SummaryMembers of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

L-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

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.

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines--T136-8, K10, and C9--which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.

Summary C‐Glycosides are characterized by their C–C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C‐Glycosides are remarkably stable, as their C–C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C‐glycosylflavonoids; however, the genes encoding for enzymes that catalyze C‐glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C‐glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C‐glucosylation of 2‐hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C‐glucosylation activity towards 2‐hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2′,4′,6′‐trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C‐glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.

Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, < 20±50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins.They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.

Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di-C-glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2-hydroxyflavanones, dihydrochalcone, and their mono-C-glucosides as sugar acceptors and produced corresponding di-C-glucosides. The K and k values of FcCGT toward phloretin were <0.5 μm and 12.0 sec , and those toward nothofagin (3'-C-glucosylphloretin) were 14.4 μm and 5.3 sec , respectively; these values are comparable with those of other glycosyltransferases reported to date. Transcripts of both CGT genes were found to concentrate in various plant organs, and particularly in leaves. Our results suggest that di-C-glucosyl flavonoid biosynthesis proceeds via a single enzyme using either 2-hydroxyflavanones or phloretin as a substrate in citrus plants. In addition, Escherichia coli cells expressing CGT genes were found to be capable of producing di-C-glucosyl flavonoids, which is promising for commercial production of these valuable compounds.