Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants

Abstract

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C 3 side chain by two carbon units, which can occur by a non–β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase ( PhCHD ), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

“…Genes involved in biosynthesis of phenylalanine and phenylalaninederived volatile compounds in petunia flowers typically display a spatial, temporal and rhythmic expression profile corresponding with floral volatile emission 16,17,25,[34][35][36] . Thus, PhPPY-AT transcript levels were investigated by qRT-PCR with genespecific primers.…”

“…3d). To biochemically confirm the cytosolic localization of PhPPY-AT, its activity was assayed in cytosolic and plastidial fractions prepared from wild-type petunia petals 34,35 (Table 2). Indeed, the cytosolic fraction showed 2.5-fold enrichment in PhPPY-AT activity, similar to the 2.6-fold enrichment in the activity of the cytosolic marker alcohol dehydrogenase, relative to crude extract.…”

Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum

“…5, 2018 identified by a functional genomics approach and has been shown to be active with p-coumaroyl-CoA ( Figure S4, Supplementary Information). 39 The next steps are expected to be catalyzed by thiolases and CoA thioesterases. A 3-ketoacyl-CoA thiolase (PhKAT1) was confirmed to be involved in the production of benzoyl-CoA from cinnamoyl-CoA in Petunia hybrid.…”

“…33 A Petunia gene encoding the bifunctional peroxisomal enzyme cinnamoyl-CoA hydratase-dehydrogenase (CHD), which is responsible for the initial two-step conversion of cinnamoyl-CoA into benzoic acid, was identified by a functional genomics approach and has been shown to be active with p-coumaroyl-CoA ( Figure S4, Supplementary Information). 39 The next steps are expected to be catalyzed by thiolases and CoA thioesterases. A 3-ketoacyl-CoA thiolase (PhKAT1) was confirmed to be involved in the production of benzoyl-CoA from cinnamoyl-CoA in Petunia hybrid.…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…1). Illuminated by the pioneering work on benzoic acid biosynthesis in petunia (van Moerkercke et al, 2009;Klempien et al, 2012;Qualley et al, 2012), this pathway was elucidated in Arabidopsis. Cytosolic transcinnamic acid, presumably as the CoA ester, is imported by PXA1 (Bussell et al, 2014).…”