Artemisinin Yield Research Articles

TRICHOME AND ARTEMISININ REGULATOR 2 positively regulates trichome development and artemisinin biosynthesis in Artemisia annua.

Glandular secretory trichomes (GSTs) are regarded as biofactories for synthesizing, storing, and secreting artemisinin. It is necessary to figure out the initiation and development regulatory mechanism of GSTs to cultivate high-yielding Artemisia annua. Here, we identified an MYB transcription factor, AaTAR2, from bioinformatics analysis of the A.annua genome database and Arabidopsis trichome development-related genes. AaTAR2 is mainly expressed in young leaves and located in the nucleus. Repression and overexpression of AaTAR2 resulted in a decrease and increase, respectively, in the GSTs numbers, leaf biomass, and the artemisinin content in transgenic plants. Furthermore, the morphological characteristics changed obviously in trichomes, suggesting AaTAR2 plays a key role in trichome formation. In addition, the expression of flavonoid biosynthesis genes and total flavonoid content increased dramatically in AaTAR2-overexpressing transgenic plants. Owing to flavonoids possibly counteracting emerging resistance to artemisinin in Plasmodium species, AaTAR2 is a potential target to improve the effect of artemisinin in clinical therapy. Taken together, AaTAR2 positively regulates trichome development and artemisinin and flavonoid biosynthesis. A better understanding of this 'multiple functions' transcription factor may enable enhanced artemisinin and flavonoids yield. AaTAR2 is a potential breeding target for cultivating high-quality A.annua.

Impact of In-vitro Propagation and Organic Farming Cultivation Practices of Artemisia annua L. on the Enhancement of Artemisinin Yield

Background : Artemisia annua is well known for its anti-malarial bio-active compound artemisinin. Development of elite planting material of A. annua and its agro-technology can fulfill the requirement of Artemisinin-based Combination Therapy (ACT) dosages worldwide. Objectives: To develop an efficient in-vitro propagation protocol for A. annua and assess the field performance of in-vitro propagated plants for their growth and artemisinin yield. Methods: The in-vitro propagation protocol of A. annua was developed using the nodal segment in four steps viz: initiation, multiplication, rooting and hardening. In-vitro propagated plants were transplanted with open-pollinated seed raised plants in an experimental field trial having soil supplementation of Farm Yard Manure (FYM), vermicompost and NPK. Result: Maximum 92% shoots were initiated in Murashige and Skoog medium (MS) with 0.44 μM 6-benzyl aminopurine (BA) and highest 281.33 ± 09.75 micro-shoots/inoculum obtained in MS with 15.54 μM BA. The maximum number of roots was found in MS with 100 mg/L activated charcoal while 78.20% of plants survived in the sand: soil: vermicompost (1:1:1) mixture. The highest dry leaf yield (6.37 t/ha) was observed in in-vitro propagated plants grown with vermicompost, while highest artemisinin content (1.11 ± 0.10) and artemisinin yield (65.05 kg/ha) was found in the in-vitro propagated plants grown with FYM after 120 days of transplantation. Conclusion: This study reports an efficient, cost-effective and rapid in-vitro propagation protocol for A. annua as well as enhanced artemisinin yield through the cultivation of in-vitro propagated plants using organic soil supplement inputs. This would lead to an increase in the production of artemisinin yield and fulfill the demand of Artemisinin-based Combination Therapy (ACT).

Salicylic acid restrains arsenic induced oxidative burst in two varieties of Artemisia annua L. by modulating antioxidant defence system and artemisinin production

