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Background: β-Caryophyllene, a sesquiterpenoid, holds considerable potential in pharmaceutical, nutraceutical, cosmetic, and chemical industries. In order to overcome the limitation of β-caryophyllene production by the extraction from plants or chemical synthesis, we aimed the microbial production of β-caryophyllene in non-conventional yeast Yarrowia lipolytica in this study., Results: Two genes, tHMG1 from S. cerevisiae to boost the mevalonate pool and QHS1 from Artemisia annua, were expressed under different promoters and copy numbers in Y. lipolytica. The co-expression of 8UAS pEYK1-QHS1 and pTEF-tHMG1 in the obese strain yielded 165.4 mg/L and 201.5 mg/L of β-caryophyllene in single and double copies, respectively. Employing the same combination of promoters and genes in wild-type-based strain with two copies resulted in a 1.36-fold increase in β-caryophyllene. The introduction of an additional three copies of 8UAS pEYK1-tHMG1 further augmented the β-caryophyllene, reaching 318.5 mg/L in flask fermentation. To maximize the production titer, we optimized the carbon source ratio between glucose and erythritol as well as fermentation condition that led to 798.1 mg/L of β-caryophyllene., Conclusions: A biosynthetic pathway of β-caryophyllene was firstly investigated in Y. lipolytica in this study. Through the modulation of key enzyme expression, we successfully demonstrated an improvement in β-caryophyllene production. This strategy suggests its potential extension to studies involving the microbial production of various industrially relevant terpenes., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Artemisinin, a potent antimalarial compound, is predominantly derived from Artemisia annua. The uniqueness of artemisinin production in A. annua lies in its complex biochemical pathways and genetic composition, distinguishing it from other plant species, even within the Asteraceae family. In this review, we investigate the potential of A. annua for artemisinin production, drawing evidence from natural populations and mutants. Leveraging high-quality whole-genome sequence analyses, we offer insights into the evolution of artemisinin biosynthesis. We also highlight current understanding of the protective functions of artemisinin in A. annua in response to both biotic and abiotic stresses. In addition, we explore the mechanisms used by A. annua to mitigate the phytotoxicity generated by artemisinin catabolism., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sweet wormwood (Artemisia annua), an annual herb belonging to the Compositae family, is the main source of the potent anti-malarial drug artemisinin, which is mainly produced in glandular trichomes of A. annua leaves. The WD40 protein family is one of the largest protein families in eukaryotes and plays crucial roles in regulating plant growth and development, stress responses, and secondary metabolite biosynthesis. However, WD40 proteins have not been comprehensively identified in A. annua. In this study, we identified 236 WD40 proteins in the A. annua genome and examined their conserved domains, motifs, and cis-regulatory elements, gene structures, chromosomal distribution, duplication events of their encoding genes. Furthermore, we isolated and characterized TRANSPARENT TESTA GLABROUS 1 (AaTTG1), a homolog of Arabidopsis TTG1, and confirmed that AaTTG1 was localized to the nucleus and cytoplasm. Indeed, AaTTG1 can rescue the glabrous phenotype of the Arabidopsis ttg1 mutant and enhanced trichome production when heterologously expressed in wild-type Arabidopsis plants. Transgenic A. annua lines overexpressing AaTTG1 displayed a significantly higher density of glandular trichomes and higher artemisinin contents. Transgenic A. annua lines with inhibited AaTTG1 function had fewer glandular trichomes and lower artemisinin levels. Moreover, we demonstrated that AaTTG1 positively regulates glandular trichome development in A. annua through interactions with AaSPL9. This study thus provides fundamental insights into the role of WD40 proteins in A. annua and introduces a promising approach to enhance artemisinin production by manipulating glandular trichome development in this valuable medicinal plant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Artemisinin, the well-known natural product for treating malaria, is biosynthesised and stored in the glandular-secreting trichomes (GSTs) of Artemisia annua. While numerous efforts have clarified artemisinin metabolism and regulation, the molecular association between artemisinin biosynthesis and GST development remains elusive. Here, we identified AaMYC3, a bHLH transcription factor of A. annua, induced by jasmonic acid (JA), which simultaneously regulates GST density and artemisinin biosynthesis. Overexpressing AaMYC3 led to a substantial increase in GST density and artemisinin accumulation. Conversely, in the RNAi-AaMYC3 lines, both GST density and artemisinin content were markedly reduced. Through RNA-seq and analyses conducted both in vivo and in vitro, AaMYC3 not only directly activates AaHD1 transcription, initiating GST development, but also up-regulates the expression of artemisinin biosynthetic genes, including CYP71AV1 and ALDH1, thereby promoting artemisinin production. Furthermore, AaMYC3 acts as a co-activator, interacting with AabHLH1 and AabHLH113, to trigger the transcription of two crucial enzymes in the artemisinin biosynthesis pathway, ADS and DBR2, ultimately boosting yield. Our findings highlight a critical connection between GST initiation and artemisinin biosynthesis in A. annua, providing a new target for molecular design breeding of traditional Chinese medicine., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Cold stress, a major abiotic factor, positively modulates the synthesis of artemisinin in Artemisia annua and influences the biosynthesis of other secondary metabolites. To elucidate the changes in the synthesis of secondary metabolites under low-temperature conditions, we conducted dynamic transcriptomic and metabolite quantification analyses of A. annua leaves. The accumulation of total organic carbon (TOC) in leaves under cold stress provided ample precursors for secondary metabolite synthesis. Short-term exposure to low temperature induced a transient increase in jasmonic acid synthesis, which positively regulates the artemisinin biosynthetic pathway, contributing to artemisinin accumulation. Additionally, transcripts of genes encoding key enzymes and transcription factors in both the phenylpropanoid and artemisinin biosynthetic pathways, including PAL, C4H, ADS, and DBR2, exhibited similar expression patterns, suggesting a coordinated effect between these pathways. Prolonged exposure to low temperature sustained high levels of phenylpropanoid synthesis, leading to significant increases in lignin, flavonoids, and anthocyanin. Conversely, the final stage of the artemisinin biosynthetic pathway is inhibited under these conditions, resulting in elevated levels of dihydroartemisinic acid and artemisinic acid. Collectively, our study provides insights into the parallel transcriptional regulation of artemisinin and phenylpropanoid biosynthetic pathways in A. annua under cold stress., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Farnesene synthase from Artemisia annua (AaFS) catalyzes the reaction from farnesyl pyrophosphate (FPP) to give the sesquiterpene β-farnesene, a key building block for the biosynthesis of vitamin E. However, an insufficient yield of β-farnesene precludes its industrialization. Understanding the mechanism would be essential for attaining β-farnesene in high yield. Guided by structure-based enzyme engineering, we designed several potent variants, among which L326I increased the β-farnesene yield from 450.65 to 3877.42 mg/L. Furthermore, we found that the function of β-farnesene synthase AaFS can be modulated at two positions; W299 is responsible for tuning the enzyme's function to give its isomeric product α-farnesene and Y402 is the key residue for diverting from the linear farnesene products to the monocyclic α-bisabolol product. These findings provide valuable insights into the catalytic mechanism and functional modulation of farnesene synthases and set the basis for rational engineering of farnesene synthases for selective biosynthesis of diverse sesquiterpene natural products.

Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua, is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes-Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)-in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to non-transgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC-MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Artemisinin-based combinational therapies (ACTs) constitute the first line of malaria treatment. However, due to its trichome-specific biosynthesis, low concentration, and poor understanding of regulatory mechanisms involved in artemisinin biosynthesis and trichome development, it becomes very difficult to meet the increased demand for ACTs. Here, we have reported that a bHLH transcription factor, AaMYC2-type, plays an important role in regulating GST development and artemisinin biosynthesis in Artemisia annua. AaMYC2-type encodes a protein that is transcriptionally active and localised to the nucleus. It is prominently expressed in aerial parts like leaves, stems, inflorescence and least expressed in roots. AaMYC2-type expression is significantly increased under different hormonal treatments. In transgenic overexpression lines, AaMYC2-type OE, a significant increase in the expression of trichome development and artemisinin biosynthesis genes was observed. While in knockdown lines, Aamyc2-type, expression of trichome development and artemisinin biosynthesis genes were significantly reduced. Yeast one-hybrid assay clearly shows that the AaMYC2-type directly binds to the E-boxes in the promoter regions of ADS and CYP71AVI. The SEM microscopy depicted the number of trichomes elevated from 11 mm -2 in AaMYC2-type OE lines to 6.1 mm -2 in Aamyc2-type. The final effect of the alteration in biosynthetic and trichome developmental genes was observed in the accumulation of artemisinin. In the AaMYC2-type OE, the artemisinin content was 12 mg g -1 DW, which was reduced to 3.2 mg g -1 DW in the Aamyc2-type. Altogether, the above findings suggest that the AaMYC2-type play a dual regulating role in controlling both trichome developmental and artemisinin biosynthetic genes., (© 2024 Scandinavian Plant Physiology Society. Published by John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

