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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.)

The medicinal herb Artemisia annua L. is prized for its capacity to generate artemisinin, which is used to cure malaria. Potentially influencing the biomass and secondary metabolite synthesis of A. annua is plant nutrition, particularly phosphorus (P). However, most soil P exist as insoluble inorganic and organic phosphates, which results to low P availability limiting plant growth and development. Although plants have developed several adaptation strategies to low P levels, genetics and metabolic responses to P status remain largely unknown. In a controlled greenhouse experiment, the sparingly soluble P form, hydroxyapatite (Ca 5 OH(PO 4 ) 3 /CaP) was used to simulate calcareous soils with low P availability. In contrast, the soluble P form KH 2 PO 4 /KP was used as a control. A. annua's morphological traits, growth, and artemisinin concentration were determined, and RNA sequencing was used to identify the differentially expressed genes (DEGs) under two different P forms. Total biomass, plant height, leaf number, and stem diameter, as well as leaf area, decreased by 64.83%, 27.49%, 30.47%, 38.70%, and 54.64% in CaP compared to KP; however, LC-MS tests showed an outstanding 37.97% rise in artemisinin content per unit biomass in CaP contrary to KP. Transcriptome analysis showed 2015 DEGs (1084 up-regulated and 931 down-regulated) between two P forms, including 39 transcription factor (TF) families. Further analysis showed that DEGs were mainly enriched in carbohydrate metabolism, secondary metabolites biosynthesis, enzyme catalytic activity, signal transduction, and so on, such as tricarboxylic acid (TCA) cycle, glycolysis, starch and sucrose metabolism, flavonoid biosynthesis, P metabolism, and plant hormone signal transduction. Meanwhile, several artemisinin biosynthesis genes were up-regulated, including DXS, GPPS, GGPS, MVD, and ALDH, potentially increasing artemisinin accumulation. Furthermore, 21 TF families, including WRKY, MYB, bHLH, and ERF, were up-regulated in reaction to CaP, confirming their importance in P absorption, internal P cycling, and artemisinin biosynthesis regulation. Our results will enable us to comprehend how low P availability impacts the parallel transcriptional control of plant development, growth, and artemisinin production in A. annua. This study could lay the groundwork for future research into the molecular mechanisms underlying A. annua's low P adaptation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

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.)

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.)

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.)

Gene annotation is essential for genome-based studies. However, algorithm-based genome annotation is difficult to fully and correctly reveal genomic information, especially for species with complex genomes. Artemisia annua L. is the only commercial resource of artemisinin production though the content of artemisinin is still to be improved. Genome-based genetic modification and breeding are useful strategies to boost artemisinin content and therefore, ensure the supply of artemisinin and reduce costs, but better gene annotation is urgently needed. In this study, we manually corrected the newly released genome annotation of A. annua using second- and third-generation transcriptome data. We found that incorrect gene information may lead to differences in structural, functional, and expression levels compared to the original expectations. We also identified alternative splicing events and found that genome annotation information impacted identifying alternative splicing genes. We further demonstrated that genome annotation information and alternative splicing could affect gene expression estimation and gene function prediction. Finally, we provided a valuable version of A. annua genome annotation and demonstrated the importance of gene annotation in future research., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microorganisms in the rhizosphere are crucial allies for plant stress tolerance. Recent research suggests that by interacting with the rhizosphere microbiome, microorganisms can aid in the revegetation of soils contaminated with heavy metal(loid)s (HMs). However, it is unknown that how Piriformospora indica influences the rhizosphere microbiome to mitigate arsenic-toxicity in arsenic-enriched environments. Artemisia annua plants were grown in the presence or absence of P. indica and spiked with low (50) and high (150 µmol/L) concentrations of arsenic (As). After inoculation with P. indica, fresh weight increased by 37.7% and 10% in control and high concentration treated plants, respectively. Transmission electron microscopy showed that cellular organelles were severely damaged by As and even disappeared under high concentration. Furthermore, As was mostly accumulated by 5.9 and 18.1 mg/kg dry weight in the roots of inoculated plants treated with low and high concentrations of As, respectively. Additionally, 16 S and ITS rRNA gene sequencing were applied to analyze the rhizosphere microbial community structure of A. annua under different treatments. A significant difference was observed in microbial community structure under different treatments as revealed by non-metric multidimensional scaling ordination. The bacterial and fungal richness and diversity in the rhizosphere of inoculated plants were actively balanced and regulated by P. indica co-cultivation. Lysobacter and Steroidobacter were found to be the As-resistant bacterial genera. We conclude that P. indica inoculation could alter rhizosphere microecology, thereby mitigating As-toxicity without harming the environment., 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.)

