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Artemisia annua , commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A . annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A . annua , which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A . annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A . annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A . annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin. [ABSTRACT FROM AUTHOR]

The plant Artemisia annua is well known due to the production of artemisinin, a sesquiterpene lactone that is widely used in malaria treatment. Phytohormones play important roles in plant secondary metabolism, such as jasmonic acid ( JA), which can induce artemisinin biosynthesis in A. annua. Nevertheless, the JA-inducing mechanism remains poorly understood., The expression of gene Aa MYC2 was rapidly induced by JA and Aa MYC2 binds the G-box-like motifs within the promoters of gene CYP71 AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway., Overexpression of Aa MYC2 in A. annua significantly activated the transcript levels of CYP71 AV1 and DBR2, which resulted in an increased artemisinin content. By contrast, artemisinin content was reduced in the RNAi transgenic A. annua plants in which the expression of Aa MYC2 was suppressed. Meanwhile, the RNAi transgenic A. annua plants showed lower sensitivity to methyl jasmonate treatment than the wild-type plants., These results demonstrate that Aa MYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production. [ABSTRACT FROM AUTHOR]

Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives. Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin. In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research. [ABSTRACT FROM AUTHOR]

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] Southern-blot analyses showed that ACR1, ACR10, ACR31 and ACR52 had low copies of the integration transgenes. [•] RT-Q-PCR showed that the expression levels of the three genes were significantly increased in transgenic A. annua plants. [•] HPLC showed that artemisinin content of ACR16 was found to contain 2.4-fold higher (15.1mg/g DW) than the control. [•] Overexpression of those three genes promoted the metabolic flux flows toward biosynthesis of artemisinin. [ABSTRACT FROM AUTHOR]

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua. [ABSTRACT FROM AUTHOR]

Plants regulate the synthesis of specialized compounds through the actions of individual transcription factors (TFs) or sets of TFs. One such compound, artemisinin from Artemisia annua, is widely used as a pharmacological product in the first-line treatment of malaria. However, the emergence of resistance to artemisinin in Plasmodium species, as well as its low production rates, have required innovative treatments such as exploiting the synergistic effects of flavonoids with artemisinin. We overview current knowledge about flavonoid and artemisinin transcriptional regulation in A. annua , and review the dual action of TFs and structural genes that can regulate both pathways simultaneously. Understanding the concerted action of these TFs and their associated structural genes can guide the development of strategies to further improve flavonoid and artemisinin production. It is increasingly recognized that different TFs act conjointly to regulate the production of plant metabolites. However, their dual functionality in regulating distinct pathways of synthesis has not been well studied. The identification of cis- regulatory elements within the promoter regions of genes encoding different pathway enzymes enables the identification of TF families that regulate their expression. Parallel regulation of the enzyme-coding genes for distinct pathways can simplify overexpression strategies. This provides an excellent starting point to unravel how individual TFs act in a coordinated manner to simultaneously regulate different pathways of specialized metabolism. [ABSTRACT FROM AUTHOR]

Artemisinin, a sesquiterpene lactone obtained from Artemisia annua , is an essential therapeutic against malaria. YABBY family transcription factor AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450–dependent hydroxylase) and AaDBR2 (double-bond reductase 2); however, the protein–protein interactions of AaYABBY5, as well as the mechanism of its regulation, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome–specific WRKY1) and AaDBR2 (double-bond reductase 2). In this study, YABBY–WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Toward the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1 and AaDBR2 promoters, respectively. In AaYABBY5 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti- AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY–WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

The glandular secretory trichomes ( GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood., To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis ( MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, Aa MIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of Aa MIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants., Transcriptome analysis and cuticular lipid profiling showed that Aa MIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that Aa MIXTA1 could directly activate the expression of genes related to cuticle biosynthesis., Taken together, Aa MIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant. [ABSTRACT FROM AUTHOR]

