Christoph Crocoll

Volatile chemicals from leaf litter are associated with invasiveness of a Neotropical weed in Asia

Some invasive plant species appear to strongly suppress neighbors in their nonnative ranges but much less so in their native range. We found that in the field in its native range in Mexico, the presence of Ageratina adenophora, an aggressive Neotropical invader, was correlated with higher plant species richness than found in surrounding plant communities where this species was absent, suggesting facilitation. However, in two nonnative ranges, China and India, A. adenophora canopies were correlated with much lower species richness than the surrounding communities, suggesting inhibition. Volatile organic compound (VOC) signals may contribute to this striking biogeographical difference and the invasive success of A. adenophora. In controlled experiments volatiles from A. adenophora litter caused higher mortality of species native to India and China, but not of species native to Mexico. The effects of A. adenophora VOCs on seedling germination and growth did not differ between species from the native range and species from the nonnative ranges of the invader. Litter from A. adenophora plants from nonnative populations also produced VOCs that differed quantitatively in the concentrations of some chemicals than litter from native populations, but there were no chemicals unique to one region. Biogeographic differences in the concentrations of some volatile compounds between ranges suggest that A. adenophora may be experiencing selection on biochemical composition in its nonnative ranges.

Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis

The aroma, flavor and pharmaceutical value of cultivated oregano (Origanum vulgare L.) is a consequence of its essential oil which consists mostly of monoterpenes and sesquiterpenes. To investigate the biosynthetic pathway to oregano terpenes and its regulation, we identified and characterized seven terpene synthases, key enzymes of terpene biosynthesis, from two cultivars of O. vulgare. Heterologous expression of these enzymes showed that each forms multiple mono- or sesquiterpene products and together they are responsible for the direct production of almost all terpenes found in O. vulgare essential oil. The correlation of essential oil composition with relative and absolute terpene synthase transcript concentrations in different lines of O. vulgare demonstrated that monoterpene synthase activity is predominantly regulated on the level of transcription and that the phenolic monoterpene alcohol thymol is derived from γ-terpinene, a product of a single monoterpene synthase. The combination of heterologously-expressed terpene synthases for in vitro assays resulted in blends of mono- and sesquiterpene products that strongly resemble those found in vivo, indicating that terpene synthase expression levels directly control the composition of the essential oil. These results will facilitate metabolic engineering and directed breeding of O. vulgare cultivars with higher quantity of essential oil and improved oil composition.

The Arabidopsis NPF3 protein is a GA transporter

Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA–ABA interaction may occur at the level of transport.

Plant defences against ants provide a pathway to social parasitism in butterflies

Understanding the chemical cues and gene expressions that mediate herbivore–host-plant and parasite–host interactions can elucidate the ecological costs and benefits accruing to different partners in tight-knit community modules, and may reveal unexpected complexities. We investigated the exploitation of sequential hosts by the phytophagous–predaceous butterfly Maculinea arion, whose larvae initially feed on Origanum vulgare flowerheads before switching to parasitize Myrmica ant colonies for their main period of growth. Gravid female butterflies were attracted to Origanum plants that emitted high levels of the monoterpenoid volatile carvacrol, a condition that occurred when ants disturbed their roots: we also found that Origanum expressed four genes involved in monoterpene formation when ants were present, accompanied by a significant induction of jasmonates. When exposed to carvacrol, Myrmica workers upregulated five genes whose products bind and detoxify this biocide, and their colonies were more tolerant of it than other common ant genera, consistent with an observed ability to occupy the competitor-free spaces surrounding Origanum. A cost is potential colony destruction by Ma. arion, which in turn may benefit infested Origanum plants by relieving their roots of further damage. Our results suggest a new pathway, whereby social parasites can detect successive resources by employing plant volatiles to simultaneously select their initial plant food and a suitable sequential host.

Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli

The methionine-derived glucosinolate glucoraphanin is associated with the health-promoting properties of broccoli. This has developed a strong interest in producing this compound in high amounts from a microbial source. Glucoraphanin synthesis starts with a five-gene chain elongation pathway that converts methionine to dihomo-methionine, which is subsequently converted to glucoraphanin by the seven-gene glucosinolate core structure pathway. As dihomo-methionine is the precursor amino acid for glucoraphanin production, a first challenge is to establish an expression system for production of dihomo-methionine. In planta, the methionine chain elongation enzymes are physically separated within the cell with the first enzyme in the cytosol while the rest are located in the chloroplast. A de-compartmentalization approach was applied to produce dihomo-methionine by expression of the respective plant genes in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health-promoting glucoraphanin.

Optimization of engineered Production of the glucoraphanin Precursor Dihomomethionine in Nicotiana benthamiana

Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g−1 fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host.

Origin and evolution of transporter substrate specificity within the NPF family

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

BackgroundThere are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria.ResultsIn this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites.ConclusionsThere is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to ‘traditional’ metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.

CB5C affects the glucosinolate profile in Arabidopsis thaliana

ABSTRACT Cytochrome b5 (CB5) proteins are small heme-binding proteins, that influence cytochrome P450 activity. While only one CB5 isoform is found in mammals, higher plants have several isoforms of these proteins. The roles of the many CB5 isoforms in plants remain unknown. We hypothesized that CB5 proteins support the cytochrome P450 enzymes of plant specialized metabolism and found CB5C from Arabidopsis thaliana to co-express with glucosinolate biosynthetic genes. We characterized the glucosinolate profiles of 2 T-DNA insertion mutants of CB5C, and found that long-chained aliphatic glucosinolates were reduced in one of the mutant lines – a phenotype that was exaggerated upon methyl-jasmonate treatment. These results support the hypothesis, that CB5C influences glucosinolate biosynthesis, however, the mode of action remains unknown. Furthermore, the mutants differed in their biomass response to methyl jasmonate treatment. Thereby, our results highlight the varying effects of T-DNA insertion sites, as the 2 analyzed alleles show different phenotypes.

Biological activity and LC-MS/MS profiling of extracts from the Australian medicinal plant Acacia ligulata (Fabaceae)

Abstract Acacia ligulata A.Cunn. ex Benth. (Fabaceae: Mimosoideae) is a native Australian plant used traditionally by Australian Aboriginal groups. This study was undertaken to investigate the bioactivity of A. ligulata extracts and to evaluate their chemical composition. Potential antibacterial, cytotoxic and enzyme inhibitory effects relevant to traditional medicinal and food uses of the species were examined and LC-MS/MS was performed to investigate the chemical composition. Antibacterial activity was observed for bark and leaf extracts with an MIC for the bark extract of 62.5 μg/mL against Streptococcus pyogenes. Pod extracts showed cytotoxic effects against cancer cells, with the highest activity against melanoma SK-MEL28 cells with IC50 values between 40.8 and 80.6 μg/mL. Further, the leaf and pod extracts also inhibited α-amylase EC-3.2.1.1 and α-glucosidase EC-3.2.1.20 with IC50 values between 9.7–34.8 and 12.6–64.3 μg/mL, respectively. The LC-MS/MS profiling indicated that several different saponins were present in the active extracts.