Arsenic is a harmful and toxic substance to the growth and development of plants. Salicylic acid (SA) acts as a signaling molecule, plays pivotal roles in the overall growth and development of plants under various environmental stresses. Artemisinin extracted from the leaves of A. annua helps in malarial treatment. The present investigation is aimed to find out the possible ameliorative role of exogenously-applied salicylic acid (SA) on two varieties of Artemisia annua L., namely ‘CIM-Arogya’ and ‘Jeevan Raksha’ under arsenic (As) stress conditions. For this, growth, physiological and biochemical characterization, and artemisinin production was assessed. The various treatments applied on the plants were Control, 10−6 M SA, 10−5 M SA, 45 mg kg−1As, 45 mg kg−1 As + 10−6 M SA, and 45 mg kg−1 As + 10−5 M SA. Arsenic at 45 mg kg−1 of soil, reducing the overall performance of both varieties at 90 and 120 DAP. However, the levels of antioxidants were enhanced in As-stressed plants, and the supplementation of SA further increased these antioxidants in SA-treated plants. It has been observed that minimum reduction in growth and yield occurs with enhanced production of artemisinin in the case of ‘CIM-Arogya’ compared to ‘Jeevan Raksha’ under As stress (45 mg kg−1 of soil). Leaf-applied SA significantly increased the content (49.0% & 43.4%) and yield (53.3% & 46.3%) of artemisinin in both tolerant and sensitive varieties as compared to their respective controls. Thus, the variety ‘CIM-Arogya’ showed tolerant behavior over ‘Jeevan Raksha’ and is much adapted to higher As stress.

Enhancing artemisinin yields through an ecologically functional community of endophytes in Artemisia annua

Studying the role of endophytic microbial diversity in plant growth is helpful to understand plant-microbe interaction. The present experiment was aimed to investigate the effect of seed endophytes of Artemisia annua on plant biomass and artemisinin content. All the ten bacterial endophytes were found to express a varying degree of plant growth-promoting ability in pots, and of these six better performing were inoculated in A. annua in monocultures and all possible combinations thereof for analyzing the contribution of species identity and species richness on plant growth and artemisinin yield. The significant effect of species identities was found on plant biomass and artemisinin yields by inoculated endophytes. Community identity was found to be a more important factor than species richness in predicting biomass and artemisinin content. Most of the microbial combinations could not enhance plant growth over their monocultures. In this way, it has been demonstrated that there is a significant role of ecological interactions in bacterial mixtures, which are directly linked with the net performance of the community. In the presence of a combination of four endophytes comprising of Bacillus subtilis, Bacillus licheniformis, Burkholderia sp. and Acinetobacter pittii, artemisinin yield was found exceeding all of these microbes as in monocultures. This microbial consortium was able to increase the artemisinin yield of about 658 % over uninoculated control. Application of endophytic microbial consortium, similar to that found in this study, may prove an effective alternative to chemical fertilizers for enhancing artemisinin yield in an economically and eco-friendly manner with the reduced cost of its production.

Kinetics study on reaction between dihydroartemisinic acid and singlet oxygen: An essential step to photochemical synthesis of artemisinin

Artemisinin is an excellent antimalarial drug widely used in clinical medicine. However, due to the limitation of natural source of artemisinin, the chemical synthesis of artemisinin has achieved substantial attention. Dihydroartemisinic acid is a key precursor for the synthesis of artemisinin. The reaction of dihydroartemisinic acid with singlet oxygen to form peroxide is a pivotal step in the photochemical preparation of artemisinin. Nevertheless, the reaction kinetics of dihydroartemisinic acid with singlet oxygen has not been investigated previously. Herein, we report the rate constants of the reaction between dihydroartemisinic acid and singlet oxygen. By directly detecting the luminescence decay kinetics of singlet oxygen at 1270 nm at room temperature, the reaction rate constants of singlet oxygen and dihydroartemisinic acid in different solvents are obtained to be 1.81×105 (mol/L)−1·s−1 in CCl4, 5.69×105 (mol/L)−1·s−1 in CH3CN, and 3.27×106 (mol/L)−1·s−1 in DMSO, respectively. It is found that the reaction rate constants of dihydroartemisinic acid with singlet oxygen increase as polarity of the solvent increases among the three solvents. These results provide fundamental knowledge to optimize experiment conditions of photochemical synthesis of artemisinin for improving the yields of artemisinin.

Oligomers of carrageenan regulate functional activities and artemisinin production in Artemisia annua L. exposed to arsenic stress.