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., (© 2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

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., (© 2024 Scandinavian Plant Physiology Society.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The leaves of Artemisia annua contain GSTs (Glandular secretory trichomes) that can secrete and store artemisinin, the drug most effective for treating uncomplicated malaria. Therefore, increasing the density of GSTs in A. annua is an efficient way to enhance artemisinin content. However, our understanding of how GSTs develop still needs to be improved. Here, we isolated an A. annua homolog of AtGL3 (GLABRA3), known as AaGL3-like, that positively regulates trichome density in A. annua. AaGL3-like is nuclear-localized and transcriptionally active. It is least expressed in roots and most prominently in aerial components like leaves, stems, and inflorescence. Under JA and GA hormonal treatments, AaGL3-like expression is significantly increased. In transgenic over-expression AaGL3-like lines, trichome developmental genes such as AaHD1 and AaGSW2 showed much increased expression. The AaGL3RNAi line exhibited considerably lower levels of AaHD1 and AaGSW2 transcripts. As a result, the AaGL3-RNAi lines showed reduced levels of artemisinin content and trichome density compared to wild-type and overexpression lines. Additionally, we have found that when co-expressed with AaJAZ8, the induction of trichome developmental genes was reduced as compared to individual OEAaGL3-like lines. Further, AaJAZ8 directly binds to AaGL3-like in the Y2H assay. These findings suggest that AaGL3-like is a jasmonate-induced bHLH transcription factor that drastically increases the final accumulation of artemisinin content by regulating trichome density in A. annua., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We investigated the effects of graphene on the model herb Artemisia annua, which is renowned for producing artemisinin, a widely used pharmacological compound. Seedling growth and biomass were promoted when A. annua was cultivated with low concentrations of graphene, an effect which was attributed to a 1.4-fold increase in nitrogen uptake, a 15%-22% increase in chlorophyll fluorescence, and greater abundance of carbon cycling-related bacteria. Exposure to 10 or 20 mg/L graphene resulted in a ∼60% increase in H 2 O 2 , and graphene could act as a catalyst accelerator, leading to a 9-fold increase in catalase (CAT) activity in vitro and thereby maintaining reactive oxygen species (ROS) homeostasis. Importantly, graphene exposure led to an 80% increase in the density of glandular secreting trichomes (GSTs), in which artemisinin is biosynthesized and stored. This contributed to a 5% increase in artemisinin content in mature leaves. Interestingly, expression of miR828 was reduced by both graphene and H 2 O 2 treatments, resulting in induction of its target gene AaMYB17, a positive regulator of GST initiation. Subsequent molecular and genetic assays showed that graphene-induced H 2 O 2 inhibits micro-RNA (miRNA) biogenesis through Dicers and regulates the miR828-AaMYB17 module, thus affecting GST density. Our results suggest that graphene may contribute to yield improvement in A. annua via dynamic physiological processes together with miRNA regulation, and it may thus represent a new cultivation strategy for increasing yield capacity through nanobiotechnology., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glandular trichomes are specialized structures found on the surface of plants to produce specific compounds, including terpenes, alkaloids, and other organic substances. Artemisia annua, commonly known as sweet wormwood, synthesizes and stores the antimalarial drug artemisinin in glandular trichomes. Previous research indicated that increasing the glandular trichome density could enhance artemisinin production, and the cuticle synthesis affected the initiation and development of glandular trichomes in A. annua. In this study, AaABCG12 and AaABCG20 were isolated from A. annua that exhibited similar expression patterns to artemisinin biosynthetic genes. Of the two, AaABCG20 acted as a specific transporter in glandular trichomes. Downregulating the expression of AaABCG20 resulted in a notable reduction in the density of glandular trichome, while overexpressing AaABCG20 resulted in an increase in glandular trichome density. GC-MS analysis demonstrated that AaABCG20 was responsible for the transport of cutin and wax in A. annua. These findings indicated that AaABCG20 influenced the initiation and development of glandular trichomes through transporting cutin and wax in A. annua. This glandular trichome specific half-size ABCG-type transporter is crucial in facilitating the transportation of cutin and wax components, ultimately contributing to the successful initiation and development of glandular trichomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The emergence of multidrug resistance (MDR) in bacterial pathogens is a serious public health concern. A significant therapeutic target for MDR infections is the quorum sensing-regulated bacterial pathogenicity. Determining the anti-quorum sensing abilities of certain medicinal plants against bacterial pathogens as well as the in-silico interactions of particular bioactive phytocompounds with QS and biofilm-associated proteins were the objectives of the present study. In this study, 6 medicinal plants were selected based on their ethnopharmacological usage, screened for Anti-QS activity and Artemisia annua leaf extract (AALE) demonstrated pigment inhibitory activity against Chromobacterium violaceum CV12472. Further, the methanol active fraction significantly inhibited the virulence factors (pyocyanin, pyoverdine, rhamnolipid and swarming motility) of Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97 at respective sub-MICs. The inhibition of biofilm was determined using a microtiter plate test and scanning electron microscopy. Biofilm formation was impaired by 70%, 72% and 74% in P. aeruginosa, C. violaceum and S. marcescens, respectively at 0.5xMIC of the extract. The phytochemical content of the extract was studied using GC-MS and 1, 8-cineole was identified as major bioactive compound. Furthermore, 1, 8-cineole was docked with quorum sensing (QS) proteins (LasI, LasR, CviR, and rhlR) and biofilm proteins (PilY1 and PilT). In silico docking and dynamics simulations studies suggested interactions with QS-receptors CviR', LasI, LasR, and biofilm proteins PilY1, PilT for anti-QS activity. Further, 1, 8-cineole demonstrated 66% and 51% reduction in violacein production and biofilm formation, respectively to validate the findings of computational analysis. Findings of the present investigation suggests that 1, 8-cineole plays a crucial role in the QS and biofilm inhibitory activity demonstrated by Artemisia annua extract. RESEARCH HIGHLIGHTS: Artemisia annua leaf extract (AALE) methanol fraction demonstrated broad-spectrum QS and biofilm inhibition Scanning electron microscopy (SEM) confirmed biofilm inhibition Molecular docking and simulation studies suggested positive interactions of 1,8-cineol with QS-receptors and biofilm proteins., (© 2023 Wiley Periodicals LLC.)

