Petunia 3-ketoacyl-CoA thiolase 1, a peroxisomal protein, is involved in the central C6–C1 floral volatile biosynthetic pathway and contributes to benzoic acid formation in petunia flowers
Petunia has been used extensively as a model system to study the biosynthesis of flavonoids, particularly those involved in floral pigmentation (Koes et al., 2005). Recently, it has emerged as a model system to study the biosynthesis of benzenoid and phenylpropanoid-related floral volatiles (Schuurink et al., 2006; Pichersky and Dudareva, 2007). Previous work, however, has concentrated on identifying genes and enzymes involved in the final 1–2 steps resulting in the synthesis of the volatiles themselves (Negre et al., 2003; Boatright et al., 2004; Kaminaga et al., 2006; Koeduka et al., 2006; Dexter et al., 2007). In this study, we used functional genomics and targeted metabolomics to identify and probe the function of PhKAT1 that encodes an enzyme potentially centrally positioned in the pathway leading to the biosynthesis of BA (Figure 1). The homology of PhKAT1 to known thiolases led to the hypothesis that it could be involved in the β-oxidative shortening of the propyl side-chain of 3-oxo-3-phenylpropionyl-CoA, leading to the production of benzoyl-CoA, which is used as a co-substrate for the production of benzylbenzoate and phenylethylbenzoate (Boatright et al., 2004). This reaction would be analogous to the shortening by two carbons of the 3-ketoacyl-CoA substrates in fatty acid degradation, catalysed by bona fide thiolases (Germain et al., 2001). Furthermore, its expression levels and patterns suggested involvement in scent biosynthesis. To test this hypothesis, we stably silenced PhKAT1 in petunia plants.
In these transgenic plants, PhKAT1 transcript levels in the petals at the peak of volatile production were decreased by as much as 75% (Figure 4b) and overall thiolase activity was reduced by 50% (Figure 5). On the metabolite levels, we observed significant decreases in internal pools of BA, methylbenzoate, benzylbenzoate, phenylethylbenzoate and benzylaldehyde (Figure 6b) and significant decrease in emission of methylbenzoate, benzylbenzoate, phenylethylbenzoate and benzylaldehyde (Figure 6a), but not of the C6–C3 compound isoeugenol, the C6–C2 compounds phenylacetaldehyde (Figure 6a), 2-phenylethanol and phenylethylacetate (Figure S3) and the p-coumaric-derived C6–C1 compound vanillin (Figure S3). Interestingly, internal pool levels of the C6–C1 compounds were more reduced than the emission levels, which can reflect a constant emission rate of these compounds, depleting the internal pools even further. Although MeSA can be produced from BA via SA, the moderate reduction in MeSA emission levels suggests that SA is also produced via (iso)chorismate, as has been shown in Arabidopsis (Wildermuth, 2001). Similar results were seen in ir-PhODO1 plants, which show dramatically reduced BA levels but only slightly reduced MeSA emission levels (Verdonk et al., 2005). The existence of this route leading to SA and MeSA has yet to be shown in petunia.
The decreased levels of compounds that are derived from BA or benzoyl-CoA, unlike those that are not derived from these two precursors (the C6–C2 and C6–C3 compounds), strongly suggest that PhKAT1 is involved in BA biosynthesis, probably through the conversion of 3-oxo-3-phenylpropionyl-CoA to benzoyl-CoA. Efforts to develop an enzymatic assay to measure 3-oxo-3-phenyl-propionyl-CoA thiolase activity were not successful. Because most thiolases have activity towards acetoacyl-CoA, measuring acetoacetyl-CoA thiolase activity in a cell represents the activity of most if not all thiolases present. Therefore, because transcript levels were reduced with 75%, the residual thiolase activity of 50% indicates that either other thiolases are still active or the PhKAT1 protein levels were less reduced than its transcripts levels. Nevertheless, this result shows that reduction of PhKAT1 transcript levels reduces overall thiolase activity in petunia petals
Silencing of PhKAT1 did not alter 2-phenylethanol levels in these lines (Figure S3). Phenylethylbenzoate emission levels, however, were significantly reduced (Figure 6a). As phenylethylbenzoate is produced from 2-phenylethanol and benzoyl-CoA by PhBPBT (Boatright et al., 2004; Dexter et al., 2007), it is likely that reduced benzoyl-CoA levels in ir-PhKAT1 lines are the cause for lower phenylethylbenzoate emission, supporting the hypothesis that benzoyl-CoA is the product of PhKAT1. Similarly, in Streptomyces maritimus, the thiolase encJ knock-out strain KJ produces less benzoyl-CoA-derived enterocin (Xiang and Moore, 2003). Our results also showed a peroxisomal localization for PhKAT1 and thus synthesis of benzoyl-CoA in this organelle. This finding is consistent with the recent report that a mutation in a gene encoding the peroxisomal protein benzoyl-CoA ligase (AT1G65880) eliminates benzoyl-CoA (or BA) biosynthesis in Arabidopsis seeds (Kliebenstein et al., 2007).