Recently, a promising technique has come forward in field of radiation-agriculture in which the natural polysaccharides are modified into useful oligomers after depolymerization. Ionizing radiation technology is a simple, pioneering, eco-friendly, and single step degradation process which is used in exploiting the efficiency of the natural polysaccharides as plant growth promoters. Arsenic (As) is a noxious and toxic to growth and development of medicinal plants. Artemisinin is obtained from the leaves of Artemisia annua L., which is effective in the treatment of malaria. The present study was undertaken to find out possible role of oligomers of irradiated carrageenan (IC) on two varieties viz. 'CIM-Arogya' (As-tolerant) and 'Jeevan Raksha' (As-sensitive) of A. annua exposed to As. The treatments applied were 0 (control), 40 IC (40mgL-1 IC), 80 IC (80mgL-1 IC), 45 As (45mgkg-1 soil As), 40 IC + 45 As (40mgL-1 IC + 45mgkg-1 soil As), and 80 IC + 45 As (80mgL-1 IC + 45mgkg-1 soil As). The present study was based on various parameters namely plant fresh weight (FW), dry weight (DW), leaf area index (LAI), leaf yield (LY), chlorophyll and carotenoid content, net photosynthetic rate (PN), stomatal conductance (Gs), carbonic anhydrase activity (CA), proline content (PRO), lipid peroxidation (TBARS), endogenous ROS production (H2O2 content), catalase activity (CAT), peroxidase activity (POX), superoxide dismutase activity (SOD), ascorbate peroxidase activity (APX), As content, and artemisinin content in leaves. Plant growth and other physiological and biochemical parameters including enzymatic activities, photosynthetic activity, and its related pigments were negatively affected under As stress. Leaf-applied IC overcame oxidative stress generated due to As in plants by activating antioxidant machinery. Interestingly, leaf-applied IC enhanced the production (content and yield) of artemisinin under high As stress regardless of varieties. The oligomers of IC and As were found to be responsible for the production of endogenous H2O2 which has a pivotal role in the biosynthesis of artemisinin in A. annua.

The Transcription Factor Aabzip9 Positively Regulates the Biosynthesis of Artemisinin in Artemisia annua.

Artemisinin-based therapies are the only effective treatment for malaria, which reached to 219 million cases and killed 435,000 people in 2017. To meet the growing demand for artemisinin and make it accessible to the poorest, genetic engineering of Artemisia annua becomes one of the most promising approaches to improve artemisinin yield. In this work, AabZIP9 transcription factor has been identified and characterized. The expression profile of AabZIP9 revealed that it was clustered with the artemisinin specific biosynthetic pathway genes ADS, CYP71AV1, DBR2, and ALDH1. Furthermore, the transiently dual-LUC analysis showed that the activation of ADS promoter was enhanced by AabZIP9. Meanwhile, yeast one-hybrid assay showed that AabZIP9 was able to bind to the “ACGT” cis-element present in both ADS and CYP71AV1 promoters. AabZIP9 gene was driven by the constitutive CaMV35S promoter and the glandular trichome specific CYP71AV1 promoter and stably transformed into A. annua plants. The transcript level of AabZIP9 was increased in both of the 35S and CYP71AV1 driven transgenic plants compared with the wild type or GUS control plants. All the transgenic A. annua plants overexpressing AabZIP9 showed elevated transcript level of ADS, but the transcription levels of CYP71AV1, DBR2, and ALDH1 have no significant change in both types of transgenic plants. The significantly upregulated ADS promoted the accumulation of artemisinin, dihydroartemisinic acid, and artemisinic acid biosynthesis in the transgenic A. annua plants. These results suggest that AabZIP9 can positively regulate the biosynthesis of artemisinin.

Advances in synthesis of artemisinin based on plant genetic engineering

Artemisinin is a kind of sesquiterpene lactone containing endoperoxide bridge,which is the most effective anti-malarial drug at present. However,low content of artemisinin in Artemisia annua,ranging from 0. 1%-1. 0% of dry weight,as well as the complicated extraction process have resulted in low yield and high cost of artemisinin,making it difficult to meet market demand.Based on the development of high-throughput sequencing and molecular biology,the related enzyme genes and transcription factors involved in the artemisinin metabolic pathway were cloned and identified. Metabolic engineering and synthetic biology methods to modify the original metabolic pathway of A. annua and genetic engineering in heterologous host cells have become one of the hotspots in this field. Therefore,the molecular mechanism of artemisin biosynthesis,different strategies of genetic modifications of A. annua,and the research status and application prospect of artemisinin synthesis in heterologous host cells( Nicotiana benthamiana,Physcomitrella patens) were summarized in our review,hoping to provide molecular basis and theoretical basis for breeding new varieties of A. annua with high artemisinin output.