Artemisia annua, a member of the Asteraceae family, remains the primary source of artemisinin. However, the artemisinin content in the existing varieties of this plant is very low. In this study, we found that the environmental factors light and phytohormone abscisic acid (ABA) could synergistically promote the expression of artemisinin biosynthetic genes. Notably, the increased expression levels of those genes regulated by ABA depended on light. Gene expression analysis found that AaABI5, a transcription factor belonging to the basic leucine zipper (bZIP) family, was inducible by the light and ABA treatment. Analysis of AaABI5-overexpressing and -suppressing lines suggested that AaABI5 could enhance artemisinin biosynthesis and activate the expression of four core biosynthetic genes. In addition, the key regulator of light-induced artemisinin biosynthesis, AaHY5, could bind to the promoter of AaABI5 and activate its expression. In conclusion, our results demonstrated that AaABI5 acts as an important molecular junction for the synergistic promotion of artemisinin biosynthesis by light and ABA signals, which provides a candidate gene for developing new germplasms of high-quality A. annua., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Natural products are a rich source of bioactive molecules that have potential pharmacotherapeutic applications. In this study, we focused on Artemisia annua (A. annua) and its enriched extracts which were biologically evaluated in vitro as virucidal, antiviral, and antioxidant agents, with a potential application against the COVID-19 infection. The crude extract showed virucidal, antiviral and antioxidant effects in concentrations that did not affect cell viability. Scopoletin, arteannuin B and artemisinic acid (single fractions isolated from A. annua) exerted a considerable virucidal and antiviral effect in vitro starting from a concentration of 50 µg/mL. Data from Surface Plasmon Resonance (SPR) showed that the inhibition of the viral infection was due to the interaction of these compounds with the 3CLpro and Spike proteins of SARS-CoV-2, suggesting that the main interaction of compounds may interfere with the viral pathways during the insertion and the replication process. The present study suggests that natural extract of A. annua and its components could have a key role as antioxidants and antiviral agents and support the fight against SARS-CoV-2 variants and other possible emerging Coronaviruses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 Mahshid Ghanbari et al., published by Sciendo.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although mainly known for producing artemisinin, Artemisia annua is enriched in phenylpropanoid glucosides (PGs) with significant bioactivities. However, the biosynthesis of A. annua PGs is insufficiently investigated. Different A. annua ecotypes from distinct growing environments accumulate varying amounts of metabolites, including artemisinin and PGs such as scopolin. UDP-glucose:phenylpropanoid glucosyltransferases (UGTs) transfers glucose from UDP-glucose in PG biosynthesis. Here, we found that the low-artemisinin ecotype GS produces a higher amount of scopolin, compared to the high-artemisinin ecotype HN. By combining transcriptome and proteome analyses, we selected 28 candidate AaUGTs from 177 annotated AaUGTs. Using AlphaFold structural prediction and molecular docking, we determined the binding affinities of 16 AaUGTs. Seven of the AaUGTs enzymatically glycosylated phenylpropanoids. AaUGT25 converted scopoletin to scopolin and esculetin to esculin. The lack of accumulation of esculin in the leaf and the high catalytic efficiency of AaUGT25 on esculetin suggest that esculetin is methylated to scopoletin, the precursor of scopolin. We also discovered that AaOMT1, a previously uncharacterized O-methyltransferase, converts esculetin to scopoletin, suggesting an alternative route for producing scopoletin, which contributes to the high-level accumulation of scopolin in A. annua leaves. AaUGT1 and AaUGT25 responded to induction of stress-related phytohormones, implying the involvement of PGs in stress responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 (AlPO 4 , AlP), using soluble monopotassium phosphate (KH 2 PO 4 , 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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., (© 2023 Scandinavian Plant Physiology Society.)