Main Conclusion: The hierarchical architecture of chromatins affects the gene expression level of glandular secreting trichomes and the artemisinin biosynthetic pathway-related genes, consequently bringing on huge differences in the content of artemisinin and its derivatives of A. annua. The plant of traditional Chinese medicine "Qinghao" is called Artemisia annua L. in Chinese Pharmacopoeia. High content and the total amount of artemisinin is the main goal of A. annua breeding, nevertheless, the change of chromatin organization during the artemisinin synthesis process has not been discovered yet. This study intended to find the roles of chromatin structure in the production of artemisinin through bioinformatics and experimental validation. Chromosome conformation capture analysis was used to scrutinize the interactions among chromosomes and categorize various scales of chromatin during artemisinin synthesis in A. annua. To confirm the effect of the changes in chromatin structure, Hi-C and RNA-sequencing were performed on two different strains to find the correlation between chromatin structure and gene expression levels on artemisinin synthesis progress and regulation. Our results revealed that the frequency of intra-chromosomal interactions was higher in the inter-chromosomal interactions between the root and leaves on a high artemisinin production strain (HAP) compared to a low artemisinin production strain (LAP). We found that compartmental transition was connected with interactions among different chromatins. Interestingly, glandular secreting trichomes (GSTs) and the artemisinin biosynthetic pathway (ABP) related genes were enriched in the areas which have the compartmental transition, reflecting the regulation of artemisinin synthesis. Topologically associated domain boundaries were associated with various distributions of genes and expression levels. Genes associated with ABP and GST in the adjacent loop were highly expressed, suggesting that epigenetic regulation plays an important role during artemisinin synthesis and glandular secreting trichomes production process. Chromatin structure could show an important status in the mechanisms of artemisinin synthesis process in A. annua., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: Eight promoters were cloned, from which AC and G-box cis-elements were identified. PAP1 enhanced the promoter activity. 2,4-D reduced the anthocyanin biosynthesis via downregulating the expression of the PAP1 transgene. Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-box cis-element. Eight promoters were cloned for activity analysis. Dual luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 µM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per Petri dish. HPLC-qTOF-MS/MS-based profiling showed that these concentrations did not alter anthocyanin profiles. Real-time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated the expression of the chalcone synthase gene either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The plant Artemisia annua L. is famous for producing "artemisinin", which is an essential component in the treatment of malaria. The glandular secretory trichomes (GSTs) on the leaves of A. annua secrete and store artemisinin. Previous research has demonstrated that raising GST density can effectively raise artemisinin content. However, the molecular mechanism of GST initiation is not fully understood yet. In this study, we identified an MYB transcription factor, the AaMYB108-like , which is co-induced by light and jasmonic acid, and positively regulates glandular secretory trichome initiation in A. annua . Overexpression of the AaMYB108-like gene in A. annua increased GST density and enhanced the artemisinin content, whereas anti-sense of the AaMYB108-like gene resulted in the reduction in GST density and artemisinin content. Further experiments demonstrated that the AaMYB108-like gene could form a complex with AaHD8 to promote the expression of downstream AaHD1 , resulting in the initiation of GST. Taken together, the AaMYB108-like gene is a positive regulator induced by light and jasmonic acid for GST initiation in A. annua.

