SCPL17 is required for GSL acylation but the effects of sng2 on GSL biosynthesis may be indirect
Side-chain modifications contribute to the structural and functional diversity of GSLs. For example, benzoylation and sinapoylation reactions generate BzGSLs (approximately 15% of total GSLs) and SnGSLs (approximately 0.06% of total GSLs) from OH-GSLs (calculated based on quantified amounts of GSLs displayed in Figure 2a,b). Investigations of the biosynthetic pathway of these GSLs have focused on BzGSLs, not only because BzGSLs are relatively abundant, but also because BzGSLs are major BA-derived compounds that may be used to dissect BA biosynthesis. Feeding experiments with isotope-labeled precursors in Arabidopsis siliques showed that the benzoyl group and aliphatic portion of BzGSLs are derived from Phe and Met, respectively (Graser et al., 2001). Simultaneous labeling of 4-methylthiobutyl GSL (4MTB), 4-methylsulfinylbutyl GSL, 4OHB and 4BZO after feeding with [1,2-14C]desulfo-4MTB suggested that 4MTB is converted to 4BZO via 4-methylsulfinylbutyl GSL and 4OHB. This hypothetical pathway has been further elucidated by identifying the flavin-dependent mono-oxygenases that convert methylthio-GSLs into methylsulfinyl GSLs (Hansen et al., 2007; Li et al., 2008), and AOP3, which converts methylsulfinyl GSLs to OH-GSLs (Kliebenstein et al., 2001b). Despite these advances, the acyltransferase(s) that convert OH-GSLs to BzGSLs and SnGSLs has not been identified.
The BAHD and the SCPL acyltransferases are good candidate families to which the acyltransferase(s) in question may belong. On one hand, it has been suggested that BAHD acyltansferase(s) may synthesize BzGSLs and SnGSLs based upon biochemical precedents and the known involvement of BZO1 in BzGSL synthesis (Kliebenstein et al., 2007; Ibdah and Pichersky, 2009; Ibdah et al., 2009; Sønderby et al., 2010). On the other hand, there are a number of examples in which sinapoylated metabolites are synthesized in Arabidopsis from the activated sinapate donor SG through the action of an SCPL acyltransferase (Lehfeldt et al., 2000; Shirley et al., 2001; Fraser et al., 2007). Co-expression analyses suggested that one or more of the several SCPL genes expressed in developing seeds may mediate these reactions (Figure 1). Previous reports have shown that AOP3 and BZO1 are abundantly expressed in seeds compared to siliques, whereas other GSL biosynthesis genes working at earlier steps display the opposite pattern (Nour-Eldin and Halkier, 2009). This indicates that precursor GSLs are synthesized in maternal tissues and that side-chain modifications leading to BzGSL biosynthesis occur in seeds. Specifically, these reactions may occur in embryos, where seed GSLs are accumulated (Kliebenstein et al., 2007). Co-expression of SCPL genes with the two reference genes suggest that these genes may function with AOP3 and BZO1 in seed embryos.
Our SCPL-mediated acylation hypothesis was supported by the observation that mutations in two SCPL genes, SNG2/SCPL19 and SCPL17, led to decreases in both BzGSLs and SnGSLs (Figure 2). While approximately 10% residual BzGSLs and SnGSLs remain in sng2, these GSLs were near the limits of detection in scpl17. In addition to these BzGSLs and SnGSLs, similar changes were observed for a pair of putative benzoylated and sinapoylated GSLs that eluted after the two known major BzGSLs and SnGSLs. The m/z values for these unknown compounds are 524 and 626 under ESI-negative mode, which match the masses of benzoylated and sinapoylated 5-methylthiobutyl GSL (5MTB), respectively. Their amounts decreased in scpl17 and sng2 mutants, while the benzoylated form decreased and the sinapoylated form increased in bzo1. Benzoylated 4-methylthiopropyl GSL, in which the benzoyl unit is linked to the thioglucose moiety, has been identified in Arabidopsis (Reichelt et al., 2002), but acylated forms of 5MTB have not. Even though their identities need to be confirmed, this finding suggests that SCPL enzyme(s) may have broad substrate specificity for benzoylating GSLs.
