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

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxygenases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis. Until recently F5H has been thought to catalyze the hydroxylation of ferulate to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derived ester in the seeds, is antinutritional and a target for elimination in canola meal. We have isolated three F5H-like genes (BNF5H1-3) from a cultivated Brassica napus, whose amphidiploid progenitor is considered to have arisen from a fusion of the diploids Brassica rapa and Brassica oleracea. Two cultivated varieties of the diploids were also found to contain BNF5H3 and additionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >90% 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 maximal F5H expression in the stems, least in the seeds, and subtle differences 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 (Liquidamber styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde or 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.