A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua.

BACKGROUND: Transcription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In Artemisia annua, various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in A. annua (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin. RESULTS: We found five AaSHI genes (AaSHI1 to AaSHI5) in the A. annua genome. The expression levels of AaSHI1, AaSHI2, AaSHI3 and AaSHI4 genes were higher in trichomes and young leaves, also induced by light and decreased when the plants were subjected to dark treatment. The expression pattern of these four AaSHI genes was consistent with the expression pattern of four structural genes of artemisinin biosynthesis and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in A. annua provided the evidence that AaSHI1 could directly bind to the promoters of structural genes AaADS and AaCYP71AV1, and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in A. annua. CONCLUSIONS: Overall, a genome-wide analysis of the AaSHI TF family of A. annua was conducted. Five AaSHIs were identified in A. annua genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of structural genes of artemisinin biosynthesis including AaADS and AaCYP71AV1. These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new A. annua germplasms.

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.

AaSEPALLATA1 integrates jasmonate and light-regulated glandular secretory trichome initiation in Artemisia annua.

Glandular secretory trichomes (GSTs) can secrete and store a variety of specific metabolites. By increasing GST density, valuable metabolites can be enhanced in terms of productivity. However, the comprehensive and detailed regulatory network of GST initiation still needs further investigation. By screening a complementary DNA library derived from young leaves of Artemisia annua, we identified a MADS-box transcription factor, AaSEPALLATA1 (AaSEP1), that positively regulates GST initiation. Overexpression of AaSEP1 in A. annua substantially increased GST density and artemisinin content. The HOMEODOMAIN PROTEIN 1 (AaHD1)-AaMYB16 regulatory network regulates GST initiation via the jasmonate (JA) signaling pathway. In this study, AaSEP1 enhanced the function of AaHD1 activation on downstream GST initiation gene GLANDULAR TRICHOME-SPECIFIC WRKY 2 (AaGSW2) through interaction with AaMYB16. Moreover, AaSEP1 interacted with the JA ZIM-domain 8 (AaJAZ8) and served as an important factor in JA-mediated GST initiation. We also found that AaSEP1 interacted with CONSTITUTIVE PHOTOMORPHOGENIC 1 (AaCOP1), a major repressor of light signaling. In this study, we identified a MADS-box transcription factor that is induced by JA and light signaling and that promotes the initiation of GST in A. annua.

Artemisia annua ZFP8L regulates glandular trichome development.

Trichomes are known to be important biofactories that contribute to the production of secondary metabolites, such as terpenoids. C2H2-zinc finger proteins (C2H2-ZFPs) are vital transcription factors of plants' trichome development. However, little is known about the function of Artemisia annua C2H2-ZFPs in trichome development. To explore the roles of this gene family in trichome development, two C2H2-ZFP transcription factors, named AaZFP8L and AaGIS3, were identified; both are hormonally regulated in A. annua. Overexpression of AaZFP8L in tobacco led to a significant increase in the density and length of glandular trichomes, and improved terpenoid content. In contrast, AaGIS3 was found to positively regulate non-glandular trichome initiation and elongation, which reduces terpenoid accumulation. In addition, ABA contents significantly increased in AaZFP8L-overexpressing tobacco lines and AaZFP8L also can directly bind the promoter of the ABA biosynthesis genes. This study lays the foundation for further investigating A. annua C2H2-ZFPs in trichome development and terpenoid accumulation.

The AaBBX21-AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21-AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1, AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5-AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

Genome-wide characterization of regulator of chromosome condensation 1 (RCC1) gene family in Artemisia annua L. revealed a conservation evolutionary pattern.

BACKGROUND: Artemisia annua is the major source for artemisinin production. The artemisinin content in A. annua is affected by different types of light especially the UV light. UVR8, a member of RCC1 gene family was found to be the UV-B receptor in plants. The gene structures, evolutionary history and expression profile of UVR8 or RCC1 genes remain undiscovered in A. annua. RESULTS: Twenty-two RCC1 genes (AaRCC1) were identified in each haplotype genome of two diploid strains of A. annua, LQ-9 and HAN1. Varied gene structures and sequences among paralogs were observed. The divergence of most RCC1 genes occurred at 46.7 - 51 MYA which overlapped with species divergence of core Asteraceae during the Eocene, while no recent novel RCC1 members were found in A. annua genome. The number of RCC1 genes remained stable among eudicots and RCC1 genes underwent purifying selection. The expression profile of AaRCC1 is analogous to that of Arabidopsis thaliana (AtRCC1) when responding to environmental stress. CONCLUSIONS: This study provided a comprehensive characterization of the AaRCC1 gene family and suggested that RCC1 genes were conserved in gene number, structures, constitution of amino acids and expression profiles among eudicots.

