Individual lipid transfer proteins from Tanacetum parthenium show different specificity for extracellular accumulation of sesquiterpenes.

KEY MESSAGE: A highly specialized function for individual LTPs for different products from the same terpenoid biosynthesis pathway is described and the function of an LTP GPI anchor is studied. Sequiterpenes produced in glandular trichomes of the medicinal plant Tanacetum parthenium (feverfew) accumulate in the subcuticular extracellular space. Transport of these compounds over the plasma membrane is presumably by specialized membrane transporters, but it is still not clear how these hydrophobic compounds are subsequently transported over the hydrophilic cell wall. Here we identified eight so-called non-specific Lipid transfer proteins (nsLTPs) genes that are expressed in feverfew trichomes. A putative function of these eight nsLTPs in transport of the lipophilic sesquiterpene lactones produced in feverfew trichomes, was tested in an in-planta transport assay using transient expression in Nicotiana benthamiana. Of eight feverfew nsLTP candidate genes analyzed, two (TpLTP1 and TpLTP2) can specifically improve extracellular accumulation of the sesquiterpene costunolide, while one nsLTP (TpLTP3) shows high specificity towards export of parthenolide. The specificity of the nsLTPs was also tested in an assay that test for the exclusion capacity of the nsLTP for influx of extracellular substrates. In such assay, TpLTP3 was identified as most effective in blocking influx of both costunolide and parthenolide, when these substrates are infiltrated into the apoplast. The TpLTP3 is special in having a GPI-anchor domain, which is essential for the export activity of TpLTP3. However, addition of the TpLTP3 GPI-anchor domain to TpLTP1 resulted in loss of TpLTP1 export activity. These novel export and exclusion assays thus provide new means to test functionality of plant nsLTPs.

The effects of several abiotic elicitors on the expression of genes of key enzymes involved in the parthenolide biosynthetic pathway and its content in feverfew plant (Tanacetum parthenium L.).

Feverfew is an herb used to treat different diseases such as migraine headaches. Due to the economic aspect of its metabolites in the pharmaceutical industry, establishing new approaches to produce the compounds on a large scale is essential. To investigate the effects of stimulators on parthenolide synthesis, feverfew plants were treated with different elicitors, including methyl jasmonate, salicylic acid, NaCl, aluminum oxide, and magnesium aluminate spinel nanoparticles. The expression of genes, E-beta-caryophyllene synthase, Germacrene A synthase, and Costunolide Synthase in the metabolite biosynthesis pathway was examined using qRT-PCR. In addition, parthenolide content, total flavonoids, and polyphenols antioxidant activity were evaluated by HPLC and spectrophotometry. Our results indicated that methyl jasmonate and salicylic acid were more effective on the final concentration of parthenolide, but magnesium aluminate spinel affected the genes' expression, positively. The results show that the elicitors can be used to increase the metabolite in the plant, commercially.

Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production.

The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3ß-hydroxy-dihydrocostunolide and 3ß-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.

Proteomic profiling of the weed feverfew, a neglected pollen allergen source.

Feverfew (Parthenium hysterophorus), an invasive weed from the Asteraceae family, has been reported as allergen source. Despite its relevance, knowledge of allergens is restricted to a partial sequence of a hydroxyproline-rich glycoprotein. We aimed to obtain the entire sequence for recombinant production and characterize feverfew pollen using proteomics and immunological assays. Par h 1, a defensin-proline fusion allergen was obtained by cDNA cloning and recombinantly produced in E. coli. Using two complementary proteomic strategies, a total of 258 proteins were identified in feverfew pollen among those 47 proteins belonging to allergenic families. Feverfew sensitized patients' sera from India revealed IgE reactivity with a pectate lyase, PR-1 protein and thioredoxin in immonoblot. In ELISA, recombinant Par h 1 was recognized by 60 and 40% of Austrian and Indian sera, respectively. Inhibition assays demonstrated the presence of IgE cross-reactive Par h 1, pectate lyase, lipid-transfer protein, profilin and polcalcin in feverfew pollen. This study reveals significant data on the allergenic composition of feverfew pollen and makes recombinant Par h 1 available for cross-reactivity studies. Feverfew might become a global player in weed pollen allergy and inclusion of standardized extracts in routine allergy diagnosis is suggested in exposed populations.

Magnesium and manganese interactively modulate parthenolide accumulation and the antioxidant defense system in the leaves of Tanacetum parthenium.

A balanced nutrient supply is a critical factor affecting accumulation of terpenoids in plants, yet data related to the interactive effects of two essential nutrients for the biosynthesis of sesquiterpenes are scarce. Here, the interactional effects between magnesium (Mg) and manganese (Mn) on plant growth, oxidative status, parthenolide accumulation and expression of key genes involved in parthenolide biosynthesis including 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR), 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR), germacrene A synthase (GAS), germacrene A oxidase (GAO), costunolide synthase (COS) and parthenolide synthase (PTS) in the leaves of feverfew plants grown at different Mn and Mn levels were assessed. Plant growth and leaf pigment concentrations were associated with the amount of applied Mg but could be modified by the Mn level. Deprivation and the addition of both Mg and Mn induce oxidative stress. Mg supply also alleviated the adverse effects of Mn excess on plant growth and oxidative status. In addition, parthenolide biosynthesis decreased under deprivation of Mg or Mn, but the addition of Mn up to 50µM under 2mM Mg supply considerably increased its accumulation. The parthenolide accumulation trend might reflect the up-regulation of terpenoid-related genes and enzyme activities as well as the oxidative status of feverfew leaves. Our data suggest a profound effect of the combined supply of Mg and Mn on parthenolide biosynthesis through the activation of terpene synthases, which concomitantly modulate by oxidative status.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

KEY MESSAGE: Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway.

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4µgg(-1) parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3ß-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug.

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.