Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers

Abstract

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the b-oxidative or nonoxidative pathways. The first step in the b-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the b-oxidative pathway.

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.…”

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…in supplying precursors for phenylpropanoid derivatives such as lignin). Recently, cDNAs encoding cinnamate:CoA ligase from Petunia hybrida and Hypericum calycinum were cloned (Gaid et al, 2012;Klempien et al, 2012). This enzyme channels the metabolic flux from the general phenylpropanoid pathway to benzenoid metabolism and further to aucuparin biosynthesis.…”

Ácido benzoico: biosíntesis, modificación y función en plantas

“…Sin embargo, se han identificado pocos genes en estas vías metabólicas (Klempien et al, 2012). Los primeros estudios apoyaron la síntesis del BA a partir de la Phe, por lo tanto, la mayoría del trabajo se ha centrado en la vía de derivados de esta.…”