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1.  Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing 
BMC Genomics  2009;10:465.
Background
Glandular trichomes produce a wide variety of commercially important secondary metabolites in many plant species. The most prominent anti-malarial drug artemisinin, a sesquiterpene lactone, is produced in glandular trichomes of Artemisia annua. However, only limited genomic information is currently available in this non-model plant species.
Results
We present a global characterization of A. annua glandular trichome transcriptome using 454 pyrosequencing. Sequencing runs using two normalized cDNA collections from glandular trichomes yielded 406,044 expressed sequence tags (average length = 210 nucleotides), which assembled into 42,678 contigs and 147,699 singletons. Performing a second sequencing run only increased the number of genes identified by ~30%, indicating that massively parallel pyrosequencing provides deep coverage of the A. annua trichome transcriptome. By BLAST search against the NCBI non-redundant protein database, putative functions were assigned to over 28,573 unigenes, including previously undescribed enzymes likely involved in sesquiterpene biosynthesis. Comparison with ESTs derived from trichome collections of other plant species revealed expressed genes in common functional categories across different plant species. RT-PCR analysis confirmed the expression of selected unigenes and novel transcripts in A. annua glandular trichomes.
Conclusion
The presence of contigs corresponding to enzymes for terpenoids and flavonoids biosynthesis suggests important metabolic activity in A. annua glandular trichomes. Our comprehensive survey of genes expressed in glandular trichome will facilitate new gene discovery and shed light on the regulatory mechanism of artemisinin metabolism and trichome function in A. annua.
doi:10.1186/1471-2164-10-465
PMCID: PMC2763888  PMID: 19818120
2.  Investigation of Glandular Trichome Proteins in Artemisia annua L. Using Comparative Proteomics 
PLoS ONE  2012;7(8):e41822.
Glandular secreting trichomes (GSTs) are called biofactories because they are active in synthesizing, storing and secreting various types of plant secondary metabolites. As the most effective drug against malaria, artemisinin, a sesquiterpene lactone is derived from GSTs of Artemisia annua. However, low artemisinin content (0.001%∼1.54% of dry weight) has hindered its wide application. We investigate the GST-expressed proteins in Artemisia annua using a comparative proteomics approach, aiming for a better understanding of the trichome proteome and arteminisin metabolism. 2D-electrophoresis was employed to compare the protein profiles of GSTs and leaves. More than 700 spots were resolved for GSTs, of which ∼93 non-redundant proteins were confidently identified by searching NCBI and Artemisia EST databases. Over 70% of these proteins were highly expressed in GTSs. Functional classification of these GSTs enriched proteins revealed that many of them participate in major plant metabolic processes such as electron transport, transcription and translation.
doi:10.1371/journal.pone.0041822
PMCID: PMC3414485  PMID: 22905110
3.  Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua 
BMC Plant Biology  2013;13:220.
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.
doi:10.1186/1471-2229-13-220
PMCID: PMC3878173  PMID: 24359620
Artemisia annua; Artemisinin; RNASeq; Glandular trichomes; Filamentous trichomes; Laser microdissection pressure catapulting; MEP pathway; Mevalonate pathway; Lipid biosynthesis; Terpene biosynthesis
4.  The effect of roots and media constituents on trichomes and artemisinin production in Artemisia annua L 
Plant cell reports  2012;32(2):207-218.
Artemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.
doi:10.1007/s00299-012-1355-4
PMCID: PMC3551342  PMID: 23085820
Auxin; Cytokinin; Trichomes; Artemisinin; Terpene; Roots
5.  Differentially Expressed Genes during Contrasting Growth Stages of Artemisia annua for Artemisinin Content 
PLoS ONE  2013;8(4):e60375.
Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling - negligible artemisinin, mature leaf - high artemisinin). The SCAR-marked artemisinin-rich (∼1.2%) Indian variety ‘CIM-Arogya’ was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.
doi:10.1371/journal.pone.0060375
PMCID: PMC3616052  PMID: 23573249
6.  Dried Whole Plant Artemisia annua as an Antimalarial Therapy 
PLoS ONE  2012;7(12):e52746.
Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality.
doi:10.1371/journal.pone.0052746
PMCID: PMC3527585  PMID: 23289055
7.  Plant Glandular Trichomes as Targets for Breeding or Engineering of Resistance to Herbivores 
Glandular trichomes are specialized hairs found on the surface of about 30% of all vascular plants and are responsible for a significant portion of a plant’s secondary chemistry. Glandular trichomes are an important source of essential oils, i.e., natural fragrances or products that can be used by the pharmaceutical industry, although many of these substances have evolved to provide the plant with protection against herbivores and pathogens. The storage compartment of glandular trichomes usually is located on the tip of the hair and is part of the glandular cell, or cells, which are metabolically active. Trichomes and their exudates can be harvested relatively easily, and this has permitted a detailed study of their metabolites, as well as the genes and proteins responsible for them. This knowledge now assists classical breeding programs, as well as targeted genetic engineering, aimed to optimize trichome density and physiology to facilitate customization of essential oil production or to tune biocide activity to enhance crop protection. We will provide an overview of the metabolic diversity found within plant glandular trichomes, with the emphasis on those of the Solanaceae, and of the tools available to manipulate their activities for enhancing the plant’s resistance to pests.
doi:10.3390/ijms131217077
PMCID: PMC3546740  PMID: 23235331
glandular trichome; plant-herbivore interactions; pathogen; Solanaceae; pest resistance; plant defense; leaf hair; tomato; plant breeding; genetic engineering
8.  Survey of artemisinin production by diverse Artemisia species in northern Pakistan 
Malaria Journal  2010;9:310.
Background
Artemisinin is the current drug of choice for treatment of malaria and a number of other diseases. It is obtained from the annual herb, Artemisia annua and some microbial sources by genetic engineering. There is a great concern that the artemisinin production at current rate will not meet the increasing demand by the pharmaceutical industry, so looking for additional sources is imperative.
Methods
In current study, artemisinin concentration was analysed and compared in the flowers, leaves, roots and stems of Artemisia annua and 14 other Artemisia species including two varieties each for Artemisia roxburghiana and Artemisia dracunculus using high performance liquid chromatography (HPLC).
Results
The highest artemisinin concentration was detected in the leaves (0.44 ± 0.03%) and flowers (0.42 ± 0.03%) of A. annua, followed by the flowers (0.34 ± .02%) of A. bushriences and leaves (0.27 ± 0%) of A. dracunculus var dracunculus. The average concentration of artemisinin varied in the order of flowers > leaves > stems > roots.
Conclusion
This study identifies twelve novel plant sources of artemisinin, which may be helpful for pharmaceutical production of artemisinin. This is the first report of quantitative comparison of artemisinin among a large number of Artemisia species.
doi:10.1186/1475-2875-9-310
PMCID: PMC2989329  PMID: 21047440
9.  Response of Sunflower (Helianthus annuus L.) Leaf Surface Defenses to Exogenous Methyl Jasmonate 
PLoS ONE  2012;7(5):e37191.
Helianthus annuus, the common sunflower, produces a complex array of secondary compounds that are secreted into glandular trichomes, specialized structures found on leaf surfaces and anther appendages of flowers. The primary components of these trichome secretions are sesquiterpene lactones (STL), a diverse class of compounds produced abundantly by the plant family Compositae and believed to contribute to plant defense against herbivory. We treated wild and cultivated H. annuus accessions with exogenous methyl jasmonate, a plant hormone that mediates plant defense against insect herbivores and certain classes of fungal pathogens. The wild sunflower produced a higher density of glandular trichomes on its leaves than the cultivar. Comparison of the profiles of glandular trichome extracts obtained by liquid chromatography–mass spectroscopy (LC-MS) showed that wild and cultivated H. annuus were qualitatively similar in surface chemistry, although differing in the relative size and proportion of various compounds detected. Despite observing consistent transcriptional responses to methyl jasmonate treatment, we detected no significant effect on glandular trichome density or LC-MS profile in cultivated or wild sunflower, with wild sunflower exhibiting a declining trend in overall STL production and foliar glandular trichome density of jasmonate-treated plants. These results suggest that glandular trichomes and associated compounds may act as constitutive defenses or require greater levels of stimulus for induction than the observed transcriptional responses to exogenous jasmonate. Reduced defense investment in domesticated lines is consistent with predicted tradeoffs caused by selection for increased yield; future research will focus on the development of genetic resources to explicitly test the ecological roles of glandular trichomes and associated effects on plant growth and fitness.
doi:10.1371/journal.pone.0037191
PMCID: PMC3356381  PMID: 22623991
10.  Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L 
PLoS ONE  2013;8(11):e80643.