Overexpression of AaPIF3 promotes artemisinin production in Artemisia annua

Artemisia annua is a Chinese traditional herbal plant that produces artemisinin, the potent anti-malarial drug. It is a common goal for the artemisinin industry to develop plants of A. annua with high yields of artemisinin. In this study, a bHLH transcription factor of A. annua (AaPIF3) was characterized and used to promote the production of artemisinin. AaPIF3 was highly expressed in flower buds and glandular secretory trichomes and its coding protein was localized to the nucleus. Dual-luciferase assays indicated that AaPIF3 was able to significantly enhance the promoter activities of artemisinin biosynthesis genes, including ADS, CYP71AV1, DBR2, and ALDH1, suggesting that AaPIF3 positively regulated artemisinin biosynthesis. Overexpression of AaPIF3 markedly upregulated artemisinin biosynthesis genes at the transcriptional level, and consequently the production of artemisinin was significantly promoted in transgenic A. annua plants. On the contrary, suppression of AaPIF3 led to significantly decreased transcript levels of artemisinin biosynthesis genes, with overall production of artemisinin significantly reduced. In summary, AaPIF3 plays a positive role in regulating artemisinin biosynthesis and transgenic plants of A. annua with high yields of artemisinin have been successfully developed through overexpression of AaPIF3.

Photosensitizer-Anchored 2D MOF Nanosheets as Highly Stable and Accessible Catalysts toward Artemisinin Production.

2D metal–organic frameworks (2D‐MOFs) have recently emerged as promising materials for gas separations, sensing, conduction, and catalysis. However, the stability of these 2D‐MOF catalysts and the tunability over catalytic environments are limited. Herein, it is demonstrated that 2D‐MOFs can act as stable and highly accessible catalyst supports by introducing more firmly anchored photosensitizers as bridging ligands. An ultrathin MOF nanosheet‐based material, Zr‐BTB (BTB = 1,3,5‐tris(4‐carboxyphenyl)benzene), is initially constructed by connecting Zr6‐clusters with the tritopic carboxylate linker. Surface modification of the Zr‐BTB structure was realized through the attachment of porphyrin‐based carboxylate ligands on the coordinatively unsaturated Zr metal sites in the MOF through strong Zr‐carboxylate bond formation. The functionalized MOF nanosheet, namely PCN‐134‐2D, acts as an efficient photocatalyst for 1O2 generation and artemisinin production. Compared to the 3D analogue (PCN‐134‐3D), PCN‐134‐2D allows for fast reaction kinetics due to the enhanced accessibility of the catalytic sites within the structure and facile substrate diffusion. Additionally, PCN‐134(Ni)‐2D exhibits an exceptional yield of artemisinin, surpassing all reported homo‐ or heterogeneous photocatalysts for the artemisinin production.

Trichoderma asperellum ACCC30536 inoculation improves soil nutrition and leaf artemisinin production in Artemisia annua

Artemisia annua L. is the main source of artemisinin, currently the most effective treatment for malaria. However, an affordable and abundant supply of artemisinin remains elusive. Trichoderma is a biocontrol agent that stimulates plant growth and defense responses, and improves soil quality. To date, relatively few studies have focused on improving leaf biomass and artemisinin production in A. annua using Trichoderma. To investigate the role of T. asperellum ACCC30536 in improving the artemisinin yield of A. annua, field-grown A. annua was inoculated with T. asperellum conidia and grown for 60 days. The results showed that leaf artemisinin concentration and dry weight were increased significantly after inoculation. The optimal inoculation dose was 200 mL of conidia suspension at 1 × 107 colony-forming units (cfu)/mL, the highest artemisinin concentration was 8.83 mg/g, and the highest artemisinin production was 70.6 g on day 50. The results of qRT-PCR revealed that expression of genes encoding key enzymes for artemisinin biosynthesis, namely HMGR1, FPS, ADS, CYP71AV1, CPR, DBR, DXS1, and DXR1, was generally upregulated during days 20–50 following induction by Trichoderma. In addition, the moisture, pH stability, organic matter content, and availability of nitrogen, phosphorus, and kalium in inoculated soil were significantly improved. Thus, application of T. asperellum ACCC30536 may offer a novel approach for improving artemisinin production by upregulating the expression of key enzymes for artemisinin biosynthesis, increasing leaf yield, and improving soil fertility.

