OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoids for the cuticle of aerial organs of Artemisia annua.

Artemisia annua is widely studied for its ability to accumulate the antimalarial sesquiterpenoid artemisinin. In addition to producing a variety of sesquiterpenoids, A. annua also accumulates mono-, di-, and triterpenoids, the majority of which are produced in the glandular trichomes. A. annua also has filamentous trichomes on its aerial parts, but little is known of their biosynthesis potential. Here, through a comparative transcriptome analysis between glandular and filamentous trichomes, we identified two genes, OSC2 and CYP716A14v2, encoding enzymes involved in the biosynthesis of specialized triterpenoids in A. annua. By expressing these genes in Saccharomyces cerevisiae and Nicotiana benthamiana, we characterized the catalytic function of these proteins and could reconstitute the specialized triterpenoid spectrum of A. annua in these heterologous hosts. OSC2 is a multifunctional oxidosqualene cyclase that produces α-amyrin, ß-amyrin, and δ-amyrin. CYP716A14v2 is a P450 belonging to the functionally diverse CYP716 family and catalyzes the oxidation of pentacyclic triterpenes, leading to triterpenes with a carbonyl group at position C-3, thereby providing an alternative biosynthesis pathway to 3-oxo triterpenes. Together, these enzymes produce specialized triterpenoids that are constituents of the wax layer of the cuticle covering the aerial parts of A. annua and likely function in the protection of the plant against biotic and abiotic stress.

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BACKGROUND: The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level. RESULTS: By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells. CONCLUSIONS: Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.

Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants.

• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.

The role of the COMT Val(158)Met polymorphism in the phenotypic expression of obsessive-compulsive disorder.

Obsessive-Compulsive Disorder (OCD) is characterized by the presence of obsessions and compulsions, and shows considerable phenotypic variability. Family and twin studies have indicated a genetic component in the etiology of OCD, and the catechol-O-methyl transferase (COMT) gene is an important candidate gene for OCD. This study investigates the influence of the functional COMT Val158Met polymorphism on the phenotypic expression of OCD, using an item-level factor-analytic approach in a large sample. The COMT Val158Met variant was genotyped in 373 patients and 462 controls. It was tested whether there was an association between the COMT Val158Met polymorphism and OCD or dimensional phenotypes such as YBOCS severity score, age of onset of obsessive-compulsive symptoms and six symptom dimensions recently found in a large item-level factor-analytic study [Katerberg et al., submitted]. We further investigated possible sex-specific associations between the COMT Val158Met polymorphism and OCD or dimensional phenotypes. There was a trend for an association of the COMT 158Met allele with OCD in males, and an interaction between the COMT Val158Met genotype and sex on the somatic and sensory phenomena symptom dimension, with females showing lower scores. In conclusion, a dimensional approach seems fruitful in detecting genes of importance for OCD.

Response to serotonin reuptake inhibitors in OCD is not influenced by common CYP2D6 polymorphisms.

The cornerstone of pharmacotherapy for OCD is serotonin reuptake inhibition, either with clomipramine or with selective serotonin reuptake inhibitors (SSRIs). In spite of the success of serotonin reuptake inhibiting drugs, nearly half of OCD patients do not respond to treatment. Treatment response may be affected by genetic polymorphisms of the P450 metabolic system. The four most common enzyme-activity reducing polymorphisms of the P450 CYP2D6 enzyme were determined in 91 outpatients with primary OCD according to DSM-IV criteria, receiving dosages titrated upward to 300 mg/day of venlafaxine or 60 mg/day of paroxetine, using a fixed dosing schedule. Our results show that the investigated CYP2D6 polymorphisms are not a decisive factor in the response to paroxetine and venlafaxine treatment in OCD in spite of their highly significant effect on the blood levels of these medicines.

Quantitation of artemisinin and its biosynthetic precursors in Artemisia annua L. by high performance liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry.

This study reports the development and validation of a rapid, sensitive and selective assay for the quantitation of artemisinin, arteannuin B, artemisitene and artemisinic acid in Artemisia annua L. by reversed phase high performance liquid chromatography (HPLC) electrospray (ESI) quadrupole time of flight (Q-TOF) tandem mass spectrometry (MS/MS). A recovery of >97% for all analytes was achieved by immersing one gram of fresh plant material in chloroform for 1 min. This result supports the hypothesis that artemisinin and some of its structural analogs present in the leaves A. annua L. are localized entirely in the subcuticular space of the glands on the surface of the leaves. We validated the use of this chloroform extract, without additional sample preparation steps, for quantitative Q-TOF MS/MS. No ion suppression (matrix effect) resulting from interference with other compounds was detected. For every concentration within the range of the standard curve (0.1 to 3.00 microg/ml), accuracy was between 85% and 115%. Within- and between-day variations for the analysis of A. annua L. samples were <20%.