In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.
doi:10.1371/journal.pone.0080643
PMCID: PMC3838408  PMID: 24278301
11.  Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry 
Journal of Experimental Botany  2009;61(4):1053-1064.
Trichomes are specialized epidermal structures that function as physical and chemical deterrents against arthropod herbivores. Aerial tissues of cultivated tomato (Solanum lycopersicum) are populated by several morphologically distinct trichome types, the most abundant of which is the type VI glandular trichome that produces various specialized metabolites. Here, the effect of the hairless (hl) mutation on trichome density and morphology, chemical composition, and resistance to a natural insect herbivore of tomato was investigated. The results show that the major effect of hl on pubescence results from structural distortion (bending and swelling) of all trichome types in aerial tissues. Leaf surface extracts and isolated type VI glands from hl plants contained wild-type levels of monoterpenes, glycoalkaloids, and acyl sugars, but were deficient in sesquiterpene and polyphenolic compounds implicated in anti-insect defence. No-choice bioassays showed that hl plants are compromised in resistance to the specialist herbivore Manduca sexta. These results establish a link between the morphology and chemical composition of glandular trichomes in cultivated tomato, and show that hl-mediated changes in these leaf surface traits correlate with decreased resistance to insect herbivory.
doi:10.1093/jxb/erp370
PMCID: PMC2826649  PMID: 20018901
Acyl sugar; herbivory; jasmonate; phenolics; plant defence; plant–insect interaction; secondary metabolite; terpene; tomato; trichome
12.  The Use of Combining Ability Analysis to Identify Elite Parents for Artemisia annua F1 Hybrid Production 
PLoS ONE  2013;8(4):e61989.
Artemisia annua is an important medicinal crop used for the production of the anti-malarial compound artemisinin. In order to assist in the production of affordable high quality artemisinin we have carried out an A. annua breeding programme aimed at improving artemisinin concentration and biomass. Here we report on a combining ability analysis of a diallel cross to identify robust parental lines for hybrid breeding. The parental lines were selected based on a range of phenotypic traits to encourage heterosis. The general combining ability (GCA) values for the diallel parental lines correlated to the positive alleles of quantitative trait loci (QTL) in the same parents indicating the presence of beneficial alleles that contribute to parental performance. Hybrids generated from crossing specific parental lines with good GCA were identified as having an increase in both artemisinin concentration and biomass when grown either in glasshouse or experimental field trials and compared to controls. This study demonstrates that combining ability as determined by a diallel cross can be used to identify elite parents for the production of improved A. annua hybrids. Furthermore, the selection of material for breeding using this approach was found to be consistent with our QTL-based molecular breeding approach.
doi:10.1371/journal.pone.0061989
PMCID: PMC3633910  PMID: 23626762
13.  Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid 
BMC Biotechnology  2008;8:83.
Background
Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.
Results
Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.
Conclusion
The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.
doi:10.1186/1472-6750-8-83
PMCID: PMC2588579  PMID: 18983675
14.  Artemisinin biosynthesis and its regulatory enzymes: Progress and perspective 
Pharmacognosy Reviews  2011;5(10):189-194.
Artemisinin is an endoperoxidized sesquiterpene from the Chinese medicinal plant Artemisia annua, used as an effective anti-malarial drug. Its biosynthesis pathway has been investigated for many years for scientific interest and for potential manufacturing applications to fulfill the market demand. A number of regulatory enzymes of its biosynthesis process, including amorpha-4,11-diene synthase, CYP71AV1 and cytochrome P450 reductase, have been obtained and utilized to increase the content of artemisinin. However, a large knowledge gap still exists, and certain points of controversy have stirred debates within the field. In this review, the progress and perspective of artemisinin biosynthesis and its regulating enzymes are described.