Optimization of artemisinin extraction from Artemisia annua L. with supercritical carbon dioxide + ethanol using response surface methodology.

Malaria is a high priority life-threatening public health concern in developing countries, and therefore there is a growing interest to obtain artemisinin for the production of artemisinin-based combination therapy products. In this study, artemisinin was extracted from the Artemisia annua L. plant using supercritical carbon dioxide (SC-CO2 ) modified with ethanol. Response surface methodology based on central composite rotatable design was employed to investigate and optimize the extraction conditions of pressure (9.9-30 MPa), temperature (33-67°C), and co-solvent (ethanol, 0-12.6 wt.%). Optimum SC-CO2 extraction conditions were found to be 30 MPa and 33°C without ethanol. Under optimized conditions, the predicted artemisinin yield was 1.09% whereas the experimental value was 0.71 ± 0.07%. Soxhlet extraction with hexane resulted in higher artemisinin yields and there was no significant difference in the purity of the extracts obtained with SC-CO2 and Soxhlet extractions. Results indicated that SC-CO2 and SC-CO2 +ethanol extraction is a promising alternative for the extraction of artemisinin to eliminate the use of organic solvents, such as hexane, and produce extracts that can be used for the production of antimalarial products.

Selection and Clonal Propagation of High Artemisinin Genotypes of Artemisia annua.

Artemisinin, produced in the glandular trichomes of Artemisia annua L. is a vital antimalarial drug effective against Plasmodium falciparum resistant to quinine-derived medicines. Although work has progressed on the semi-synthetic production of artemisinin, field production of A. annua remains the principal commercial source of the compound. Crop production of artemisia must be increased to meet the growing worldwide demand for artemisinin combination therapies (ACTs) to treat malaria. Grower artemisinin yields rely on plants generated from seeds from open-pollinated parents. Although selection has considerably increased plant artemisinin concentration in the past 15 years, seed-generated plants have highly variable artemisinin content that lowers artemisinin yield per hectare. Breeding efforts to produce improved F1 hybrids have been hampered by the inability to produce inbred lines due to self-incompatibility. An approach combining conventional hybridization and selection with clonal propagation of superior genotypes is proposed as a means to enhance crop yield and artemisinin production. Typical seed-propagated artemisia plants produce less than 1% (dry weight) artemisinin with yields below 25 kg/ha. Genotypes were identified producing high artemisinin levels of over 2% and possessing improved agronomic characteristics such as high leaf area and shoot biomass production. Field studies of clonally-propagated high-artemisinin plants verified enhanced plant uniformity and an estimated gross primary productivity of up to 70 kg/ha artemisinin, with a crop density of one plant m-2. Tissue culture and cutting protocols for the mass clonal propagation of A. annua were developed for shoot regeneration, rooting, acclimatization, and field cultivation. Proof of concept studies showed that both tissue culture-regenerated plants and rooted cutting performed better than plants derived from seed in terms of uniformity, yield, and consistently high artemisinin content. Use of this technology to produce plants with homogeneously-high artemisinin can help farmers markedly increase the artemisinin yield per cultivated area. This would lead to increased profit to farmers and decreased prices of ACT.