In plants, β-oxidation is not only essential for fatty acid catabolism (Germain et al., 2001), but also for the production of indole acetic acid (Zolman et al., 2001), jasmonic acid (Afitlhile et al., 2005) and valine (Lange et al., 2004). In Arabidopsis, there are three 3-ketoacyl-CoA thiolase (KAT) genes, but only for AtKAT2 (AT2G33150) it is known that it is important for fatty acid β-oxidation, jasmonic acid biosynthesis and indole acetic acid production (Hayashi et al., 1998; Germain et al., 2001). In silico co-expression analysis groups AtKAT5 with genes of the flavonoid pathway, suggesting it has a role different from that in general fatty acid β-oxidation (Carrie et al., 2007). Because PhKAT1 is not expressed in seedlings (Figure 2a), it is not likely to be involved in fatty acid β-oxidation. In addition, it has been hypothesised that in some plant species biosynthesis of vanillin, which is a minor volatile in petunia, occurs by a process that mirrors fatty acid β-oxidation (Loscher and Heide, 1994; Podstolski et al., 2002). However, the silencing of PhKAT1 did not affect the emission of vanillin (Figure S3), excluding a role for PhKAT1 in vanillin biosynthesis in petunia flowers. It is likely that PhKAT2, which is already expressed early during flower development (Figure 2c), when no volatile benzenoids are produced (Verdonk et al., 2003) and that lacks rhythmic expression in petals (Figure S1), is involved in fatty acid degradation or the synthesis of other metabolites, but not in the synthesis of floral scent compounds.
What is the proportional contribution of β-oxidation to the synthesis of benzenoids in petunia flowers?
While elucidating the specific biosynthetic pathways leading to BA in plants has been a long and difficult process that is still incomplete, feeding experiments previously suggested the contribution of both the β-oxidative and the non-β-oxidative pathway with benzoyl-CoA and benzaldehyde as intermediates, respectively (Ribnicky et al., 1998; Abd El-Mawla and Beerhues, 2002; Boatright et al., 2004). Silencing of PhBPBT and subsequent metabolic flux analysis following feeding of labelled Phe to flowers of these transgenic plants indicated that both β-oxidative and the non-β-oxidative pathways contribute to the synthesis of benzenoid compounds in petunia (Boatright et al., 2004). Flux analysis of ir-PhBPBT transgenic flowers suggested that benzylbenzoate is a precursor to some of the free BA found in the petals (Orlova et al., 2006). Our results strongly support a major role for a peroxisomal thiolase in the synthesis of benzoyl-CoA and consequently for the synthesis of benzenoids. From our results it is clear that mainly the β-oxidative pathway contributes to BA formation in petunia flowers during the night (Figure 6b) although isotope labelling and modelling studies suggested a greater flux through the non-β-oxidative pathway (Orlova et al., 2006). The reduced internal BA pool levels (four-fold reduction) correlate perfectly with the reduced PhKAT1 transcript levels (four-fold reduction) in the dark period, when BA levels (Kolosova et al., 2001a), PhKAT1 transcript levels (Figure 2b) and volatile emission (Verdonk et al., 2005) are high. Our results however, do not exclude the contribution of additional routes leading to the biosynthesis of BA (Boatright et al., 2004; Orlova et al., 2006). Related to this outcome, an aldehyde oxidase that converts benzaldehyde to BA was identified in Arabidopsis recently (Ibdah et al., 2009).
