Functional characterisation of Artemisia annua jasmonic acid carboxyl methyltransferase: a key enzyme enhancing artemisinin biosynthesis.

MAIN CONCLUSION: Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-L-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC-MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content.

Molecular understanding of anthocyanin biosynthesis activated by PAP1 and regulated by 2, 4-dichlorophenoxyacetic acid in engineered red Artemisia annua cells.

MAIN CONCLUSION: Eight promoters were cloned, from which AC and G-box cis-elements were identified. PAP1 enhanced the promoter activity. 2,4-D reduced the anthocyanin biosynthesis via downregulating the expression of the PAP1 transgene. Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-box cis-element. Eight promoters were cloned for activity analysis. Dual luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 µM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per Petri dish. HPLC-qTOF-MS/MS-based profiling showed that these concentrations did not alter anthocyanin profiles. Real-time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated the expression of the chalcone synthase gene either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua.

AaWRKY6 contributes to artemisinin accumulation during growth in Artemisia annua.

Artemisinin, which is extracted from the plant Artemisia annua L., is a crucial drug for curing malaria and has potential applications for treating cancer, diabetes, pulmonary tuberculosis, and other conditions. Demand for artemisinin is therefore high, and enhancing its yield is important. Artemisinin dynamics change during the growth cycle of A. annua; however, the regulatory networks underlying these changes are poorly understood. Here, we collected A. annua leaves at different growth stages and identified target genes from transcriptome data. We determined that WRKY6 binds to the promoters of the artemisinin biosynthesis gene artemisinic aldehyde Δ11(13) reductase (DBR2). In agreement, overexpression of WRKY6 in A. annua resulted in higher expression levels of genes in the artemisinin biosynthesis pathway and greater artemisinin contents than in the wild type. When expression of WRKY6 was down-regulated, artemisinin biosynthesis pathway genes were also down-regulated and the content of artemisinin was lower. WRKY6 mediates the transcriptional activation of artemisinin biosynthesis by binding to the promoter of DBR2, making it a key regulator for modulating the dynamics of artemisinin changes during the A. annua growth cycle.

RNA sequencing in Artemisia annua L explored the genetic and metabolic responses to hardly soluble aluminum phosphate treatment.

Artemisia annua L. is a medicinal plant valued for its ability to produce artemisinin, a molecule used to treat malaria. Plant nutrients, especially phosphorus (P), can potentially influence plant biomass and secondary metabolite production. Our work aimed to explore the genetic and metabolic response of A. annua to hardly soluble aluminum phosphate (AlPO4, AlP), using soluble monopotassium phosphate (KH2PO4, KP) as a control. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze artemisinin. RNA sequencing, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to analyze the differentially expressed genes (DEGs) under poor P conditions. Results showed a significant reduction in plant growth parameters, such as plant height, stem diameter, number of leaves, leaf areas, and total biomass of A. annua. Conversely, LC-MS analysis revealed a significant increase in artemisinin concentration under the AlP compared to the KP. Transcriptome analysis revealed 762 differentially expressed genes (DEGs) between the AlP and the KP. GH3, SAUR, CRE1, and PYL, all involved in plant hormone signal transduction, showed differential expression. Furthermore, despite the downregulation of HMGR in the artemisinin biosynthesis pathway, the majority of genes (ACAT, FPS, CYP71AV1, and ALDH1) were upregulated, resulting in increased artemisinin accumulation in the AlP. In addition, 12 transcription factors, including GATA and MYB, were upregulated in response to AlP, confirming their importance in regulating artemisinin biosynthesis. Overall, our findings could contribute to a better understanding the parallel transcriptional regulation of plant hormone transduction and artemisinin biosynthesis in A. annua L. in response to hardly soluble phosphorus fertilizer.

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1-1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

The truncated AaActin1 promoter is a candidate tool for metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Malaria is a devastating parasitic disease with high levels of morbidity and mortality worldwide. Artemisinin, the active substance against malaria, is a sesquiterpenoid produced by Artemisia annua. To improve artemisinin content in the native A. annua plants, considerable efforts have been attempted, with genetic transformation serving as an effective strategy. Although, the most frequently-used cauliflower mosaic virus (CaMV) 35S (CaMV35S) promoter has proved to be efficient in A. annua transgenic studies, it appears to show weak activity in peltate glandular secretory trichomes (GSTs) of A. annua plants. Here, we characterized the 1727 bp fragment upstream from the translation start codon (ATG) of AaActin1, however, found it was inactive in tobacco. After removal of the 5' intron, the truncated AaActin1 promoter (tpACT) showed 69% and 50% activity of CaMV35S promoter in transiently transformed tobacco and stably transformed A. annua, respectively. ß-glucuronidase (GUS) staining analysis showed that the tpACT promoter was capable of directing the constant expression of a foreign gene in peltate GSTs of transgenic A. annua, representing higher activity than CaMV35S promoter. Collectively, our study provided a novel promoter available for metabolic engineering of artemisinin biosynthesis in A. annua.

Enhancing artemisinin content in and delivery from Artemisia annua: a review of alternative, classical, and transgenic approaches.