JA-Regulated AaGSW1-AaYABBY5/AaWRKY9 Complex Regulates Artemisinin Biosynthesis in Artemisia annua.

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

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.

Profiling of phytohormone-specific microRNAs and characterization of the miR160-ARF1 module involved in glandular trichome development and artemisinin biosynthesis in Artemisia annua.

MicroRNAs (miRNAs) play crucial roles in plant development and secondary metabolism through different modes of sequence-specific interaction with their targets. Artemisinin biosynthesis is extensively regulated by phytohormones. However, the function of phytohormone-responsive miRNAs in artemisinin biosynthesis remains enigmatic. Thus, we combined the analysis of transcriptomics, small RNAs, and the degradome to generate a comprehensive resource for identifying key miRNA-target circuits involved in the phytohormone-induced process of artemisinin biosynthesis in Artemisia annua. In total, 151 conserved and 52 novel miRNAs and their 4132 targets were determined. Based on the differential expression analysis, miR160 was selected as a potential miRNA involved in artemisinin synthesis. Overexpressing MIR160 significantly impaired glandular trichome formation and suppressed artemisinin biosynthesis in A. annua, while repressing its expression resulted in the opposite effect, indicating that miR160 negatively regulates glandular trichome development and artemisinin biosynthesis. RNA ligase-mediated 5' RACE and transient transformation assays showed that miR160 mediates the RNA cleavage of Auxin Response Factor 1 (ARF1) in A. annua. Furthermore, ARF1 was shown to increase artemisinin synthesis by activating AaDBR2 expression. Taken together, our results reveal the intrinsic link between the miR160-ARF1 module and artemisinin biosynthesis, and may expedite the innovation of metabolic engineering approaches for high and stable production of artemisinin in the future.

AabHLH113 integrates jasmonic acid and abscisic acid signaling to positively regulate artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is in huge market demand due to its efficient antimalarial action, especially after the COVID-19 pandemic. Many researchers have elucidated that phytohormones jasmonic acid (JA) and abscisic acid (ABA) positively regulate artemisinin biosynthesis via types of transcription factors (TFs). However, the crosstalk between JA and ABA in regulating artemisinin biosynthesis remains unclear. Here, we identified a novel ABA- and JA-induced bHLH TF, AabHLH113, which positively regulated artemisinin biosynthesis by directly binding to the promoters of artemisinin biosynthetic genes, DBR2 and ALDH1. The contents of artemisinin and dihydroartemisinic acid increased by 1.71- to 2.06-fold and 1.47- to 2.23-fold, respectively, in AabHLH1113 overexpressed A. annua, whereas they decreased by 14-36% and 26-53%, respectively, in RNAi-AabHLH113 plants. Furthermore, we demonstrated that AabZIP1 and AabHLH112, which, respectively, participate in ABA and JA signaling pathway to regulate artemisinin biosynthesis, directly bind to and activate the promoter of AabHLH113. Collectively, we revealed a complex network in which AabHLH113 plays a key interrelational role to integrate ABA- and JA-mediated regulation of artemisinin biosynthesis.

Synergistic interaction of two bHLH transcription factors positively regulates artemisinin biosynthetic pathway in Artemisia annua L.