Although benzylbenzoate, phenylethylbenzoate and BA levels are reduced in both the ir-PhBPBT and our ir-PhKAT1 plants, there are also several differences, not in the least because PhBPBT concerns an enzyme in a final step, i.e. making the volatiles benzylbenzoate and phenylethylbenzoate. The most striking difference is that silencing of PhBPBT increased benzaldehyde emission (Orlova et al., 2006; Dexter et al., 2008) and internal pools (Orlova et al., 2006) whereas benzaldehyde emission and internal pools decreased as a consequence of PhKAT1 silencing (Figure 6a,b). The immediate precursor of benzylbenzoate, benzylalcohol, was reduced in ir-PhKAT1 plants as well but increased in ir-PhBPBT plants Labelling studies indicated that benzylalcohol can be produced from both benzaldehyde and benzylbenzoate in petunia flowers (Boatright et al., 2004). Because the exact nature of the enzymes that are involved in benzaldehyde and benzylalcohol production in plants is not known, it is difficult to explain these different results. One interpretation is that accumulation of intermediates upstream of benzoyl-CoA in the ir-PhKAT1 plants could inhibit the activity of benzylalcohol and/or benzaldehyde-producing enzymes, as is the case for PhBPBT inhibition by coniferyl aldehyde (Dexter et al., 2007). Alternatively, accumulated intermediates could inhibit transcription of biosynthetic genes, as is the case for PhBSMT by Phe (Boatright et al., 2004). Finally, reduced benzoyl-CoA and/or BA levels in ir-PhKAT1 plants could enhance the formation of BA from benzaldehyde through the non-β-oxidative pathway in the dark period (Figure 1) as suggested for ir-PhBPBT plants in the light period (Orlova et al., 2006). This enhancement would deplete the benzaldehyde internal pool and consequently reduce benzylalcohol internal pool and emission levels. These ir-PhKAT1 lines can be important tools for future labelling studies to investigate the proportional contributions of the β-oxidative and non-β-oxidative pathway in more detail.
Regulation of floral scent production and the role of compartmentalization
In this study, we have shown that the expression pattern of PhKAT1 (Figure 2) is characteristic for a floral benzenoid-related gene (Kolosova et al., 2001a; Negre et al., 2003; Boatright et al., 2004; Underwood et al., 2005; Verdonk et al., 2005; Kaminaga et al., 2006; Koeduka et al., 2006; Dexter et al., 2007). We have further shown that expression of PhKAT1 peaks approximately 3–6 h earlier in petals than PhODO1 during the day/night cycle (Figure 2b), which precedes peak volatile emission by approximately 2–3 h (Verdonk et al., 2005). Reanalysis of our microarray experiments with ir-PhODO1 plants (Verdonk et al., 2005) showed that PhKAT1 expression is not influenced by PhODO1 silencing (ratio 1.04; P = 0.44). Apparently, the expression of PhODO1 and PhKAT1 are under different transcriptional control mechanisms.
Volatile benzenoid/phenylpropanoid production and emission are spatially, developmentally and diurnally regulated. This fact makes sense as these volatiles have Phe as precursor in common with many other primary and secondary metabolites. Also methyl donors, supplied by the SAM cycle (Verdonk et al., 2003, 2005), are shared with other pathways (Negre et al., 2003; Schuurink et al., 2006). It is therefore expected that channelling and the existence of different substrate pools are part of the regulatory machinery of the benzenoid pathway. When fluxes are perturbed, for instance by the accumulation of intermediates as a consequence of petunia acetyl-CoA:coniferyl alcohol acetyltransferase (PhCFAT) silencing, this outcome has a direct effect on PhBSMT and PhBPBT enzymatic activity (Dexter et al., 2007). PhBSMT is likely to be localised in the cytosol in analogy to the snapdragon S-adenosyl-l-methionine:BA carboxyl methyltransferase (AmBAMT) (Kolosova et al., 2001b). However, it was modelled by Boatright et al. (2004) that a second, large pool of MeBA is stored in the vacuole. Here we show that PhKAT1 localises to the peroxisome, adding another layer of regulation. In analogy with fatty acid β-oxidation, CoA-activated compounds like benzoyl-CoA could be transported across the peroxisomal membrane by ABC-transporters (Footitt et al., 2002). Both in the cytosol and the peroxisomes, benzoyl-CoA can be converted to BA by the action of thioesterases (Figure 1), which would implicate the existence of different BA and benzoyl-CoA pools in the cell. Tilton et al. (2004) identified a peroxisomal acyl-CoA thioesterase that is likely not involved in fatty acid β-oxidation, but in another process in plants. The substrate for this thioesterase has not been identified yet and it remains to be seen whether multiple thioesterases that can act on benzoyl-CoA exist in petunia petals. Finally, the snapdragon benzaldehyde dehydrogenase, involved in the non-β-oxidative pathway, was recently shown to be located in the mitochondria (Long et al., 2009). Our finding that PhKAT1 and thus the β-oxidative pathway localises to the peroxisomes means that not only distinct routes with different enzymes are involved in the production of BA and volatile benzenoids, but that these enzymes are active in different cellular compartments.