Benzoylation and sinapoylation of glucosinolate R-groups in Arabidopsis.

Glucosinolates (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.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ß-oxidative or nonoxidative pathways. The first step in the ß-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 ß-oxidative pathway.

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes.

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

Benzoic 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 beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-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.

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation.

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 (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.

Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells.

Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to various glucosides. The glucosylation efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures prior to curcumin administration. Two cDNAs encoding UDP-glucosyltransferases (CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C. roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases. The sequence identity between their deduced amino acid sequences was 27%. The expression of both genes was up-regulated by addition of MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than that of CaUGT2. The corresponding cDNAs were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant CaUGT1 exhibited no glucosylation activity with either curcumin or curcumin monoglucoside as substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin monoglucoside from curcumin and also conversion of curcumin monoglucoside to curcumin diglucoside. The use of the recombinant CaUGT2 may provide a useful new route for the production of curcumin glucosides.

Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus.

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 efficiency 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 micromol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4',4"-O-beta-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.