Artemisinin is a type of sesquiterpene lactone well known as an antimalarial drug, and is specifically produced in glandular trichomes of Artemisia annua. However, the regulatory network for the artemisinin biosynthetic pathway remains poorly understood. Exploration of trichome-specific transcription factors would facilitate the elucidation of regulatory mechanism of artemisinin biosynthesis., The WRKY transcription factor GLANDULAR TRICHOME- SPECIFIC WRKY 1 ( Aa GSW1) was cloned and analysed in A. annua. Aa GSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ ERF transcription factor Aa ORA. A β-glucuronidase (GUS) staining assay further demonstrated that Aa GSW1 is a glandular trichome-specific transcription factor., Aa GSW1 positively regulates CYP71 AV1 and Aa ORA expression by directly binding to the W-box motifs in their promoters. Overexpression of Aa GSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, Aa GSW1 can be directly regulated by Aa MYC2 and Aab ZIP1, which are positive regulators of jasmonate ( JA)- and abscisic acid ( ABA)-mediated artemisinin biosynthetic pathways, respectively., These results demonstrate that Aa GSW1 is a glandular trichome-specific WRKY transcription factor and a positive regulator in the artemisinin biosynthetic pathway. Moreover, we propose that two trifurcate feed-forward pathways involving Aa GSW1, CYP71 AV1 and Aa MYC2/ Aab ZIP1 function in the JA/ ABA response in A. annua. [ABSTRACT FROM AUTHOR]

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-β-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. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA) was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase), CYP71AV1 (cytochrome P450 dependent hydroxylase) and DBR2 (double bond reductase 2) were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A.annua. [ABSTRACT FROM AUTHOR]

Artemisinin has attracted interest due to its medicinal value in treating malaria and its potential for use against certain cancers and viral diseases. Trichome density and capacity determine artemisinin content in Artemisia annua plants. Thus, the ATP-binding cassette transporter G (ABCG) subfamily involved in trichome cuticle development may also influence artemisinin accumulation. In this study, putative A. annua ABC transporter unigenes were identified and classified from the unigene sequences up to date in the National Center for Biotechnology Information database, and nine putative A. annua ABCG transporter unigenes that may be involved in cuticle development were selected for expression analyses. Two of them, AaABCG6 and AaABCG7, showed parallel expression pattern as two artemisinin biosynthesis-specific genes (amorpha-4, 11-diene synthase and a cytochrome P450-dependent hydroxylase, CYP71AV1) in different tissues and different leaf development stages and also showed similar induction in the plants after methyl jasmonate or abscisic acid treatments. Identification of these putative A. annua ABCG transporter unigenes could provide the basis for cloning of the full-length genes and further functional investigation to find the artemisinin relevant transporters, which could be used for improving artemisinin yield in both A. annua plants and heterologous systems using transgenic technology. [ABSTRACT FROM AUTHOR]

emisinin is currently the best therapeutic against both drug-resistant and cerebral-malaria-causing strains of Plasmodium falciparum. Allene oxide cyclase (AOC) is the key enzyme in the biosynthetic pathway of jasmonates. In this study, a full-length cDNA of AOC gene (named as AaAOC) was cloned from Artemisia annua L. AaAOC was 1,007 bp, containing an open reading frame (750 bp) encoding 249 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of AaAOC was highly homologous to AOC from other plant species. Phylogenetic analysis indicated that AaAOC was clustered in a closely related subgroup with AOC of Camptotheca acuminata. Southern blot analysis revealed that AaAOC was a multicopy gene. Reverse transcriptase-polymerase chain reaction (RT-PCR) and QPCR analysis showed that AaAOC mRNA accumulated most abundantly in alabastrums, in which the content of artemisinin was previously proven to be the highest. RT-PCR analysis revealed that MeJA, ABA, and ethylene treatments significantly enhanced AaAOC transcript expression. And the results of HPLC showed that the contents of artemisinin were greatly increased after the treatments of ABA and MeJA. [ABSTRACT FROM AUTHOR]

Summary: Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1 , thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90–120% and decreased by 20–60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production. [ABSTRACT FROM AUTHOR]

Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions. [ABSTRACT FROM AUTHOR]

