Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis

SUMMARYBenzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primarymetabolites to secondary products. Benzoic acid biosynthesis from L -phenylalanine requires shortening of thepropyl side chain by two carbons, which can occur via a b-oxidative pathway or a non-b-oxidative pathway,with benzaldehyde as a key intermediate. The non-b-oxidative route requires benzaldehyde dehydrogenase(BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identiﬁed an Antirrhinummajus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript proﬁling, biochemicalcharacterization of the puriﬁed recombinant protein, molecular homology modeling, in vivo stable isotopelabeling,andtransientexpressioninpetuniaﬂowersrevealthatBALDHiscapableofoxidizingbenzaldehydetoBA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in themitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.Keywords: benzoic acid, benzenoids, aldehyde dehydrogenase, metabolic engineering, ﬂoral volatiles,snapdragon.INTRODUCTIONBenzoic acid (BA) is a weak acid with anti-microbial prop-erties mediated in part by the inhibition of glycolysis inmicro-organisms (Krebs et al., 1983). Benzoic acid is pro-duced in many plants and animals, and is thus a naturalconstituent of numerous foods including milk products(Sieber et al., 1995). In plants, BA and its derivatives areimportant building blocks in a wide spectrum of com-pounds varying from primary metabolites [e.g. aromaticcytokinins (Werbrouck et al., 1996) and salicylic acid] tosecondary products with pharmacological activities such asthe anti-cancer agent taxol (Walker and Croteau, 2000) andthe local anesthetic cocaine (Bjorklund and Leete, 1992).They also play critical roles in plant survival and repro-ductive success in natural ecosystems. Plant–insect andplant–plant interactions are often mediated by volatilebenzenoids (Qualley and Dudareva, 2008), which are highlyrepresented within volatile natural products (Knudsen andGershenzon, 2006). Benzenoids emitted from many fruitscontribute to the overall aroma and taste, which are valu-able attributes for consumers and animal seed dispersers(Schwab et al., 2008; Negre-Zakharov et al., 2009). Inaddition, defensive non-volatile compounds, such asbenzoyloxyglucosinolates in cruciferous plants, also con-tain the benzoyl moiety derived from BA (Graser et al.,2001; Kliebenstein et al., 2007).Despite the simple structure of BA, its widespreaddistribution and its importance for the plant life cycle, thebiochemical pathways leading to its formation remainlargely unknown. Benzoic acid biosynthesis has attractedthe attention of scientists for nearly half a century (Ibrahimand Towers, 1959), mainly due to its involvement insynthesis of the ubiquitous plant hormone salicylic acid(2-hydroxy BA). The limited understanding of these path-ways is the result of the existence of multiple routes withina plant or between plant species, and an inability to trackpathway intermediates. Recently, it has been shown thatpathogen-infected Arabidopsis (Wildermuth et al., 2001)and Nicotiana benthamiana (Catinot et al., 2008) are ableto synthesize salicylic acid predominantly from isochoris-mate without BA involvement. However, a soluble mono-