Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

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.

Comprehensive Map of the Artemisia annua Proteome and Quantification of Differential Protein Expression in Chemotypes Producing High versus Low Content of Artemisinin.

Artemisia annua is well known for biosynthesizing the antimalarial drug artemisinin. Here, a global proteomic profiling of A. annua is conducted with identification of a total of 13 403 proteins based on the genome sequence annotation database. Furthermore, a spectral library is generated to perform quantitative proteomic analysis using data independent acquisition mass spectrometry. Specifically, proteins between two chemotypes that produce high (HAP) and low (LAP) artemisinin content, respectively, are comprehensively quantified and compared. 182 proteins are identified with abundance significantly different between these two chemotypes means after the statistic use the p-value and fold change it is found 182 proteins can reach the demand conditions which represent the expression are significantly different between the high artemisnin content plants (HAPs) and the low artemisnin content plants (LAPs). Data are available via ProteomeXchange with identifier PXD015547. Overall, this current study globally identifies the proteome of A. annua and quantitatively compares the targeted sub-proteomes between the two cultivars of HAP and LAP, providing systematic information on metabolic pathways of A. annua.

A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua.

Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as 'biofactories' for producing valuable natural products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive. Here we identify a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua. We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle-related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA-like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes. Our results reveal a molecular mechanism by which a novel HD-ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation.

The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua.

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, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 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 AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis. Taken together, AaMIXTA1 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.

GLANDULAR TRICHOME-SPECIFIC WRKY 1 promotes artemisinin biosynthesis in Artemisia annua.

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 (AaGSW1) was cloned and analysed in A. annua. AaGSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ERF transcription factor AaORA. A ß-glucuronidase (GUS) staining assay further demonstrated that AaGSW1 is a glandular trichome-specific transcription factor. AaGSW1 positively regulates CYP71AV1 and AaORA expression by directly binding to the W-box motifs in their promoters. Overexpression of AaGSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, AaGSW1 can be directly regulated by AaMYC2 and AabZIP1, which are positive regulators of jasmonate (JA)- and abscisic acid (ABA)-mediated artemisinin biosynthetic pathways, respectively. These results demonstrate that AaGSW1 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 AaGSW1, CYP71AV1 and AaMYC2/AabZIP1 function in the JA/ABA response in A. annua.

HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua.

Glandular trichomes are generally considered biofactories that produce valuable chemicals. Increasing glandular trichome density is a very suitable way to improve the productivity of these valuable metabolites, but little is known about the regulation of glandular trichome formation. Phytohormone jasmonate (JA) promotes glandular trichome initiation in various plants, but its mechanism is also unknown. By searching transcription factors regulated by JA in Artemisia annua, we identified a novel homeodomain-leucine zipper transcription factor, HOMEODOMAIN PROTEIN 1 (AaHD1), which positively controls both glandular and nonglandular trichome initiations. Overexpression of AaHD1 in A. annua significantly increased glandular trichome density without harming plant growth. Consequently, the artemisinin content was improved. AaHD1 interacts with A. annua jasmonate ZIM-domain 8 (AaJAZ8), which is a repressor of JA, thereby resulting in decreased transcriptional activity. AaHD1 knockdown lines show decreased sensitivity to JA on glandular trichome initiation, which indicates that AaHD1 plays an important role in JA-mediated glandular trichome initiation. We identified a new transcription factor that promotes A. annua glandular trichome initiation and revealed a novel molecular mechanism by which a homeodomain protein transduces JA signal to promote glandular trichome initiation. Our results also suggested a connection between glandular and nonglandular trichome formations.

Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua.