The wonder drug artemisinin, a sesquiterpene lactone endoperoxide from Artemisia annua is the million-dollar molecule required to curb the deadliest disease, Malaria. One of the major challenges even today is to increase the concentration of artemisinin within plants. The transcription factors are important regulators of plant secondary metabolites and have the potential to regulate key steps or the whole biosynthetic pathway. In this study, we have identified and characterised two bHLH transcription factors (Aa6119 and Aa7162) from A. annua. Both the transcription factors turned out to be transcriptionally active and nuclear-localised typical bHLH proteins. In our study, we found that Aa6119 specifically binds to the E-box element present on the promoter of artemisinin biosynthetic gene, AMORPHA-4,11-DIENE SYNTHASE (ADS). The protein-DNA interaction confirmed by Yeast one-hybrid assay was specific as Aa6119 was unable to bind to the mutated E-boxes of ADS. Further, Aa6119 interacted physically with Aa7162, which was confirmed in vitro by Yeast two-hybrid assay and in vivo by Bimolecular Fluorescent complementation assay. Our quantitative expression studies have confirmed that Aa6119 and Aa7162 act synergistically in the regulation of artemisinin biosynthetic and trichome developmental genes. The higher accumulation of artemisinin content in the transient co-transformed transgenic plants than in the individual over-expression transgenic plants has further validated that Aa6119 and Aa7162 act positively and synergistically to regulate artemisinin accumulation.

Effects of exogenous indole-3-acetic acid on the density of trichomes, expression of artemisinin biosynthetic genes, and artemisinin biosynthesis in Artemisia annua.

Artemisinin is the most practical medication for the treatment of malaria, but is only very minimally synthesized in Artemisia annua, significantly less than the market needs. In this study, indole-3-acetic acid (IAA) was used to investigate its effects on trichomes, artemisinin accumulation, and biosynthetic gene expression in A. anuua. The results showed that exogenous IAA could contribute to the growth and development of A. annua and increase the density of trichomes. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that artemisinin and dihydroartemisinic acid (DHAA) contents were increased by 1.9-fold (1.1 mg/g) and 2.1-fold (0.51 mg/g) after IAA treatment in comparison with control lines (CK), respectively. Furthermore, quantitative real-time PCR results showed that AaADS, AaCYP71AV1, AaALDH1, and AaDBR2, four critical enzyme genes for the biosynthesis of artemisinin, had relatively high transcription levels in leaves of A. annua treated with IAA. In summary, this study indicated that exogenous IAA treatment was a feasible strategy to enhance artemisinin production, which paves the way for further metabolic engineering of artemisinin biosynthesis.

Oxidative stress in liver of streptozotocin-induced diabetic mice fed a high-fat diet: A treatment role of Artemisia annua L.

Objective. The aim of this study was the investigation of a treatment role of Artemisia annua L. (AA) on liver dysfunction and oxidative stress in high-fat diet/streptozotocin-induced diabetic (HFD/STZ) mice. Methods. Sixty mice were divided into 12 groups including control, untreated diabetic, and treated diabetic ones with metformin (250 mg/kg), and doses of 100, 200, and 400 mg/kg of water (hot and cold) and alcoholic (methanol) extracts of AA. Type 2 diabetes mellitus (T2DM) was induced in mice by high-fat diet for 8 weeks and STZ injection in experimental animals. After treatment with doses of 100, 200 or 400 mg/kg of AA extracts in HFD/STZ diabetic mice for 4 weeks, oxidative stress markers such as malondialdehyde (MDA), glutathione (GSH), and free radicals (ROS) were determined in the liver tissue in all groups. Results. Diabetic mice treated with metformin and AA extracts showed a significant decrease in ROS and MDA concentrations and a notable increase in GSH level in the liver. Effectiveness of higher doses of AA extracts (200 and 400 mg/kg), especially in hot-water and alcoholic ones, were similar to and/or even more effective than metformin. Conclusion. Therapeutic effects of AA on liver dysfunction showed that antioxidant activity of hot-water and alcoholic AA extracts were similar or higher than of metformin.

Bio Prospecting of Endophytes and PGPRs in Artemisinin Production for the Socio-economic Advancement.