doi:10.4103/0973-7847.91118
PMCID: PMC3263054  PMID: 22279377
Artemisinin; biosynthesis pathway; progress and perspective; regulatory enzymes
15.  Artemisinin production in Artemisia annua: studies in planta and results of a novel delivery method for treating malaria and other neglected diseases 
Artemisia annua L. produces the sesquiterpene lactone, artemisinin, a potent antimalarial drug that is also effective in treating other parasitic diseases, some viral infections and various neoplasms. Artemisinin is also an allelopathic herbicide that can inhibit the growth of other plants. Unfortunately, the compound is in short supply and thus, studies on its production in the plant are of interest as are low cost methods for drug delivery. Here we review our recent studies on artemisinin production in A. annua during development of the plant as it moves from the vegetative to reproductive stage (flower budding and full flower formation), in response to sugars, and in concert with the production of the ROS, hydrogen peroxide. We also provide new data from animal experiments that measured the potential of using the dried plant directly as a therapeutic. Together these results provide a synopsis of a more global view of regulation of artemisinin biosynthesis in A. annua than previously available. We further suggest an alternative low cost method of drug delivery to treat malaria and other neglected tropical diseases.
doi:10.1007/s11101-010-9166-0
PMCID: PMC3106422  PMID: 21643453
Artemisinin pharmacokinetics; ROS; DMSO; Artemisia annua development; Trichomes
16.  Coartem®: the journey to the clinic 
Malaria Journal  2009;8(Suppl 1):S3.
Artemisinin, from which the artemether component of Coartem®(artemether/lumefantrine, AL) is derived, is obtained from the plant sweet wormwood (Artemisia annua) which has been used for over 2,000 years as a Chinese herbal remedy. Artemisinin was first identified by Chinese researchers as the active anti-malarial constituent of A. annua and its derivatives were found to be the most potent of all anti-malarial drugs. Artemether acts rapidly, reducing the infecting parasite biomass by approximately 10,000-fold per asexual life cycle. Lumefantrine, the other active constituent of AL, acts over a longer period to eliminate the residual 100-100,000 parasites that remain after artemether is cleared from the body and thus minimizes the risk of recrudescence. The two agents have different modes of action and act at different points in the parasite life cycle and show a synergistic action against Plasmodium falciparum in vitro. The combination of artemether and lumefantrine reduces the risk of resistance developing to either agent, and to date there are no reports of resistance to AL combined therapy in the malaria parasite that infects humans. Following a unique partnership agreement between Chinese authorities and Novartis, the manufacturer of AL, over 20 sponsored clinical studies have been undertaken in various malaria endemic regions and in travellers. These trials have involved more than 3,500 patients (including over 2,000 children), and led to identification of a six-dose, three-day regimen as the optimal dosing strategy for AL in uncomplicated falciparum malaria. AL has consistently shown 28-day polymerase chain (PCR)-corrected cure rates greater than 95% in the evaluable population, meeting WHO recommendations. More recently, Novartis and the Medicines for Malaria Venture have worked in partnership to develop Coartem® Dispersible, a new formulation designed specifically to meet the specific needs of children with malaria. The dispersible tablets have shown similar high response rates to those observed with crushed standard tablets of AL. A partnership agreement between Novartis and WHO has seen over 250 million AL (Coartem®) treatments (75% for children) being distributed to malaria patients in developing countries without profit, supported by training programmes and educational resources.
doi:10.1186/1475-2875-8-S1-S3
PMCID: PMC2760238  PMID: 19818170
17.  Promoter of a cotton fibre MYB gene functional in trichomes of Arabidopsis and glandular trichomes of tobacco 
Journal of Experimental Botany  2008;59(13):3533-3542.