Oxidative stress is an important contributor to the pathogenesis of Alzheimer's disease (AD). The overproduction of reactive oxygen species observed in AD patients results in the loss of mitochondrial function, altered metal ion homeostasis, lipopolysaccharide metabolism disorder, reduced anti-oxidant defense, increased release of inflammatory factors, and the aggravation and accumulation of amyloid-beta and tau hyper-phosphorylation, which directly cause synaptic and neuronal loss and lead to cognitive dysfunction. Thus, oxidative stress proves to be a fundamental part of AD development and progression, suggesting the potential benefits of anti-oxidant-based therapies for AD. In this study, we found that a water-soluble extract of Artemisia annua ( WSEAA ), a traditional Chinese herbal medicine, has a strong anti-oxidant function. We also found that WSEAA is able to improve the cognitive function of 3xTg AD mice. However, the mechanisms and molecular targets underlying WSEAA action are still not known. In order to uncover the potential molecular mechanisms involved, we used a combination of network pharmacology and different experimental approaches. Obtained results revealed key genes (such as AKT1, BCL2, IL-6, TNF-[Formula: see text] and BAX) and signaling pathways (like PI3K-AKT and BCL2/BAX) are closely associated with the biological processes responding to oxidative stress. Further verification of the survival/anti-oxidant effects of WSEAA in vitro and in vivo showed that the extract has anti-oxidatant/neuronal survival action against H 2 O 2 -induced damage, and is thus able to prevent the cognitive decline and pathological changes of 3xTg transgenic (3xTg) mice via the regulation of key target-genes and pathways, such as PI3K-AKT and BCL2/BAX, related to survival/apoptosis. Our findings strongly indicate the potential of WSEAA for the prevention and treatment of AD.

Chronic liver disease(CLD) is a slow-developing and long-term disease that can cause serious damage to the liver. Thus far, it has been associated with viral hepatitis, non-alcoholic fatty liver disease(NAFLD), alcoholic liver disease(ALD), hepatic fibrosis(HF), liver cirrhosis (LC), and liver cancer. Qinghao Biejia Decoction (QBD) is a classic ancient Chinese herbal prescription with strong immune-enhancing, anti-inflammatory, and anti-tumor effects. In this study, we used a network pharmacology approach to investigate the molecular mechanisms of QBD in the inflammation-carcinoma transformation process of chronic liver disease. Two key drug targets, MAPK1 and PIK3CA, were screened using network pharmacology and molecular docking techniques, revealing dihydroartemisinin, artesunate, 12-O-Nicotinoylisolineolone, caffeic acid, and diincarvilone A as active ingredients involved in QBD mechanisms. The main signaling pathways involved were the PI3K-AKT signaling pathway and MAPK signaling pathway. In summary, our results indicated that QBD affects the inflammatory transformation of chronic liver disease through MAPK1 and PIK3CA and signaling pathways MAPK and PI3K/AKT. These data provide research direction for investigating the mechanisms underlying the inflammation-carcinoma transformation process in QBD for chronic liver disease., Competing Interests: The authors declare that they have no conflicts of interest in this paper., (Copyright © 2022 Xin Cheng et al.)

