Artemisinin Biosynthesis in Non-glandular Trichome Cells of Artemisia annua.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Active phloem sap collection by Trigona spinipes (Fabricius) (Hymenoptera, Apidae)in Artemisia annuaLinn (Asteraceae)

Trigona spinipes Fabricius (Apidae: Meliponini), is a common stingless bee widely seen in urban and rural areas in Brazil, popularly known as irapuá, arapuá or bee-dog. Although these bees are considered pollinators of some cultivated plants, they are better known for the damage they cause in different crops. During experimental agroecological cultivation of Artemisia (Artemisia annuaLinn, Asteraceae), in Jaguariúna (SP, Brazil), stingless bees Trigona spinipes (Fabricius) were observed sucking phloem sap directly from the plant, a phenomenon not yet described in scientific literature. This study aimed to register and describe the phloem sap-sucking behavior performed by T. spinipesfor the first time, as well as to assess the potential impact of this behavior on A. annuacultivation. The behavior and the material collected by bees and the severity of attack were also analyzed. The aging and premature death of observed A. annuaspecimens occurred because of extensive lesions caused by T. spinipes, confirming the negative consequence of sap-sucking attacks of T. spinipesbees on the plants. Factors that could induce this unusual behavior were presented, pointing out the need for future studies on the development of strategies to protect plants, without causing damage to the T. spinipesbee populations, which are elements of Brazilian bee fauna and, therefore, protected by law.

Four New Compounds Obtained from Cultured Cells of Artemisia annua.

Four new compounds obtained from cultured cells of Artemisia annua were reported. Products were detected by HPLC-ELSD/GC-MS and isolated by chromatographic methods. The structures of four new compounds, namely 6-hydroxy arteannuin I (1), 1-hydroxy arteannuin I (2), 2-hydroxy arteannuin J (3), and 14-hydroxy arteannuin J (4), were elucidated using their physico-chemical properties by NMR and MS data analyses. The results from the spontaneous oxidative experiment indicated that the biosynthesis of the new compounds was enzyme-catalyzed. Interestingly, the enzymes in the cultured cells of A. annua showed the abilities of substrate-selective and region-selective hydroxylation of the sesquiterpene lactone. Furthermore, the artemisinin contents were increased by 50% and 80% compared to the control group after the addition of arteannuin I/J to the suspension-cultured cells of A. annua under light and dark culture conditions, respectively.

T-shaped trichome-specific expression of monoterpene synthase ADH2 using promoter-ß-GUS fusion in transgenic Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (sweet wormwood), is extensively used in the treatment of malaria. In order to better understand the metabolism of terpenes in A. annua and the influence of terpene synthases on artemisinin yield, the expression pattern of a monoterpene alcohol dehydrogenase (ADH2) has been studied using transgenic plants expressing promoter-ß-glucuronidase (GUS) fusion. ADH2 played a major role in monoterpenoid biosynthesis including carveol, borneol, and artemisia ketone through in vitro biochemical analysis. In this study, the ADH2 promoter was cloned by the genome walking method. A number of putative cis-acting elements were predicted in promoter region, suggesting that the ADH2 is driven by a complex regulation mechanism. ADH2 gene was highly expressed in old leaves, whereas the artemisinin biosynthetic genes were mainly expressed in bud and young leaves. The expression of ADH2 gene increased quickly during leaf development, revealed by qRT-PCR. GUS expression analysis in different tissues of transgenic A. annua demonstrates that ADH2 expression is exclusively located to T-shaped trichome, not glandular secretory trichome.

Transcriptome responses involved in artemisinin production in Artemisia annua L. under UV-B radiation.

Artemisinin, an endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from Artemisia annua L. In this study, a low dose (1.44 kJm(-2)d(-1)) of UV-B radiation (280-320 nm) for short-term (1h per day for 10 days) was applied to A. annua seedlings to stimulate artemisinin production. UV-B treatment not only induced the generation of reactive oxygen species (ROS), enhanced peroxidase activity and endogenous content of abscisic acid (ABA), but stimulated the biosynthesis of artemisinin in the seedlings. Here, transcriptomic changes during UV-B radiation in A. annua were detected using an Agilent GeneChip with 43,692 probe sets. In total, 358 transcripts were identified as differentially expressed under UV-B stress, of which 172 transcripts increased and 186 transcripts decreased in abundance. In terms of biological processes, gene ontology (GO) terms including primary carbohydrate and nitrogen compound metabolic processes were enriched in UV-B-repressed genes. The up-regulated genes were enriched in response to stress, ROS generation, hormone (ethylene, ABA) stimulus and cell cycle control. The expression of key enzymes such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 dependent monooxygenase/hydroxylase (CYP71AV1), and related WRKY transcription factors was up-regulated significantly for artemisinin biosynthesis. This profile of global gene expression patterns during UV-B stress will be valuable for further identification of the enzymes involved in artemisinin biosynthesis.

Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua.

Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (ß-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.

Efficient method for Agrobacterium mediated transformation of Artemisia annua L.

Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

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.

Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua suspension cultures.

Artemisia annua L. is a herb traditionally used for treatment of fevers. The glandular trichomes of this plant accumulate, although at low levels, artemisinin, which is highly effective against malaria. Due to the great importance of this compound, many efforts have been made to improve knowledge on artemisinin production both in plants and in cell cultures. In this study, A. annua suspension cultures were established in order to investigate the effects of methyl jasmonate (MeJA) and miconazole on artemisinin biosynthesis. Twenty-two micro molar MeJA induced a three-fold increase of artemisinin production in around 30 min; while 200 µm miconazole induced a 2.5-fold increase of artemisinin production after 24 h, but had severe effects on cell viability. The influence of these treatments on expression of biosynthetic genes was also investigated. MeJA induced up-regulation of CYP71AV1, while miconazole induced up-regulation of CPR and DBR2.

[Biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua].

OBJECTIVE: To investigate the biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua. METHODS: Artemisinic acid was added into to the media of the suspension cells of Cephalotaxus fortunei and Artemisia annua in their logarithmic growth phase. The biotransfromed product was detected with HPLC and isolated by silica gel column, Sephadex LH20 and ODS chromatography methods. The chemical structure of biotransformed product was elucidated on the basis of physical-chemical properties and spectroscopic data. Otherwise, the influence of co-cultured time on conversion ratio was investigated with HPLC. RESULTS: One biotransformed product, 3-alpha-hydroxyartemisinic acid, was obtained after two days of artemisinic acid administration to the suspension cells of Cephalotaxus fortunei and Artemisia annua. The optimal co-cultured time in suspension cells of Cephalotaxus fortunei was 2 days with the highest biotransformation rate of 8.42%, and in the case of Artemisia annua, it was 3 days and 3.95% respectively. CONCLUSION: It was the first time for the biotransformation of artemisinic acid to 3-alpha-hydroxyartemisinic acid by using cell suspension cultures of Cephalotaxus fortunei and Artemisia annua.

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua.

Artemisinin, isolated from the shrub-Artemisia annua, is a sesquiterpene lactone used to treat multi-drug resistant strains of falciparum malaria. It is also effective against a wide variety of cancers such as leukemia and colon cancer. To counter the present low content in leaves and uneconomical chemical synthesis, alternate ways to produce artemisinin have been sought. But this compound remains elusive in cell cultures of A. annua despite the extensive studies undertaken. This work reports the first successful approach for production of artemisinin by cell cultures of Indian variety of A. annua. In the present study, an integrated yield enhancement strategy, developed by addition of selected precursor (mevalonic acid lactone) and elicitor (methyl jasmonate) at optimized concentrations, resulted in 15.2g/l biomass and 110.2mg/l artemisinin, which was 5.93 times higher in productivity in comparison to control cultures.

Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.

Growth dynamics of Artemisia annua hairy roots in three culture systems.

The transient growth of Artemisia annua hairy roots was compared for cultures grown in shake flasks and in bubble column and mist reactors. Instantaneous growth rates were obtained by numerically differentiating the transient biomass measurements. Specific sugar consumption rates showed good agreement with literature values. From the growth rate and sugar consumption rate, the specific yield and maintenance coefficient for sugar were determined for all three culture systems. These values were statistically indistinguishable for roots grown in shake flasks and bubble columns. In contrast, the values for roots grown in bubble columns and mist reactors were statistically different, suggesting that sugar utilization by roots grown in these two systems may be different. By measuring respiration rates in the bubble column reactor we also determined the actual biomass yield and maintenance coefficient for O(2) and CO(2). Together with an elemental analysis of the roots, this allowed us to obtain a reasonable carbon balance.

Adhesion of plant roots to poly-L-lysine coated polypropylene substrates.

The ability to immobilize plant tissue in a bioreactor is an important process tool. We have shown that roots of several species rapidly attach to poly-L-lysine coated polypropylene mesh in a liquid environment. Using transformed roots of Artemisia annua as a model, the attachment process was found to be enhanced by sheep serum, but not BSA and inhibited by excess Mn(2+), but unaffected by Ca(2+) or Mg(2+). Attempts to characterize the molecule(s) responsible for binding using lectins and antibodies showed that the binding site does not appear to be glycosylated or vitronectin-like. This method of rapid attachment should prove useful for controlled immobilization of roots in bioreactors.