Cotton fibres are unicellular seed trichomes. Our previous study suggested that the cotton R2R3 MYB transcript factor GaMYB2 is a functional homologue of the Arabidopsis trichome regulator GLABRA1 (GL1). Here, the GaMYB2 promoter activity is reported in cotton (Gossypium hirsutum), tobacco (Nicotiana tabacum), and Arabidopsis plants. A 2062 bp promoter of GaMYB2 was isolated from G. arboreum, and fused to a β-glucuronidase (GUS) reporter gene. In cotton, the GaMYB2 promoter exhibited activities in developing fibre cells and trichomes of other aerial organs, including leaves, stems and bracts. In Arabidopsis the promoter was specific to trichomes. Different from Arabidopsis and cotton that have unicellular non-glandular simple trichomes, tobacco plants contain more than one type of trichome, including multicellular simple and glandular secreting trichomes (GSTs). Interestingly, in tobacco plants the GaMYB2 promoter directed GUS expression exclusively in glandular cells of GSTs. A series of 5′-deletions revealed that a 360 bp fragment upstream to the translation initiation codon was sufficient to drive gene expression. A putative cis-element of the T/G-box was located at -233 to -214; a yeast one-hybrid assay showed that Arabidopsis bHLH protein GLABRA3 (GL3), also a trichome regulator, and GhDEL65, a GL3-like cotton protein, had high binding activities to the T/G-box motif. Overexpression of GL3 or GhDEL65 enhanced the GaMYB2 promoter activity in transgenic Arabidopsis plants. A comparison of GaMYB2 promoter specificities in trichomes of different plant species with different types of trichomes provides a tool for further dissection of plant trichome structure and development.
doi:10.1093/jxb/ern204
PMCID: PMC2561158  PMID: 18711121
Cotton fibre; glandular; MYB; promoter; tobacco; trichome
18.  The presence of amorpha-4, 11-diene synthase, a key enzyme in artemisinin production in ten Artemisia species 
Background and the purpose of the study
Artemisinin is one of the most effective medicine against malaria, which is produced naturally by Artemisia annua in low yield. It is produced in a metabolic pathway, in which several genes and gene products are involved. One of the key genes in this pathway is am1, which encodes amorpha-4, 11-diene synthase (ADS), a key enzyme in artemisinin biosynthesis pathway. The aim of this study was to determine the presence of this gene in ten Artemisia species in order to increase the yield of production of Artemisinin.
Methods
The experiments were carried out using PCR. Specific primers were designed based on the published am1 gene sequence obtained from A. annua (NCBI, accession number AF327527).
Results
The amplification of this gene by the specific primers was considered as a positive sign for the potentiality of artemisinin production. Since the entire am1 gene was not amplified in any of the 10 species used, four parts of the gene, essential in ADS enzyme function, corresponding to a) pair site of Arg10-Pro12 in the first 100 amino acids, b) aspartate rich motif (DDXXD), c) active site final lid and d) active site including farnesyl diphosphate (FDP) ionization sites and catalytic site in the ADS enzyme, were investigated.
Major conclusion
The sequence corresponding to ADS active site was amplified only in A. annua, A. aucheri and A. chamaemelifolia. The negative results obtained with other species could be due to some sequence alteration, such as point mutations or INDELs. We propose A. aucheri and A. chamaemelifolia as two potential candidate species for further characterization, breeding and transferring am1 gene for artemisinin overproduction.
PMCID: PMC3304396  PMID: 22615678
amorpha-4,11-diene synthase; Artemisia (sweet wormwood); artemisinin; Asteraceae; PCR
19.  An Endophytic Pseudonocardia Species Induces the Production of Artemisinin in Artemisia annua 
PLoS ONE  2012;7(12):e51410.
Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants.
doi:10.1371/journal.pone.0051410
PMCID: PMC3520919  PMID: 23251523
20.  Isolation, characterization and structural studies of amorpha ― 4, 11­diene synthase (ADS3963) from Artemisia annua L. 
Bioinformation  2010;4(9):421-429.
With the escalating prevalence of malaria in recent years, artemisinin demand has placed considerable stress on its production worldwide. At present, the relative low­yield of artemisinin (0.01­1.1 %) in the source plant (Artemisia annua L. plant) has imposed a serious limitation in commercializing the drug. Amorpha­4, 11­diene synthase (ADS) has been reported a key enzyme in enhancing the artemisinin level in Artemisia annua L. An understanding of the structural and functional correlations of Amorpha­4, 11­diene synthase (ADS) may therefore, help in the molecular up­regulation of the enzyme. In this context, an in silico approach was used to study the ADS3963 (3963 bp) gene cloned by us, from high artemisinin (0.7­0.9% dry wt basis) yielding strain of A. annua L. The full­length putative gene of ADS3963 was found to encode a protein consisting of 533 amino acid residues with conserved aspartate rich domain. The isoelectric point (pI) and molecular weight of the protein were 5.25 and 62.2 kDa, respectively. The phylogenetic analysis of ADS genes from various species revealed evolutionary conservation. Homology modeling method was used for prediction of the 3D structure of ADS3963 protein and Autodock 4.0 version was used to study the ligand binding. The predicted 3D model and docking studies may further be used in characterizing the protein in wet laboratory.