The growing demand for Artemisia annua plants in healthcare, food, and pharmaceutical industries has led to increased cultivation efforts to extract a vital compound, Artemisinin. The efficacy of Artemisinin as a potent drug against malaria disease is well established but its limited natural abundance. However, the common practice of using chemical fertilizers for maximum yield has adverse effects on plant growth, development, and the quality of phytochemicals. To address these issues, the review discusses the alternative approach of harnessing beneficial rhizosphere microbiota, particularly plant growth-promoting rhizobacteria (PGPR). Microbes hold substantial biotechnological potential for augmenting medicinal plant production, offering an environmentally friendly and cost-effective means to enhance medicinal plant production. This review article aims to identify a suitable endophytic population capable of enabling Artemisia sp. to thrive amidst abiotic stress while simultaneously enhancing Artemisinin production, thereby broadening its availability to a larger population. Furthermore, by subjecting endophytes to diverse combinations of harsh conditions, this review sheds light on the modulation of essential artemisinin biosynthesis pathway genes, both up regulated and down regulated. The collective findings suggest that through the in vitro engineering of endophytic communities and their in vivo application to Artemisia plants cultivated in tribal population fields, artemisinin production can be significantly augmented. The overall aim of this review to explore the potential of harnessing microbial communities, their functions, and services to enhance the cultivation of medicinal plants. It outlines a promising path toward bolstering artemisinin production, which holds immense promise in the fight against malaria.

Isolation and characterization of AaZFP1, a C2H2 zinc finger protein that regulates the AaIPPI1 gene involved in artemisinin biosynthesis in Artemisia annua.

MAIN CONCLUSION: AaZFP1, a C2H2-type transcription factor, was found to bind the AGT-N1-10-AGT box of AaIPPI1pro and activate the expression of AaIPPI1 involved in artemisinin biosynthesis. Artemisinin, an endoperoxide sesquiterpene lactone, is a widely used antimalarial drug isolated from Artemisia annua L. Isopentenyl pyrophosphate isomerase (AaIPPI1) catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate and is the key gene involved in the biosynthesis of artemisinin. However, the AaIPPI1 gene regulation network remains largely unknown. Here, we isolated the AaIPPI1 promoter (AaIPPI1pro) and predicted that it contains cis-elements involved in stress responses, including the TGACG motif (a methyl jasmonate-responsive element), GARE motif (a gibberellin-responsive element), ABRE (an abscisic acid-responsive element), TC-rich repeats (a stress-responsive element), and the AGT-N1-10-AGT box, which is the binding site of Cys/His2 zinc finger protein (C2H2 ZFP). The C2H2 ZFP gene AaZFP1 was discovered by screening a cDNA library using AaIPPI1pro as bait in yeast. AaZFP1 contains two conserved C2H2 regions, a nuclear localization domain (B box), a Leu-rich domain (L box), and a conserved DLN sequence (DLN box) close to its C terminus. A subcellular localization assay indicated that AaZFP1 protein is localized in the nucleus and cytoplasm. An electrophoretic mobility shift assay demonstrated that AaZFP1 binds to the AGT-N1-10-AGT box of AaIPPI1pro. A dual-luciferase assay indicated that AaZFP1 enhanced the promoter activity of AaIPPI1 in vivo. Transient overexpression of AaZFP1 in A. annua increased the expression of AaIPPI1 and the content of artemisinin. Our data demonstrated that AaZFP1 functions as a transcriptional activator that regulates the expression of AaIPPI1 by directly binding to its promoter. The present study provides insights into the transcriptional regulation of genes involved in artemisinin biosynthesis in A. annua.

Assessment of rhizosphere bacterial diversity and composition in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) in an antimony mine.

AIMS: Heavy metal hyperaccumulators are widely used in mining restoration due to their ability to accumulate and transport heavy metals, compared to nonaccumulators. Rhizosphere bacteria in metal hyperaccumulators play a key role in the uptake of heavy metals from soil; however, assessments of the differences of rhizosphere bacteria between metal hyperaccumulators and nonaccumulator are scarce. METHODS AND RESULTS: To understand the difference of bacterial composition between hyperaccumulator and nonaccumulator in rhizosphere, the diversity and composition of rhizosphere bacteria in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) grown in the same field in Xikuangshan were evaluated using Illumina Miseq high-throughput sequencing technology. Boehmeria nivea and A. annua had 3926 overlapping OTUs, 19,736 and 17,579 unique OTUs, respectively. Boehmeria nivea had lower Chao1 index, Shannon index and Pielou index than A. annua. The dominant phyla and genera of rhizosphere bacteria in B. nivea and A. annua were similar, but some rhizosphere bacterial communities with heavy metal remediation ability mainly appeared in the rhizosphere of the hyperaccumulator. Compared to A. annua, B. nivea showed a significantly higher relative abundance of rhizosphere bacteria, such as Acidobacteria and Bacteroidete at the phylum level and RB41 at the genus level. Some specific rhizosphere bacteria with the ability to bind metal, such as Leifsonia and Kibdelosporangium, were only found in the rhizosphere of B. nivea. CONCLUSION: Results indicated that B. nivea, as a metal hyperaccumulator, has a key function in governing metal-resistant rhizosphere bacteria in response to antimony compound pollution stress. SIGNIFICANCE AND IMPACT OF STUDY: Understanding the diversity of rhizosphere bacteria between hyperaccumulators and nonaccumulators is beneficial to formulate strategies to improve metal uptake efficiency by selecting specific plant species and rhizosphere bacteria grown on polluted soil.