MAIN CONCLUSION: This review analyses the most recent scientific research conducted for the purpose of enhancing artemisinin production. It may help to devise better artemisinin enhancement strategies, so that its production becomes cost effective and becomes available to masses. Malaria is a major threat to world population, particularly in South-East Asia and Africa, due to dearth of effective anti-malarial compounds, emergence of quinine resistant malarial strains, and lack of advanced healthcare facilities. Artemisinin, a sesquiterpene lactone obtained from Artemisia annua L., is the most potent drug against malaria and used in the formulation of artemisinin combination therapies (ACTs). Artemisinin is also effective against various types of cancers, many other microbes including viruses, parasites and bacteria. However, this specialty metabolite and its derivatives generally occur in low amounts in the source plant leading to its production scarcity. Considering the importance of this drug, researchers have been working worldwide to develop novel strategies to augment its production both in vivo and in vitro. Due to complex chemical structure, its chemical synthesis is quite expensive, so researchers need to devise synthetic protocols that are economically viable and also work on increasing the in-planta production of artemisinin by using various strategies like use of phytohormones, stress signals, bioinoculants, breeding and transgenic approaches. The focus of this review is to discuss these artemisinin enhancement strategies, understand mechanisms modulating its biosynthesis, and evaluate if roots play any role in artemisinin production. Furthermore, we also have a critical analysis of various assays used for artemisinin measurement. This may help to develop better artemisinin enhancement strategies which lead to decreased price of ACTs and increased profit to farmers.

Lipid Transfer Proteins (AaLTP3 and AaLTP4) Are Involved in Sesquiterpene Lactone Secretion from Glandular Trichomes in Artemisia annua.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.

Enhanced arsenic tolerance and secondary metabolism by modulation of gene expression and proteome profile in Artemisia annua L. after application of exogenous salicylic acid.

This study was designed to investigate the effect of exogenous application of salicylic acid (SA) on proteins pattern and secondary metabolites in arsenic (As) stressed Artemisia annua. A. annua was treated by As 100 µM, SA 100 µM and combined treatment of SA 100 µM + As 100 µM upto 3 days. Significant accumulation of As was observed in roots than shoots at As 100 µM treatment. Under As treatment, oxidative stress was induced as indicated by increased TBARS content. Biomass, carotenoid, flavonoids were enhanced whereas total chlorophyll pigment was reduced under As treatment. Combined treatment of SA 100 µM + As 100 µM was more effective for increment of biomass, total chlorophyll content, and flavonoids as compared to As 100 µM treatment. Protein profiling revealed 20 differentially abundant proteins by 2-DE PAGE and MALDI-TOF-MS analysis. Identified proteins were related to photosynthesis, energy metabolism, transcriptional regulators, secondary metabolism, lipid metabolism, transport proteins and unknown/hypothetical proteins. All identified proteins were significantly increased in abundance under combined treatments of SA 100 µM + As 100 µM. The expression analysis of key genes involved in biosynthesis of lipid metabolism, signal molecule, transcriptional regulators, artemisinin biosynthetic genes, isoprenoids pathway, terpenes and flavonoids pathway were significantly upregulated under combined treatments of SA 100 µM + As 100 µM, suggesting a fine linkage in regulation of primary and secondary metabolism to modulate tolerance capacity and to improve phytoremediation property of A. annua against arsenic toxicity.

Deciphering transcriptome profiles of tetraploid Artemisia annua plants with high artemisinin content.

To investigate on the effects of autopolyploidization on growth and artemisinin biosynthesis in Artemisia annua, we performed a comprehensive transcriptomic characterization of diploid and induced autotetraploid A. annua. The polyploidization treatment not only enhanced photosynthetic capacity and endogenous contents of indole-3-acetic acid (IAA), abscisic acid (ABA) and jasmonic acid (JA), oxidative stress, but increased the average level of artemisinin in tetraploids from 42.0 to 63.6%. The obvious phenotypic alterations in tetraploids were observed including shorter stems, larger size of stomata and glandular secretory trichomes (GSTs), larger leaves, more branches and roots. A total of 8763 (8.85%) differentially expressed genes (DEGs) were identified in autotetraploids and mainly involved in carbohydrate metabolic processes, cell wall organization and defense responses. Both the up-regulated expression of DNA methylation unigenes and enhanced level of DNA methylation in autotetraploids indicated a possible role of DNA methylation on transcriptomic remodeling and phenotypic alteration. The up-regulated genes were enriched in response to extracellular protein biosynthesis, photosynthesis and hormone stimulus for cell enlargement and phenotypic alteration. The genomic shock induced by chromosome duplication stimulated the expression of transcripts related to oxidative stress, biosynthesis and signal transduction of ABA and JA, and key enzymes in artemisinin biosynthetic pathway, leading to the increased accumulation of artemisinin. This is the first transcriptomic research that identifies DEGs involved in the polyploidization of A. annua. The results provide novel information for understanding the complexity of polyploidization and for further identification of the factors and genes involve in artemisinin biosynthesis.

[Molecular cloning and characterization of CMK from Artemisia annua].