PMCID: PMC2951637  PMID: 20975893
Artemisia annua; artemisinin; ADS3963 gene; homology modeling; phylogenetic tree; docking
21.  Antimicrobial Activity of Artemisinin and Precursor Derived from In Vitro Plantlets of Artemisia annua L. 
BioMed Research International  2014;2014:215872.
Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities.
doi:10.1155/2014/215872
PMCID: PMC3915762  PMID: 24575401
22.  Mapping the potential distribution of high artemisinin-yielding Artemisia annua L. (Qinghao) in China with a geographic information system 
Chinese Medicine  2010;5:18.
Background
Artemisia annua L. is an important source for artemisinin, a potent drug for treating malaria. This study aims to map and predict the potential geographic distribution of A. annua L. in China.
Methods
The Geographic Information System for traditional Chinese medicine (TCM-GIS) was developed and used to map the potential geographic distribution of A. annua L.
Results
Climatic, edaphic and topographic characteristics of A. annua L. microhabitats in Youyang County were mapped to find distribution patterns. The maps identified that certain habitats in the Chongqing region and some potential regions, especially in Guizhou Province, possess similarity indices of ≥98%. In particular, high quality microhabitats A. annua L. were found in the Wuling mountains region.
Conclusion
The present study demonstrates a GIS approach to predict potential habitats for A. annua L. TCM-GIS is a powerful tool for assessing bioclimatic suitability for medicinal plants.
doi:10.1186/1749-8546-5-18
PMCID: PMC2886037  PMID: 20470438
23.  Recent Advances in Artemisinin Production Through Heterologous Expression 
Current medicinal chemistry  2008;15(27):2886.
Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases.
PMCID: PMC2821817  PMID: 18991643
Amorpha diene; CYP71 AV1; terpenoid; genetic engineering; malaria
24.  Whole plant extracts versus single compounds for the treatment of malaria: synergy and positive interactions 
Malaria Journal  2011;10(Suppl 1):S4.
Background
In traditional medicine whole plants or mixtures of plants are used rather than isolated compounds. There is evidence that crude plant extracts often have greater in vitro or/and in vivo antiplasmodial activity than isolated constituents at an equivalent dose. The aim of this paper is to review positive interactions between components of whole plant extracts, which may explain this.
Methods
Narrative review.
Results
There is evidence for several different types of positive interactions between different components of medicinal plants used in the treatment of malaria. Pharmacodynamic synergy has been demonstrated between the Cinchona alkaloids and between various plant extracts traditionally combined. Pharmacokinetic interactions occur, for example between constituents of Artemisia annua tea so that its artemisinin is more rapidly absorbed than the pure drug. Some plant extracts may have an immunomodulatory effect as well as a direct antiplasmodial effect. Several extracts contain multidrug resistance inhibitors, although none of these has been tested clinically in malaria. Some plant constituents are added mainly to attenuate the side-effects of others, for example ginger to prevent nausea.
Conclusions
More clinical research is needed on all types of interaction between plant constituents. This could include clinical trials of combinations of pure compounds (such as artemisinin + curcumin + piperine) and of combinations of herbal remedies (such as Artemisia annua leaves + Curcuma longa root + Piper nigum seeds). The former may enhance the activity of existing pharmaceutical preparations, and the latter may improve the effectiveness of existing herbal remedies for use in remote areas where modern drugs are unavailable.
doi:10.1186/1475-2875-10-S1-S4
PMCID: PMC3059462  PMID: 21411015
25.  Effect of Sugars on Artemisinin Production in Artemisia annua L.: Transcription and Metabolite Measurements 
Molecules (Basel, Switzerland)  2010;15(4):2302-2318.
The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.
doi:10.3390/molecules15042302
PMCID: PMC3939791  PMID: 20428043
terpene; sugar signal; secondary metabolism

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