Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.

Benzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants.

SummaryUridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana. A phylogenetic analysis of the conserved amino acids encoded by these Arabidopsis genes reveals the presence of 14 distinct groups of UGTs in this organism. Genes encoding UGTs have also been identified in several other higher plant species. Very little is yet known about the regulation of plant UGT genes or the localization of the enzymes they encode at the cellular and subcellular levels. The substrate specificities of these UGTs are now beginning to be established and will provide a foundation for further analysis of this large enzyme superfamily as well as a platform for future biotechnological applications.

SummaryAn analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg À1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg À1 FW compared to approximately 15 pg mg À1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.

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.