Artemisinin is a preferred medicine in the treatment of malaria. In this study, AaCMK, a key gene involved in the upstream pathway of artemisinin biosynthesis, was cloned and characterized from Artemisia annua for the first time. The full-length cDNA of AaCMK was 1 462 bp and contained an ORF of 1 197 bp that encoded a 399-anomo-acid polypeptide. Tissue expression pattern analysis showed that AaCMK was expressed in leaves, flowers, roots and stems, but with higher expression level in glandular secretory trichomes. In addition, the expression of AaCMK was markedly increased after MeJA treatment. Subcellular localization showed that the protein encoded by AaCMK was localized in chloroplast. Overexpression of AaCMK in Arabidopsis increased the contents of chlorophyll a, chlorophyll b and carotenoids. These results suggest that AaCMK plays an important role in the biosynthesis of terpenoids in A. annua and this research provids a candidate gene that could be used for engineering the artemisinin biosynthesis.

Red and Blue Light Promote the Accumulation of Artemisinin in Artemisia Annua L.

Artemisinin, which has been isolated from Artemisiaannua L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in A.annua is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in A.annua, but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich A.annua.

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua.

Prolonged exposure to salt stress affects specialized metabolites-artemisinin and essential oil accumulation in Artemisia annua L.: metabolic acclimation in preferential favour of enhanced terpenoid accumulation accompanying vegetative to reproductive phase transition.

Artemisia annua accumulates substantial quantities of unique and highly useful antimalarial sesquiternoid artemisinin and related phytomolecules as well as its characteristic essential oil in its glandular trichomes. The phytomolecules are mainly produced in its leaves and inflorescences. Artemisia annua plants were grown under NaCl salinity (50, 100 and 200 mM) stress conditions imposed throughout the entire life cycle of the plant. Results revealed that specialized metabolites like artemisinin, arteannuin-B, artemisinic acid + dihydroartemisinic acid and essential oil accumulation were positively modulated by NaCl salinity stress. Interestingly, total content of monoterpenoids and sesquiterpenoids of essential oil was induced by NaCl salinity treatment, contrary to previous observations. Production of camphor, the major essential oil constituent was induced under the influence of treatment. The metabolic acclimation and manifestations specific to terpenoid pathway are analysed vis-a-vis vegetative to reproductive periods and control of the modulation. WRKY and CYP71AV1 play a key role in mediating the responses through metabolism in glandular trichomes. The distinctness of the salinity induced responses is discussed in light of differential mechanism of adaptation to abiotic stresses and their impact on terpenoid-specific metabolic adjustments in A. annua. Results provide potential indications of possible adaptation of A. annua under saline conditions for agrarian techno-economic benefaction.

Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L.

This study aims to elucidate the underlying molecular mechanisms of artemisinin accumulation induced by Cd. The effects of different Cd concentrations (0, 20, 60, and 120 µmol/L) on the biosynthesis of Artemisia annua L. were examined. Intermediate and end products were quantified by HPLC-ESI-MS/MS analysis. The expression of key biosynthesis enzymes was also determined by qRT-PCR. The results showed that the application of treatment with 60 and 120 µmol/L Cd for 3 days significantly improved the biosynthesis of artemisinic acid, arteannuin B, and artemisinin. The concentrations of artemisinic acid, arteannuin B, and artemisinin in the 120 µmol/L Cd-treated group were 2.26, 102.08, and 33.63 times higher than those in the control group, respectively. The concentrations of arteannuin B and artemisinin in 60 µmol/L Cd-treated leaves were 61.10 and 26.40 times higher than those in the control group, respectively. The relative expression levels of HMGR, FPS, ADS, CYP71AV1, DBR2, ALDH1, and DXR were up-regulated in the 120 µmol/L Cd-treated group because of increased contents of artemisinic metabolites after 3 days of treatment. Hence, appropriate doses of Cd can increase the concentrations of artemisinic metabolites at a certain time point by up-regulating the relative expression levels of key enzyme genes involved in artemisinin biosynthesis.

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.

Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 µM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering.

Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua.

Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future.

Effect of Rol Genes on Polyphenols Biosynthesis in Artemisia annua and Their Effect on Antioxidant and Cytotoxic Potential of the Plant.

Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8-5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.

[Mitogen-activated protein kinase genes of Artemisia annua and their expression analysis under Cadmium stress].

In order to study Artemisia annua under cadmium stress, whether there are corresponding MAPK genes involved in transduction of the cadmium signal. 17 AaMAPK genes, named AaMAPK1-AaMAPK17 repectively, were finally obtained by using Trinity method for de novo assembly of transcripts from SRA database and BLAST search against AtMAPK genes and determing conserved domain using a series of bioinformatics tools. There exist 16 MAPK genes contained T[D/E]Y conserved domains among the obtained genes. The expressions of these genes were analyzed by Real-time PCR under cadmium stress. The results showed that the expressions level of AaMAPK3 and AaMAPK10 were down-regulated and MAPK7, MAPK9 and MAPK12 were up-regulated. These indicated that there exist corresponding MAPK genes involved in transduction of the cadmium signal.