Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavors, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data-mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).

Background: In the Caryophyllaceae, cyclic peptides (CP) are biosynthesized from linear precursors via an unknown pathway. Results: Two protease-like enzymes are involved in precursor processing. Conclusion: A serine protease-like enzyme was recruited for the cyclization step in CP biosynthesis. Significance: This represents a very significant advance in our understanding of the mode and evolution of CP biosynthesis in plants.

Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA’s modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for Triticum aestivum (bread wheat) and Brachypodium distachyon (a model cereal) are reported. A number of these were cloned for recombinant expression and their functionality as ABA receptors confirmed by in vitro assays against protein phosphatases Type 2Cs. Ligand selectivity profiling of one of the wheat receptors (Ta_PYL2DS_FL) highlighted unique activities compared to Arabidopsis AtPYL5. Mutagenic analysis showed Ta_PYL2DS_FL amino acid D180 as being a critical contributor to this selectivity. Subsequently, a virus induced gene silencing (VIGS) approach was used to knockdown wheat Ta_PYL4AS_A (and similar) in planta, yielding plants with increased early stage resistance to FHB progression and decreased mycotoxin accumulation. Together these results confirm the existence of a family of ABA receptors in wheat and Brachypodium and present insight into factors modulating receptor function at the molecular level. That knockdown of Ta_PYL4AS_A (and similar) leads to early stage FHB resistance highlights novel targets for investigation in the future development of disease resistant crops.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography.