Glucosinolates (GSLs) are nitrogen- and sulfur-containing secondary metabolites derived from amino acids that protect plants from pests and in some plants are involved in auxin biosynthesis (Agerbirk et al., 2009; Bender and Celenza, 2009; Hopkins et al., 2009). From a human perspective, GSLs are important as contributors of flavor to certain foods, as cancer prevention agents, and as biofumigants (Traka and Mithen, 2009). Because of the economic importance of GSLs, their biosynthetic pathways have been intensively studied using molecular genetics and systems biology approaches in Arabidopsis (Hirai, 2009; Albinsky et al., 2010; Sønderby et al., 2010).
In Arabidopsis, approximately 40 GSLs have been identified, most of which are derived from methionine (Met) and tryptophan (Trp) (Hogge et al., 1988; Kliebenstein et al., 2001a, 2007; Reichelt et al., 2002). In some Met-derived GSLs, the R-group is hydroxylated by the 2-oxo acid-dependent dioxygenase AOP3 (Kliebenstein et al., 2001b). In Arabidopsis Col-0, the two resulting hydroxylated GSLs (OH-GSLs), 3-hydroxypropyl GSL (3OHP) and 4-hydroxybutyl GSL (4OHB), are further benzoylated to give 3-benzoyloxypropyl GSL (3BZO) and 4-benzoyloxybutyl GSL (4BZO) or sinapoylated to give the corresponding 3-sinapoyloxypropyl GSL (3SIN) and 4-sinapoyloxybutyl GSL (4SIN) (Hogge et al., 1988; Kliebenstein et al., 2007). In addition, similar benzoylated GSLs (BzGSLs) with longer chain lengths, 5-benzoyloxypentyl GSL and 6-benzoyloxyhexyl GSL, have been detected, but the corresponding OH-GSLs and sinapoylated GSLs (SnGSLs) have not (Hogge et al., 1988). Further, two additional types of BzGSLs have been identified, i.e. BzGSLs in which benzoylation occurs on the glucose ring of GSL core structure (Reichelt et al., 2002), and 2-benzoyloxybut-3-enyl GSL, the only Arabidopsis GSL in which hydroxylation and benzoylation occur after alkenylation (Kliebenstein et al., 2001b). 3BZO and 4BZO accumulate abundantly in Col-0 seeds, but 2-benzoyloxybut-3-enyl GSL has not been detected in this ecotype and the amounts of other BzGSLs are small. Therefore, most studies that have been performed have focused on the changes of 3BZO or 4BZO, and hereafter the term BzGSLs refers these two BzGSLs only.
Previous isotope-labeling experiments together with genetic and biochemical studies in various plant species have identified two major biosynthetic routes to benzoate (BA), the β-oxidative and non-β-oxidative pathways, both of which begin with phenylalanine (Phe) (Ribnicky et al., 1998; Abd El-Mawla and Beerhues, 2002; Orlova et al., 2006). In the β-oxidative pathway, Phe is converted into cinnamate, which is in turn activated to its corresponding CoA thioester (Ribnicky et al., 1998; Beuerle and Pichersky, 2002). Cinnamoyl CoA is then converted to benzoyl CoA by a series of side chain-shortening reactions shared with fatty acid degradation (Hertweck et al., 2001). Finally, BA is released from benzoyl CoA by a thioesterase (Van Moerkercke et al., 2009). In the non-β-oxidative pathway, benzaldehyde is synthesized first from cinnamate or cinnamoyl CoA and is then converted to BA by an aldehyde oxidase or benzaldehyde dehydrogenase (Schnitzler et al., 1992; Abd El-Mawla and Beerhues, 2002; Boatright et al., 2004). In Arabidopsis, BA biosynthesis has been studied mainly in the seeds because BzGSLs are abundant there and can be used as marker metabolites to measure BA biosynthesis activity. For example, BzGSLs decrease in the aao4 mutant and are barely detectable in the chy1 mutant. AAO4 displays aldehyde oxidase activity (Ibdah and Pichersky, 2009; Ibdah et al., 2009), and Chy1 has been suggested to be involved in β-oxidation (Ibdah and Pichersky, 2009).
Another Arabidopsis gene, BZO1, has been identified by forward genetics screening using BzGSLs as reporter molecules for BA metabolism. Kliebenstein et al. (2007) showed that Ler bzo1 mutants lack 3BZO and have elevated levels of 3OHP in their seeds. Further, enzymatic assays with recombinant BZO1 revealed that the protein is capable of forming benzoyl CoA from BA and coenzyme A. Although these findings implicate benzoyl CoA as the acyl donor in benzoylated GSL synthesis, the identities of the acyltransferases involved in the benzoylation and sinapoylation reactions have not yet been determined. The benzoyltransferase may belong to the BAHD family (named on the basis of the first letter of the names of the first four enzymes recognized as belonging to this family: BEAT (benzylalcohol O-acetyltransferase), AHCT (anthocyanin O-hydroxycinnamoyltransferase), HCBT (N-hydroxycinnamoyl/benzoyltransferase) and DAT (deacetylvindoline 4-O-acetyltransferase); St-Pierre and De Luca, 2000), because these enzymes use acyl CoAs as donors, and benzoyl CoA was shown to be the acyl donor in synthesis of benzylbenzoate and taxol (D’Auria et al., 2002; Walker et al., 2002; Boatright et al., 2004). An alternative to the BAHD acyltransferases are the serine carboxypeptidase-like (SCPL) acyltransferases, which use glucose esters such as sinapoylglucose (SG) as acyl donors to generate esters including sinapoylmalate, sinapoylcholine, 1,2-disinapoylglucose and sinapoylated anthocyanins (Lehfeldt et al., 2000; Shirley et al., 2001; Fraser et al., 2007). Recently it was reported that SCPL1 from oat is required to transfer a benzoyl group to des-acyl avenacins, compounds that contribute to broad-spectrum disease resistance in this species (Mugford et al., 2009).
To identify the enzyme(s) required to acylate OH-GSLs, we targeted these two representative acyltransferase families that can potentially utilize activated forms of BA and sinapate as acyl donors. Here we report that the acyltransferase responsible for formation of the benzoylated and sinapoylated GSLs in Arabidopsis seeds belongs to the SCPL family, and probably uses benzoylglucose (BG) and SG as substrates. We further show that the enzyme encoded by BZO1 is a cinnamoyl CoA ligase that catalyzes the formation of cinnamoyl CoA to provide the substrate for the pathway(s) by which this phenylpropanoid derivative is converted to BA.