The NAC (NAM, ATAF and CUC) superfamily is one of the largest plant-specific transcription factor families. NAC transcription factors always play important roles in response to various abiotic stresses. A NAC transcription factor gene AaNAC1 containing a complete open reading frame (ORF) of 864 bp was cloned from Artemisia annua. The expression of AaNAC1 could be induced by dehydration, cold, salicylic acid (SA) and methyl jasmonate (MJ), suggesting that it might be a key regulator of stress signaling pathways in A. annua. AaNAC1 was shown to be localized to the nuclei by transforming tobacco leaf epidermal cells. When AaNAC1 was overexpressed in A. annua, the content of artemisinin and dihydroartemisinic acid was increased by 79% and 150%, respectively. The expression levels of artemisinin biosynthetic pathway genes, i.e. amorpha-4,11-diene synthase (ADS), artemisinic aldehyde Δ11(13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1), were increased. Dual luciferase (dual-LUC) assays showed that AaNAC1 could activate the transcription of ADS in vivo. The transgenic A. annua exhibited increased tolerance to drought and resistance to Botrytis cinerea. When AaNAC1 was overexpressed in Arabidopsis, the transgenic Arabidopsis were markedly more tolerant to drought. The transgenic Arabidopsis showed increased resistance to B. cinerea. These results indicate that AaNAC1 can potentially be used in transgenic breeding for improving the content of artemisinin and drought tolerance in A. annua.

Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin.

There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at branching points and the artemisinin biosynthetic pathway, ß-caryophyllene, ß-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of ß-caryophyllene synthase (CPS), ß-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by ß-caryophyllene, ß-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of ß-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while ß-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.

The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua.

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 AaMYC2 was rapidly induced by JA and AaMYC2 binds the G-box-like motifs within the promoters of gene CYP71AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway. Overexpression of AaMYC2 in A. annua significantly activated the transcript levels of CYP71AV1 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 AaMYC2 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 AaMYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production.

Investigation of the chemomarkers correlated with flower colour in different organs of Catharanthus roseus using NMR-based metabolomics.

INTRODUCTION: Flower colour is a complex phenomenon that involves a wide range of secondary metabolites of flowers, for example phenolics and carotenoids as well as co-pigments. Biosynthesis of these metabolites, though, occurs through complicated pathways in many other plant organs. The analysis of the metabolic profile of leaves, stems and roots, for example, therefore may allow the identification of chemomarkers related to the final expression of flower colour. OBJECTIVE: To investigate the metabolic profile of leaves, stems, roots and flowers of Catharanthus roseus and the possible correlation with four flower colours (orange, pink, purple and red). METHODS: (1) H-NMR and multivariate data analysis were used to characterise the metabolites in the organs. RESULTS: The results showed that flower colour is characterised by a special pattern of metabolites such as anthocyanins, flavonoids, organic acids and sugars. The leaves, stems and roots also exhibit differences in their metabolic profiles according to the flower colour. Plants with orange flowers featured a relatively high level of kaempferol analogues in all organs except roots. Red-flowered plants showed a high level of malic acid, fumaric acid and asparagine in both flowers and leaves, and purple and pink flowering plants exhibited high levels of sucrose, glucose and 2,3-dihydroxy benzoic acid. High concentrations of quercetin analogues were detected in flowers and leaves of purple-flowered plants. CONCLUSIONS: There is a correlation between the metabolites specifically associated to the expression of different flower colours and the metabolite profile of other plant organs and it is therefore possible to predict the flower colours by detecting specific metabolites in leaves, stems or roots. This may have interesting application in the plant breeding industry.

Development of efficient Catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants.

BACKGROUND: As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. RESULTS: To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report ß-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 µM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. CONCLUSIONS: In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.

Basic Helix-Loop-Helix Transcription Factors AabHLH2 and AabHLH3 Function Antagonistically With AaMYC2 and Are Negative Regulators in Artemisinin Biosynthesis.