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.

Artemisia annua L. ( A. annua ) contains artemisinin, which attracts attention on account of its anti-inflammatory and anti-oxidant effects. Increased intestinal inflammation, oxidative stress, and hypoimmunity commonly occur in neonatal and early-weaning piglets. Abundant evidence suggests that maternal nutritional interventions during pregnancy modify the offspring's long-term gut development. The present study was conducted to investigate the effects of maternal A. annua extract (AAE) supplementation on the offspring's intestinal inflammation and redox status. A total of 90 pregnant sows were assigned randomly and equally into the control (CON) group (fed with a basal diet) and the 0.1% (AAE) group (basal diet with 1.0 g kg -1 AAE) during late gestation and lactation. The results showed that 0.1% AAE supplementation significantly decreased the contents and relative mRNA expressions of interleukin (IL)-1β, IL-6, and IL-12, and tumor necrosis factor-α in the small intestine of the newborn and weaned piglets (offspring) ( P < 0.05). There were higher activities of total antioxidant capacity and total superoxide dismutase, whereas a lower concentration of malondialdehyde in the small instestine of offspring in the 0.1% AAE group than that in the CON group ( P < 0.05). Furthermore, the 0.1% AAE group decreased the mRNA and protein expressions of Toll-like receptor 4 (TLR4) and inhibited the activation of TLR4-mediated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways ( P < 0.05). The mRNA expression of peroxisome proliferator activated receptor γ (PPARγ), porcine beta-defensin (PBD)-1, PBD-2, PBD-3, mucin (MUC)-1, MUC-2 and MUC-4 was significantly enhanced in the small intestine of both neonatal and weanling piglets ( P < 0.05). Together, these results showed that maternal 0.1% AAE supplementation improved the redox status and attenuated the neonatal and early-weaning associated inflammatory response in the offspring's small intestine, possibly by suppressing the activation of the TLR4/NF-κB and MAPK inflammatory pathways, and stimulated expressions of beta-defensins, mucins, and PPARγ to promote inflammation resolution and innate immunity response.

Artemisia annua is the major natural source of artemisinin, an anti-malarial medicine commonly used worldwide. Here, we present chromosome-level haploid maps for two A. annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production. High-fidelity sequencing, optical mapping, and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A. annua. Approximately 50,000 genes were annotated for each haplotype genome, and a triplication event that occurred approximately 58.12 million years ago was examined for the first time in this species. A total of 3,903,467-5,193,414 variants (SNPs, indels, and structural variants) were identified in the 1.5-Gb genome during pairwise comparison between haplotypes, consistent with the high heterozygosity of this species. Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-diene synthase genes. This correlation was further confirmed by resequencing of 36 A. annua samples with varied artemisinin contents. Circular consensus sequencing of transcripts facilitated the detection of paralog expression. Collectively, our study provides chromosome-level allele-aware genome assemblies for two A. annua strains and new insights into the biosynthesis of artemisinin and its regulation, which will contribute to conquering malaria worldwide., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

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. β-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., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

A supply of plant micronutrients (some of which are metals) is necessary to regulate many plant processes; their excess, however, can have detrimental consequences and can hamper plant growth, physiology and metabolism. Artemisia annua is an important crop plant used in the treatment of malaria. In this investigation, the physio-biochemical mechanisms involved in exogenous hydrogen sulphide-mediated (H 2 S) alleviation of copper (Cu) stress in A. annua were assessed.. Two different levels of Cu (20, 40 mg·kg -1 ), one H 2 S treatment (200 µm) and their combinations were introduced while one set of plants was retained as control. Results showed that the presence of excess Cu in the soil reduced growth and biomass, photosynthetic parameters, chlorophyll content and fluorescence, gas exchange parameters and induced antioxidant enzyme activity. Copper stress enhanced the production of thiobarbituric acid reactive substances (TBARS) and increased Cu content in both roots and shoots of affected plants. Exogenous application of H 2 S restored the physio-biochemical characteristics of Cu-treated A. annua plants by reducing lipid peroxidation and enhancing the activity of antioxidant enzymes in Cu-stressed plants as compared with the controls. Hydrogen sulphide also reduced the Cu content in different plant parts, increased photosynthetic efficiency, trichome density, average area of trichomes and artemisinin content. Therefore, our results provide a comprehensive assessment of the defensive role of H 2 S in Cu-stressed A. annua., (© 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

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., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

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-N 1-10 -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-N 1-10 -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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (© 2022 Society for Applied Microbiology.)