The fact that mutations in both SCPL17 and SNG2 strongly reduce BzGSL and SnGSL accumulation suggests two possible models for involvement of the corresponding proteins. According to a sequential model, SCT synthesizes sinapoylated and benzoylated intermediates, possibly choline esters, which are then used to acylate OH-GSLs by SCPL17. Consistent with this model, SCT, which is known to synthesize sinapoylcholine (Shirley et al., 2001), is required for BC accumulation in vivo (Figures 3 and 6), and can also generate BC when assayed in vitro (Figure 4). Further, BC accumulates in scpl17 mutants, suggesting that it may be a substrate for the enzyme that SCPL17 encodes. On the other hand, choline esters are expected to be poor benzoyl and sinapoyl donors in comparison to 1-O-glucose esters, which are known to have a high free energy of hydrolysis (Mock and Strack, 1993).
In the second and preferred indirect model, SCPL17 catalyzes both benzoylation and sinapoylation of OH-GSLs using BG and SG as donors, but the impact of sng2 mutations is indirect (Figure 9). This model is consistent with the stronger GSL phenotype of scpl17 versus sng2 mutants, and the observation that BzGSL accumulation is enhanced when sng2 siliques, but not scpl17 siliques, are provided with exogenous BA (Figure 6c). In this model, BC accumulation in scpl17 mutants occurs only because a pool of BG accumulates when BzGSL synthesis is blocked, and this pool becomes available to SCT for trans-esterification. Unfortunately, attempts to directly test the ability of SCPL17 heterologously expressed in Nicotiana benthamiana to acylate OH-GSLs using BG (and BC) in vitro were unsuccessful even though control experiments measuring SCT and sinapoylglucose:malate sinapoyltransferase (SMT) (Lehfeldt et al., 2000) activities using previously established assay conditions (Lehfeldt et al., 2000; Shirley et al., 2001; Shirley and Chapple, 2003) were not problematic. For these transient expression studies in tobacco, we used the same CsMV:SCPL17 vector that we used to complement scpl17 mutants, and thus would expect successful protein transient expression in tobacco as well. Although it is unclear why these experiments were unsuccessful, previous attempts to heterologously express SCPL proteins have been challenging (Lehfeldt et al., 2000; Shirley et al., 2001; Shirley and Chapple, 2003), perhaps due to the large number of disulfide cross-links that need to be made correctly in order for this class of proteins to function. Alternatively, these data may indicate that the substrate for the enzyme is not the mature glucosinolate, or that the enzyme requires specific assay conditions, essential co-factors or partner proteins for activity that we have not yet identified.
Figure 9. Preferred model explaining BzGSL and SnGSL biosyntheses. SCPL17 may mediate final acyltransferase reactions by using glucose esters as acyl donors. BZO1 works as a cinnamoyl CoA ligase and provides benzoate for BzGSL biosynthesis.
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Although our second model appears more likely at this time, it is unclear how the postulated indirect effect of sng2 mutations on BzGSL and SnGSL accumulation is mediated. Mutations in SNG2 are known to dramatically alter soluble phenylpropanoid pools, and it is possible that the accumulation of SG has a direct impact on SCPL17-mediated catalysis. Alternatively, these alterations in pool sizes may act through the same mechanism that leads to alterations in BzGSL levels in brt1, ref1 and fah1 (Figure 7). Finally, previous studies have demonstrated interactions between the glucosinolate and phenylpropanoid biosynthetic pathways (Hemm et al., 2003), and it is possible that the same metabolic interplay extends to the level of the OH-GSL acyltransferase encoded by SCPL17.
BZO1 functions as a cinnamoyl CoA ligase
The observation that BA feeding restores BzGSL accumulation in bzo1 indicates that the benzoyl CoA ligase activity of BZO1 is not required for BzGSL synthesis and that BZO1 must have another catalytic function elsewhere in the pathway. Our results showing that SCPL17 is the acyltransferase involved in transferring the benzoyl moiety from BG to OH-GSLs, and the kinetic characterization of BZO1 in vitro (Table 1) showing that the enzyme prefers cinnamate to BA, demonstrate that both of these conclusions are correct. These findings place BZO1 at the beginning of the BA synthetic pathway where it functions in the peroxisome as a cinnamoyl CoA ligase as recently reported in petunia (Klempien et al., 2012). These findings shed more light on the BA biosynthetic pathway in Arabidopsis. The near absence of BA-derived compounds such as BC, BG and BzGSLs in bzo1 and chy1 mutants (Ibdah and Pichersky, 2009) suggests that BA biosynthesis in Arabidopsis seeds occurs exclusively via cinnamoyl CoA, and that the non-β-oxidative pathway mediated by direct conversion of cinnamate into benzaldehyde is of negligible importance, at least in developing seeds. Although either BA or benzaldehyde is the likely end product of cinnamoyl CoA metabolism, synthesis of BzGSLs probably occurs in the vacuole where SCPL proteins are thought to be localized (Fraser et al., 2005).