Biotechnological Approaches for Production of Artemisinin, an Anti-Malarial Drug from Artemisia annua L.

Artemisinin is an anti-malarial sesquiterpene lactone derived from Artemisia annua L. (Asteraceae family). One of the most widely used modes of treatment for malaria is an artemisinin-based combination therapy. Artemisinin and its associated compounds have a variety of pharmacological qualities that have helped achieve economic prominence in recent years. So far, research on the biosynthesis of this bioactive metabolite has revealed that it is produced in glandular trichomes and that the genes responsible for its production must be overexpressed in order to meet demand. Using biotechnological applications such as tissue culture, genetic engineering, and bioreactor-based approaches would aid in the upregulation of artemisinin yield, which is needed for the future. The current review focuses on the tissue culture aspects of propagation of A. annua and production of artemisinin from A. annua L. cell and organ cultures. The review also focuses on elicitation strategies in cell and organ cultures, as well as artemisinin biosynthesis and metabolic engineering of biosynthetic genes in Artemisia and plant model systems.

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 transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua.

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density; however, the factors affecting trichome development are largely unknown. Here, we demonstrate that the A. annua R2R3 MYB transcription factor TrichomeLess Regulator 1 (TLR1) negatively regulates trichome development. In A. annua, TLR1 overexpression lines had 44.7%-64.0% lower trichome density and 11.5%-49.4% lower artemisinin contents and TLR1-RNAi lines had 33%-93.3% higher trichome density and 32.2%-84.0% higher artemisinin contents compared with non-transgenic controls. TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A. annua. When heterologously expressed in Arabidopsis thaliana, TLR1 interacts with GLABROUS3a, positive regulator of trichome development, and represses trichome development. Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox (WOX) protein AaWOX1, which interacts with the LEAFY-like transcription factor TLR2. TLR2 overexpression in Arabidopsis and A. annua showed that TLR2 reduces trichome development by reducing gibberellin levels. Furthermore, artemisinin contents were 19%-43% lower in TLR2-overexpressing A. annua plants compared to controls. These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

An HD-ZIP-MYB complex regulates glandular secretory trichome initiation in Artemisia annua.

Plant glandular secretory trichomes (GSTs) produce various specialized metabolites. Increasing GST density represents a strategy to enhance the yield of these chemicals; however, the gene regulatory network that controls GST initiation remains unclear. In a previous study of Artemisia annua L., we found that a HD-ZIP IV transcription factor, AaHD1, promotes GST initiation by directly regulating AaGSW2. Here, we identified two AaHD1-interacting transcription factors, namely AaMIXTA-like 2 (AaMYB16) and AaMYB5. Through the generation and characterization of transgenic plants, we found that AaMYB16 is a positive regulator of GST initiation, whereas AaMYB5 has the opposite effect. Notably, neither of them regulates GST formation independently. Rather, they act competitively, by interacting and modulating AaHD1 promoter binding activity. Additionally, the phytohormone jasmonic acid (JA) was shown to be associated with the AaHD1-AaMYB16/AaMYB5 regulatory network through transcriptional regulation via a JASMONATE-ZIM DOMAIN (JAZ) protein repressor. These results bring new insights into the mechanism of GST initiation through regulatory complexes, which appear to have similar functions in a range of vascular plant taxa.