Glandular trichomes can secrete and store a large number of secondary metabolites in plants, some of which are of high medicinal and commercial value. For example, artemisinin, isolated from Artemisia annua L. plants, and its derivatives have great high medicinal value. Previous research indicated that artemisinin was synthesized in the glandular trichomes on the leaves of A. annua. It is an important study direction to improve artemisinin yield by promoting the initiation and development of glandular trichome. In this study, SQUAMOSA promoter-binding protein-like 9 (AaSPL9) was identified. In AaSPL9 overexpression transgenic plants, the glandular trichomes density was increased by 45-60 %, and the content of artemisinin was increased by 33-60 %, indicating that AaSPL9 positively regulate the glandular trichomes initiation. Yeast one-hybrid(Y1H), Dual-luciferase (Dual-Luc), Electrophoretic Mobility Shift Assay (EMSA) demonstrated that AaSPL9 activated the expression of AaHD1 by combining directly the GTAC-box of the AaHD1 promoter. Taken together, we identified AaSPL9, a positive transcription factor, regulating the glandular trichome initiation in A. annua, and revealed a novel molecular mechanism by which a SPL protein to promote glandular trichome initiation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

ChIP-seq (Chromatin immunoprecipitation with sequencing) is the gold standard for determining genome-wide in vivo transcription factor binding sites, the first step for targets prediction and network construction. For non-model plants, it is challenging to perform ChIP-seq due to the difficulty in generating stable transgenic plants. AaHY5 is a positive regulator in artemisinin biosynthesis, whose detailed mode of action remains elusive. Here, we established a protoplast transformation procedure for Artemisia annua by optimizing different conditions in protoplast isolation and transfection. We then performed AaHY5 ChIP-seq based on the established transient expression system. Combining RNA-seq data for various tissues, we identified four transcription factors (one MYB and three WRKY family members) in AaHY5 targets that potentially regulated artemisinin biosynthesis. The three WRKY transcription factors could be induced by light and the overexpression of AaHY5 and upregulate two artemisinin biosynthetic genes, ADS and CYP71AV1. Furthermore, AaWRKY14 showed transcriptional activation activity on artemisinin biosynthetic gene CYP71AV1. Together, AaWRKY14 was identified as a potential transcription factor linking AaHY5 and the artemisinin biosynthetic gene regulation., (Copyright © 2021. Published by Elsevier Masson SAS.)

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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Artemisinin is a secondary metabolite extracted from Artemisia annua. As an effective antimalarial component certified by WHO, artemisinin has extensive economical values. Numerous studies about transcription factors positively regulating artemisinin biosynthesis have been published while negative regulators are rarely reported. In the present study, we identified AaMYB15 as the first R2R3-MYB that negatively regulates artemisinin biosynthesis in A. annua. Experimental evidences showed that AaMYB15 is a transcription factor within nucleus and predominantly expressed in glandular secretory trichomes (GSTs) in A. annua where artemisinin is synthesized and accumulated. The expression of AaMYB15 was induced by dark and JA treatment. Overexpression of AaMYB15 led to a significant decline in the expression levels of key enzyme genes ADS, CYP, DBR2, and ALDH1 and a significant decrease in the artemisinin contents of transgenic A. annua. AaMYB15 directly bound to the promoter of AaORA, a reported positive regulator of artemisinin biosynthesis in JA signaling pathway, to repress its transcriptional activity, thus downregulating the expression levels of downstream key enzyme genes and negatively regulating the artemisinin biosynthesis. Our study provides candidate gene for improvement of A. annua germplasm and new insights into the artemisinin biosynthesis regulation network mediated by light and JA., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