The Artemisia annua FLOWERING LOCUS T Homolog 2, AaFT2, is a key regulator of flowering time

Artemisia annua is a typical short-day photoperiod (SD) and belongs to Asteraceae family. FLOWERING LOCUS T Homolog genes play important roles in regulation of flowering time. Two FT-like genes AaFT1 and AaFT2 were isolated from cDNA libraries constructed from leaves of A. annua. The gene expression analysis revealed that AaFT1 and AaFT2 mRNAs were expressed predominantly in leaves and could be induced by GA3. Subcellular localization results indicated that AaFT1 and AaFT2 proteins were both localized to the nuclei and cytoplasm. The Y2H assay results indicated that AaFT1 and AaFT2 could interact with a bZIP transcription factor, AaFD. AaFT2 was induced by SDs condition and may play an important role in modulation of flowering. In the present study, we performed RNA interference (RNAi) experiments to inhibit the mRNA level of AaFT2 in A. annua for detecting AaFT2 gene function. Our findings suggest that inhibition of AaFT2 may delay the flowering time and increase the accumulation of artemisinin (ART) in transgenic A. annua. Collectively, these results indicate that AaFT2 is a key regulator of regulation flowering time and can be used for genetic engineering to improve artemisinin yield.

Influence of harvesting stage on herb, essential oil and artemisinin yields in the antimalarial herb, Artemisia annua L.

The investigation was carried out to standardize the harvesting stage for maximum oil and essential oil yield in Artemisia annua L. in the semi arid region of Gujarat. The experiment was laid out in randomized block design with seedlings planted at the age of six weeks and harvested at five different phonological stages.viz., early vegetative (March), late vegetative (May), pre bloom (September), bloom (October) and post bloom (November) stages. The results revealed that the maximum herb could be obtained in the late vegetative stage (31.74 t/ha) followed by bloom stage (30.19 t/ha). The leaf yield was maximum in the bloom stage (6.39t/ha). The essential oil content and yield was maximum in the bloom stage (0.61% and 61.30 kg/ha, respectively) followed by post bloom stage (0.55% and 38.87 kg/ha) and minimum in early vegetative stage (0.18% and 8.29 kg/ha). However, the artemisinin content was maximum in the late vegetative stage (0.48%) followed by prebloom stage (0.44%). Similarly, the artemisinin yield per hectare was maximum in the crop harvested during late vegetative stage followed by the prebloom stage. The essential oil obtained during different harvest stages were subjected to GC-MS analysis in which, the main compounds were camphor (28.6–31.7%), 1, 8cineole (2.1–20.8%), germacrene D (3.8–12.0%), β-caryophyllene (2.8–6.9%), trans-β-farnesene (0.7–4.5%), α-pinene (0.5–2.4%), p-cymene (2.1–2.3%) and terpinen-4-ol (0.9–2.1%). Camphor was found to be a major compound in the oil at all growth stages.

The application of biotic elicitor on Artemisia annua L. to increase artemisinin content

Artemisinin-based Combination Therapy (ACT) has been recommended by WHO as an alternative to treat malaria overcoming drug resistance. The secondary metabolic products in plants, including artemisinin, can be increased by utilizing biotic elicitor from fungi. The research was conducted in Gunung Putri Research Installation, Cipanas, West Java from 2010 to 2011. Phytophthora sp. from eggplant and Colletotrichum sp. from Artemisia annua were applied as biotic elicitor. The types of biotic elicitor applied to the plants were 1) the medium of potato dextrose broth were inoculated with fungi and harvested after 10 days (filtrate), 2) powdery mycelium of both fungi. There were 16 treatments: control negative, control positive (uninoculated medium) 1%, 2%, 3% (v/v)], Phytophthora sp. filtrate [1, 2% and 3% (v/v)], Colletotrichum sp. filtrate [1, 2% and 3% (v/v)], Phytophthora sp. mycelium [1%, 2% and 3% (w/v)], Colletotrichum sp mycelium [1%, 2% and 3% (w/v)]. The elicitor application increased plant production by 26.21% and artemisinin yield by 72% compared to control. Furthermore, the artemisinin production of the plants treated with medium inoculated with 2% filtrate of Phytophthora sp (FP2) (25.19 kg/ha) and 1% powdery mycelium of Colletotrichum sp (MC1) (26.42 kg/ha) were higher than control (K) (11.17 kg/ha).

