5.3 +/- 0.3 mg g(-1 ) seed. F5H from Arabidopsis and a similar enzyme from sweetgum (Liquidambe r styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde o r 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.