Plants have evolved sophisticated systems for regulating the biosynthesis of specialized phytochemicals. Artemisinin, which is a sesquiterpene lactone widely used in anti-malaria treatment, is produced by the Artemisia annua L. plant. However, the artemisinin content in A. annua is low and difficult to meet market demands. Studies have shown that artemisinin biosynthesis in A. annua has complex temporal and spatial specificity and is under tightly transcriptional regulation. However, the mechanism of transcriptional regulation of artemisinin biosynthesis remains unclear. In this study, we identified two MYC-type bHLH transcription factors (AabHLH2 and AabHLH3) as novel regulators of artemisinin biosynthesis. These bHLH TFs act as transcription repressors and function redundantly to negatively regulate artemisinin biosynthesis. Furthermore, AabHLH2 and AabHLH3 are nuclear proteins that bind to DNA elements with similar specificity to that of AaMYC2, but lack the conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Together, our findings reveal a novel artemisinin biosynthesis regulatory network, provide new insight into how specialized metabolites are modulated in plants, and propose a model in which different bHLH TFs coordinated in regulating artemisinin production in the plant. Finally, this study provides some useful target genes for metabolic engineering of artemisinin production via CRISPR/Cas9 gene editing.

Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-O-acetyltransferase gene promoter.

Madagascar periwinkle (Catharanthus roseus) produces many therapeutically valuable terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine derived from the monomers vindoline and catharanthine. Deacetylvindoline-4-O-acetyltransferase (DAT) is a key enzyme for the terminal step of vindoline biosynthesis. In this research, the DAT gene promoter was cloned, sequenced, and analyzed. An approximately 1,773 bp genomic DNA fragment of DAT promoter was obtained. Sequence analysis revealed that DAT promoter contained several potential regulatory elements which were involved in the regulation of gene expression. To investigate its function, the promoter fragments with 5' deletions and gain-of-function deletions were fused to GUS reporter gene, and their expressions were analyzed by transient expression in C. roseus cell suspensions. The regulatory activity of DAT promoter was identified with fluorescence quantitative assays. Three TGACG motifs and one inverted motif (CGTCA) between -808 and -1,086 bp in the DAT promoter were found to be involved in methyljasmonate signal transduction pathway.

AaABCG40 Enhances Artemisinin Content and Modulates Drought Tolerance in Artemisia annua.

The phytohormone Abscisic acid (ABA) regulates plant growth, development, and responses to abiotic stresses, including senescence, seed germination, cold stress and drought. Several kinds of researches indicate that exogenous ABA can enhance artemisinin content in A. annua. Some transcription factors related to ABA signaling are identified to increase artemisinin accumulation through activating the artemisinin synthase genes. However, no prior study on ABA transporter has been performed in A. annua. Here, we identified a pleiotropic drug resistance (PDR) transporter gene AaPDR4/AaABCG40 from A. annua. AaABCG40 was expressed mainly in roots, leaves, buds, and trichomes. GUS activity is primarily observed in roots and the vascular tissues of young leaves in proAaABCG40: GUS transgenic A. annua plants. When AaABCG40 was transferred into yeast AD12345678, yeasts expressing AaABCG40 accumulated more ABA than the control. The AaABCG40 overexpressing plants showed higher artemisinin content and stronger drought tolerance. Besides, the expression of CYP71AV1 in OE-AaABCG40 plants showed more sensitivity to exogenous ABA than that in both wild-type and iAaABCG40 plants. According to these results, they strongly suggest that AaABCG40 is involved in ABA transport in A. annua.

High-Level Patchoulol Biosynthesis in Artemisia annua L.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 µg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 µg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

CrERF5, an AP2/ERF Transcription Factor, Positively Regulates the Biosynthesis of Bisindole Alkaloids and Their Precursors in Catharanthus roseus.

