We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (ϳ65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent K m of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO 2 , ammonia, and hydrogen peroxide in stoichiometric amounts.

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.

Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far. The e⁄ciency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin. The glucoside yield was 2.5 W Wmol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4P P,4Q Q-O-L L-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin. ß

SUMMARYBenzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a b-oxidative pathway or a non-b-oxidative pathway, with benzaldehyde as a key intermediate. The non-b-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

SUMMARYGlucosinolates (GSLs) are nitrogen-and sulfur-containing metabolites that contribute to human health and plant defense. The biological activities of these molecules are largely dependent on modification of the GSL R-groups derived from their corresponding amino acid precursors. In Arabidopsis seeds, esterification of the R-group of hydroxylated GSLs (OH-GSLs) leads to the accumulation of benzoylated GSLs (BzGSLs) and sinapoylated GSLs (SnGSLs). BzGSLs were thought to be synthesized from OH-GSLs and benzoyl CoA by a BAHD acyltransferase, but no BAHD gene is strongly co-expressed with the two reference genes BZO1 and AOP3 that are required for BzGSL biosynthesis. In contrast, three genes encoding serine carboxypeptidase-like (SCPL) acyltransferases [SCPL5, SCPL17 and SCPL19 (SNG2)] do exhibit strong co-expression. Using a reverse genetic approach, we found that the GSL profile of the scpl5 mutant was identical to that of wild-type, but both BzGSLs and SnGSLs were barely detectable in scpl17 mutants and their amounts were decreased in the sng2 mutant. In addition, both scpl17 and sng2 mutants accumulate the putative BzGSL precursors OH-GSLs and benzoylglucose. The results of further GSL analyses in other phenylpropanoid mutants and benzoate feeding experiments suggested that SCPL17 mediates the acyltransferase reaction directly, while the mutation in sng2 causes a decrease in BzGSLs and SnGSLs via an unknown indirect mechanism. Finally, benzoate feeding experiments using bzo1 mutants and BZO1 biochemical characterization indicated that the in vivo role of BZO1 is to synthesize the benzoate precursor cinnamoyl CoA rather than to generate benzoyl CoA from benzoate and CoA as previously predicted.