UV-B radiation is a pivotal photomorphogenic signal and positively regulates plant growth and metabolite biosynthesis. In order to elucidate the transcriptional regulation mechanism underlying UV-B-induced artemisinin and flavonoid biosynthesis in Artemisia annua, the transcriptional responses of A. annua L. leaves to UV-B radiation were analyzed using the Illumina transcriptome sequencing. A total of 10705 differentially expressed genes (DEGs) including 533 transcription factors (TFs), were identified. Based on the expression trends of the differentially expressed TFs as well as artemisinin and flavonoid biosynthesis genes, we speculated that TFs belonging to 6 clusters were most likely to be involved in the regulation of artemisinin and/or flavonoid biosynthesis. The regulatory relationship between TFs and artemisinin/flavonoid biosynthetic genes was further studied. Dual-LUC assays results showed that AaMYB6 is a positive regulator of AaLDOX which belongs to flavonoid biosynthesis pathway. In addition, we identified an R2R3 MYB TF, AaMYB4 which potentially mediated both artemisinin and flavonoid biosynthesis pathways by activating the expression of AaADS and AaDBR2 in artemisinin biosynthesis pathway and AaUFGT in flavonoid biosynthesis pathway. Overall, our findings would provide an insight into the elucidation of the parallel transcriptional regulation of artemisinin and flavonoid biosynthesis in A. annua L. under UV-B radiation., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Artemisinin (ART) is the most effective component in malaria treatment, however, the extremely low content restricts its clinical application. Therefore, it is urgent to increase the yield of ART. ART gradually accumulates with aging, small RNA (sRNA) and transcriptome analysis were applied on the leaves of 2-week-old (2 w) and 3-month-old (3 m) A. annua respectively. Among all the annotated sRNAs, 125 were upregulated and 128 downregulated in the 3 m sample compared to the 2 w one. Whereas 2183 genes were upregulated and 2156 downregulated. Notably, the level of miR156 and several annotated miRNAs gradually decreased while SPLs increased. In addition, the genes on ART biosynthesis pathway were significantly upregulated including ADS, CYP71AV1, ADH1, DBR2 and ALDH1, and so were the positive transcription factors like AaERF1, AaORA and AaWRKY1 indicating that age influences the ART biosynthesis by activating the expression of the synthesizing genes as well as positive transcription factors. This study contributes to reveal the regulatory effects of age on ART biosynthesis both in sRNA and transcription levels.

Nootkatone is a valuable sesquiterpene widely used in the food, fragrance, and flavor industries. Its price is very high due to its limited production in grapefruit peels or Alaska cypress heartwoods. Chemical synthesis of nootkatone uses heavy metals, highly flammable compounds, and strong oxidants, which cause severe damage to the environment. In this study, nootkatone is synthesized in Artemisia annua , using synthetic biology methods. Engineered Artemisia annua coexpressing valencene synthase (VS) and valencene oxidase (VO) in the cytosol produced nootkatone ranging from 0.89 to 8.52 μg/g fresh weight (FW). Furthermore, transgenic Artemisia annua coexpressing farnesyl diphosphate synthase (FPS), VS, and VO in plastids produced nootkatone ranging from 12.11 to 47.80 μg/g FW. These results indicated that engineering nootkatone biosynthesis in plastids was superior to that in the cytosol. Meanwhile, artemisinin production was unaltered in nootkatone-producing Artemisia annua . Our study developed a green approach for producing nootkatone in Artemisia annua with great market potential.

Artemisinin, an effective antimalarial compound, is isolated from the medicinal plant Artemisia annua L. However, because of the low content of artemisinin in A. annua, the demand of artemisinin exceeds supply. Previous studies show that the artemisinin biosynthesis is promoted by light in A. annua. Cryptochrome1 (CRY1) is involved in many processes in the light response. In this study, AaCRY1 was cloned from A. annua. Overexpressing AaCRY1 in Arabidopsis thaliana cry1 mutant resulted in blue-light-dependent short hypocotyl phenotype and short coleoptile under blue light. Yeast two-hybrid and subcellular colocalization showed that AaCRY1 interacted with AtCOP1 (ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1). Overexpression of AaCRY1 in transgenic A. annua increased the artemisinin content. When AaCRY1 was overexpressed in A. annua driven by the CYP71AV1 (cytochrome P450 dependent amorpha-4,11-diene 12-hydroxylase) promoter, the artemisinin content was 1.6 times higher than that of the control. Furthermore, we expressed the C terminal of AaCRY1(CCT) involved a GUS-CCT fusion protein in A. annua. The results showed that the artemisinin content was increased to 1.7- to 2.4-fold in GUS-CCT transgenic A. annua plants. These results demonstrate that overexpression of GUS-CCT is an effective strategy to increase artemisinin production in A. annua., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA's effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro., Competing Interests: The authors have declared that no competing interests exist.