Box–Behnken experimental design for extraction of artemisinin from Artemisia annua and validation of the assay method

Obtaining artemisinin and its derivatives is very costly, which limits access to low-income people. Some hydroalcoholic extract of Artemisia annua L., Asteraceae, which has shown comparable antimalarial activity could be an alternative to the purified compound, especially if the cultivars have higher content of artemisinin. The objective of this study was to evaluate the effects of the extraction parameters (ethanol graduation, previous shaking time in an ultrasound bath and drug/solvent ratio) on the yield of artemisinin in the liquid extract obtained by percolation from A. annua and then optimize the extraction efficiency of this compound. The Box–Behnken (33) factorial design was used in association with response surface methodology. The derivatization reaction of artemisinin was used in an analytical method which was submitted to validation, after reaching the specification of the selectivity, linearity, precision, accuracy and robustness. Detection and quantification limits were 1.3 and 4.0μg/ml, respectively. The largest amount of this compound of interest was obtained without any ultrasound bath, with an ethanol graduation of 95% and a drug/solvent ratio of 2%. Drug/solvent ratio was the factor which most influenced extraction efficiency. The maximum range of artemisinin yield was 1.21%. Information obtained in this study can be used for future approaches to determining and extracting artemisinin from A. annua.

AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana.

The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA9 into the bioactive GA4 as well as the enzymes involved in the degradation of GA4 . Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.

Overexpression of the gibberellin 20-oxidase gene from Torenia fournieri resulted in modified trichome formation and terpenoid metabolities of Artemisia annua L.

Gibberellins (GAs) are diterpenoid hormones, control various physiological developments in plants. The role of gibberellins on morphology and secondary metabolite production was examined in Artemisia annua, a medicinal plant that has been acknowledged as a source of artemisinin, an antimalarial compound. Subsequently, the GA20ox gene from Torenia fournieri (TfGA20ox2) was transferred to A. annua by Agrobacterium-mediated transformation. Compared with wild type plants, all nine transgenic plants showed significantly higher plant heights and artemisinin contents. The highest artemisinin content and yield in TfGA20ox2-overexpressing plants was around two-fold higher than wild type. Moreover, transgenic plants had higher numbers of branches (52.4%) and greater branch lengths (60–203%), but smaller leaf size (77.6%). Interestingly, relative to wild type the number and size of glandular trichomes in transgenic leaves was about 30 and 35% higher, respectively. From GC–MS analysis, the proportion of diterpenes in transgenic plant extracts was 1.5-fold lower than those noticed in wild type, while the proportion of sesquiterpenes was increased about 1.6 times when compared to wild type. However, the content proportion of monoterpenes showed a slightly increase, whereas the level of triterpenes showed no variation. In addition, two monoterpenes (eucalyptol and borneol), four sesquiterpenes (α-caryophyllene, β-guaiene, δ-cadinene and β-cubebene) and one triterpenes (isomultiflorenone) were detected only in transgenic extract, whereas d-α-tocopherol, a diterpenoid compound was found only in wild type but not transgenic plant. These results suggested that gibberellins play a significant role in regards to morphology, trichome formation and terpenoid metabolite production in A. annua.

Promotion of artemisinin content in Artemisia annua by overexpression of multiple artemisinin biosynthetic pathway genes

Artemisinin, isolated from an annual herbaceous plant Artemisia annua L., is an effective antimalarial compound. However, artemisinin is accumulated in small amounts (0.01–0.1% leaf dry weight) in A. annua, resulting in constant high artemisinin price. Although metabolic engineering of partial artemisinin metabolic pathway in yeast achieved great success, artemisinin from A. annua is still the important business resource. Here, we report on the generation of transgenic plants with simultaneously overexpressing four artemisinin biosynthetic pathway genes, amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene 12-monooxygenase gene (CYP71AV1), cytochrome P450 reductase gene (CPR), and aldehyde dehydrogenase 1 gene (ALDH1) via Agrobacterium-mediated transformation. The qRT-PCR analysis demonstrated that the introduced four genes of the transgenic lines were all highly expressed. Through high-performance liquid chromatography analysis, the artemisinin contents were increased markedly in transformants, with the highest being 3.4-fold higher compared with non-converter. These results indicate that overexpression of multiple artemisinin biosynthetic pathway genes is a promising approach to improve artemisinin yield in A. annua.