Artemisia annua is the only natural source of the sesquiterpenoid artemisinin, which is widely used to treat malaria. The phytohormone jasmonic acid (JA) can significantly promote artemisinin biosynthesis in A. annua. AabHLH1 can bind and activate artemisinin biosynthetic genes, such as AaADS and AaCYP71AV1. In this study, we proved that AabHLH1 was responsive to MeJA treatment and highly expressed in glandular trichome‐enriched tissues, and that its expression profile was similar to that of AaADS. Yeast two‐hybrid assays showed that AabHLH1 interacted with all nine AaJAZ proteins in A. annua. Functional analysis with transgenic plants showed that several artemisinin biosynthetic genes were upregulated in AabHLH1‐OE transgenic A. annua lines and downregulated in AabHLH1‐EAR lines; furthermore, the artemisinin content was increased in the AabHLH1‐OE lines and decreased in the AabHLH1‐EAR lines. These results demonstrate that the JA‐induced AabHLH1 positively regulates artemisinin biosynthesis by regulating the biosynthetic genes, and thus provide new insight into the regulatory mechanism of JA‐induced artemisinin biosynthesis in A. annua. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua , which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A . annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1 . Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1 , which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua . Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production. [ABSTRACT FROM AUTHOR]

Amorpha-4,11-diene synthase (ADS) is the first key enzyme of artemisinin biosynthetic pathway in Artemisia annua L. In this study, the promoter region of the ADS gene has been cloned and used to demonstrate the expression of GUS reporter gene in both glandular trichomes of A. annua and non-glandular trichomes of Arabidopsis thaliana following homologous and heterologous expression of ADS promoter- GUS fusion. Subsequently, 5′ sequential deletion analysis of the ADS promoter revealed that a short sequence, −350 upstream of the transcription start site, was sufficient for trichome-specific expression in A. thaliana and that the region from −350 to −300 contained essential elements for this observed specificity. However, frequencies of transgenic A. thaliana plants displaying trichome-specific expressions varied between different lines, and all the lines with deleted fragments of the ADS promoter showed lower frequencies than the line with full-length ADS promoter. Most lines with deleted ADS promoter- GUS fusions showed GUS expressions in the guard cells of stomata as well, which was not observed in A. thaliana plants transformed with the full-length ADS promoter. GUS activities varied among different transgenic lines as well, both in transiently transformed Nicotiana benthamiana and stably transformed A. thaliana, with promoter-deletion lines exhibiting higher GUS activities than the full-length ADS promoter line. [ABSTRACT FROM AUTHOR]

Artemisinin is widely used as an antimalarial drug around the world. Artemisinic aldehyde Δ11(13) reductase (DBR2) is a key enzyme which reduces artemisinic aldehyde to dihydroartemisinic aldehyde in the biosynthesis of artemisinin. In this study, two fragments encompassing a putative promoter of DBR2, designated as DBR2pro1 and DBR2pro2, were isolated using genomic DNA walking. The transcription start site and the putative cis-elements of each version of promoter were predicted using bioinformatic analysis. In order to study the function of the cloned promoter, Artemisia annua was transformed with β-glucuronidase (GUS) reporter gene driven by DBR2pro1 and DBR2pro2, respectively. GUS staining results demonstrated that both DBR2pro1 and DBR2pro2 were strongly expressed in glandular secretory trichomes (GSTs) of leaf primordia and flower buds, but were not obviously expressed in roots, stems, old leaves, and fully developed flowers, thus indicating that the two versions of promoter were functional and specifically expressed in GSTs. [ABSTRACT FROM AUTHOR]

Six transcription factors of APETALA2/ethylene-response factor ( AP2/ ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction ( RT- Q- PCR) showed that Aa ORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene ( ADS), cytochrome P450-dependent hydroxylase gene ( CYP71AV1) and double bond reductase 2 gene ( DBR2) in different tissues of A. annua., Aa ORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes ( GSTs) and nonglandular T-shaped trichomes ( TSTs) of A. annua. The result of subcellular localization shows that Aa ORA is targeted to the nuclei and the cytoplasm., Overexpression and RNA interference ( RNAi) of Aa ORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71 AV1, DBR2 and Aa ERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that Aa ORA is a positive regulator in the biosynthesis of artemisinin., Overexpression of Aa ORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 ( PDF1.2), HEVEIN-LIKE PROTEIN ( HEL) and BASIC CHITINASE ( B-CHI). After inoculation with Botrytis cinerea, the phenotypes of Aa ORA overexpression in A. thaliana and Aa ORA RNAi in A. annua demonstrate that Aa ORA is a positive regulator of disease resistance to B. cinerea. [ABSTRACT FROM AUTHOR]

Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway. [ABSTRACT FROM AUTHOR]