Catharanthus roseus contains a variety of monoterpenoid indole alkaloids (MIAs), among which bisindole alkaloids vinblastine and vincristine are well-known to have antitumor effects and widely used in clinical treatment. However, their contents in C. roseus is extremely low and difficult to meet market demands. Therefore, it is of great significance to study the transcriptional regulation mechanism of MIAs biosynthesis for high yielding of bisindole alkaloids in C. roseus. Studies have shown that MIAs biosynthesis in C. roseus has complex temporal and spacial specificity and is under tight transcriptional regulation, especially bisindole alkaloids. In this study, an AP2/ERF transcription factor CrERF5 was selected by RNA-seq of C. roseus organs, and its full-length sequence was cloned and characterized. CrERF5 responds to both ethylene and JA signals and is localized in the nucleus. CrERF5 could activate the transcriptional activity of the TDC promoter. Transient overexpressing CrERF5 in C. roseus petals caused a significant increase of the expression levels of key genes in both the upstream and downstream pathways of MIAs biosynthesis while silencing CrERF5 resulted in a decrease of them. Accordingly, the contents of bisindole alkaloids anhydrovinblastine and vinblastine, monoindole alkaloids ajmalicine, vindoline, and catharanthine were strongly enhanced in CrERF5-overexpressing petals while their contents decreased in CrERF5-silenced plants. These results suggested that CrERF5 is a novel positive ethylene-JA-inducible AP2/ERF transcription factor upregulating the MIAs biosynthetic pathway leading to the bisindole alkaloids accumulation.

The YABBY Family Transcription Factor AaYABBY5 Directly Targets Cytochrome P450 Monooxygenase (CYP71AV1) and Double-Bond Reductase 2 (DBR2) Involved in Artemisinin Biosynthesis in Artemisia Annua.

Artemisinin is an effective antimalarial sesquiterpene lactone synthesized in Artemisia annua. Various transcription factors have been previously reported that can influence the biosynthesis of artemisinin; however, the effect of YABBY family transcription factors on artemisinin biosynthesis was unknown. In the present study, we cloned and characterized AaYABBY5: a homolog of MsYABBY5 in Mentha spicata which is involved in modulating the monoterpenes, as a positive regulator of artemisinin biosynthesis in A. annua. AaYABBY5 was found localized to the nucleus, and its expression was found to be induced by exogenous methyl jasmonic acid (MeJA) treatment. In the dual-luciferase reporter assay, it was found that AaYABBY5 significantly increased the activities of promoters of amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1) genes. Yeast one hybrid assay showed that AaYABBY5 directly bonds to the promoters of CYP71AV1 and DBR2 genes. Quantitative real-time polymerase chain reaction (qPCR) of AaYABBY5 overexpression and AaYABBY5 antisense plants revealed a significant increase in the expression of ADS, CYP71AV1, DBR2, and ALDH1 in AaYABBY5 overexpression plants and a significant decrease in the expression of these genes in AaYABBY5 antisense A. annua, respectively. Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin and its precursor dihydroartemisinic acid were significantly increased in the AaYABBY5 overexpression plants while AaYABBY5 downregulation resulted in a significant decrease in the concentration of artemisinin. Taken together, these results explicitly represent that AaYABBY5 is a positive regulator of artemisinin biosynthesis in A. annua.

AaABF3, an Abscisic Acid-Responsive Transcription Factor, Positively Regulates Artemisinin Biosynthesis in Artemisia annua.

Artemisinin is well known for its irreplaceable curative effect on the devastating parasitic disease, Malaria. This sesquiterpenoid is specifically produced in Chinese traditional herbal plant Artemisia annua. Earlier studies have shown that phytohormone abscisic acid (ABA) plays an important role in increasing the artemisinin content, but how ABA regulates artemisinin biosynthesis is still poorly understood. In this study, we identified that AaABF3 encoded an ABRE (ABA-responsive elements) binding factor. qRT-PCR analysis showed that AaABF3 was induced by ABA and expressed much higher in trichomes where artemisinin is synthesized and accumulated. To further investigate the mechanism of AaABF3 regulating the artemisinin biosynthesis, we carried out dual-luciferase analysis, yeast one-hybrid assay and electrophoretic mobility shift assay. The results revealed that AaABF3 could directly bind to the promoter of ALDH1 gene, which is a key gene in artemisinin biosynthesis, and activate the expression of ALDH1. Functional analysis revealed that overexpression of AaABF3 in A. annua enhanced the production of artemisinin, while RNA interference of AaABF3 resulted in decreased artemisinin content. Taken together, our results demonstrated that AaABF3 played an important role in ABA-regulated artemisinin biosynthesis through direct regulation of artemisinin biosynthesis gene, ALDH1.

The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis.

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.