Detection of an O-methyltransferase synthesising acetosyringone in methyl jasmonate-treated tobacco cell-suspensions cultures

Abstract

Acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) is a well-known and very effective inducer of the virulence genes of Agrobacterium tumefaciens but the precise pathway of its biosynthesis in plants is still unknown. We have used two tobacco cell lines, cultured in suspension and exhibiting different patterns of accumulation of acetosyringone in their culture medium upon treatment with methyl jasmonate, to study different steps of acetosyringone biosynthesis. In the two cell lines studied, treatment with 100μM methyl jasmonate triggered a rapid and transient increase in acetovanillone synthase activity followed by a progressive increase in S-adenosyl-L-methionine: 5-hydroxyacetovanillone 5-O-methyltransferase activity which paralleled the rise in acetosyringone concentration in the culture medium. This O-methyltransferase displayed Michaelis–Menten kinetics with an apparent Km value of 18μM for 5-hydroxyacetovanillone and its activity was magnesium-independent. Its molecular mass was estimated by gel permeation on an FPLC column and was found to be of ca. 81kDa. 5-Hydroxyacetovanillone was the best substrate among the different o-diphenolic compounds tested as methyl acceptors in the O-methyltransferase assay. No formation of 5-hydroxyacetovanillone could be detected in vitro from 5-hydroxyferuloyl-CoA and NAD in the extracts used to measure acetovanillone synthase activity, indicating that 5-hydroxyacetovanillone is probably formed by direct hydroxylation of acetovanillone rather than by β-oxidation of 5-hydroxyferulic acid. Taken together our results strongly support the hypothesis that acetosyringone biosynthesis in tobacco proceeds from feruloyl-CoA via acetovanillone and 5-hydroxyacetovanillone.