Background: Artemisia annua, a well-known traditional Chinese medicine, is the main source for production of artemisinin, an anti-malaria drug. A. annua is distributed globally, with great diversity of morphological characteristics and artemisinin contents. Diverse traits among A. annua populations impeded the stable production of artemisinin, which needs an efficient tool to identify strains and assess population genetic homogeneity., Purpose: In this study, ribosomal DNA (rDNA), were characterized for A. annua for strains identification and population genetic homogeneity assessment., Methods: The ribosomal RNA (rRNA) genes were identified using cmscan and assembled using rDNA unit of LQ-9 as a reference. rDNA among Asteraceae species were compared performing with 45S rDNA. The rDNA copy number was calculated based on sequencing depth. The polymorphisms of rDNA sequences were identified with bam-readcount, and confirmed by Sanger sequencing and restriction enzyme experiment. The ITS2 amplicon sequencing was used to verify the stability of ITS2 haplotype analysis., Results: Different from other Asteraceae species, 45S and 5S linked-type rDNA was only found in Artemisia genus. Rich polymorphisms of copy number and sequence of rDNA were identified in A. annua population. The haplotype composition of internal transcribed spacer 2 (ITS2) region which had moderate sequence polymorphism and relative short size was significantly different among A. annua strains. A population discrimination method was developed based on ITS2 haplotype analysis with high-throughput sequencing., Conclusion: This study provides comprehensive characteristics of rDNA and suggests that ITS2 haplotype analysis is ideal tool for A. annua strain identification and population genetic homogeneity assessment., 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 GmbH. 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.)

β-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. β-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of β-caryophyllene synthase (CPS) is one of the main restrictive factors for β-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the β-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in V max and K cat . The K cat / K m of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in β-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to β-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6 T1025N to improve the transmembrane transport of β-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of β-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a β-caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of β-caryophyllene production by yeast.

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.)

Trichomes are attractive cells for terpenoid biosynthesis and accumulation in Artemisia annua . However, the molecular process underlying the trichome of A. annua is not yet fully elucidated. In this study, an analysis of multi-tissue transcriptome data was performed to examine trichome-specific expression patterns. A total of 6646 genes were screened and highly expressed in trichomes, including artemisinin biosynthetic genes such as amorpha-4,11-diene synthase ( ADS ) and cytochrome P450 monooxygenase ( CYP71AV1 ). Mapman and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that trichome-specific genes were mainly enriched in lipid metabolism and terpenoid metabolism. These trichome-specific genes were analyzed by a weighted gene co-expression network analysis (WGCNA), and the blue module linked to terpenoid backbone biosynthesis was determined. Hub genes correlated with the artemisinin biosynthetic genes were selected based on TOM value. ORA , Benzoate carboxyl methyltransferase ( BAMT ), Lysine histidine transporter-like 8 ( AATL1 ), Ubiquitin-like protease 1 ( Ulp1 ) and TUBBY were revealed as key hub genes induced by methyl jasmonate (MeJA) for regulating artemisinin biosynthesis. In summary, the identified trichome-specific genes, modules, pathways and hub genes provide clues and shed light on the potential regulatory mechanisms of artemisinin biosynthesis in trichomes in A. annua .

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.)

Exploring the genetic network of glandular trichomes and manipulating genes relevant to secondary metabolite biosynthesis are of great importance and value. Artemisinin, a key antimalarial drug ingredient, is synthesized and stored in glandular secretory trichomes (GSTs) in Artemisia annua. WIN/SHN proteins, a clade of AP2/ERF family, are known as regulators for cuticle biosynthesis. However, their function in glandular trichome development is less unknown. In this study, we identified a WIN/SHN gene from A. annua and named it as AaWIN1. AaWIN1 was predominantly expressed in buds, flowers and trichomes, and encoded a nuclear-localized protein. Overexpressing AaWIN1 in A. annua significantly increased the density of GST as well as the artemisinin content. Furthermore, AaGSW2 was reported to play an important role in promoting GST initiation, and the expression of AaGSW2 was induced in AaWIN1-overexpression lines. AaMIXTA1, a MYB protein positively regulating trichome initiation and cuticle biosynthesis, was confirmed to interact with AaWIN1. In addition, the ectopic expression of AaWIN1 resulted in slender and curled leaves, fewer trichomes, and rising expressions of cuticle biosynthesis genes in Arabidopsis thaliana. Taken together, based on phenotype observations, content measurements and gene expression detections, AaWIN1 was considered as a positive regulator for GST initiation 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 © 2023 Elsevier B.V. All rights reserved.)

Artemisinin, a sesquiterpene compound synthesized and stored in the glandular trichome of Artemisia annua leaves, has been used to treat malaria. Previous studies have shown that both light and jasmonic acid (JA) can promote the biosynthesis of artemisinin, and the promotion of artemisinin by JA is dependent on light. However, the specific molecular mechanism remains unclear. Here, we report a MYB transcription factor, AaMYB108, identified from transcriptome analysis of light and JA treatment, as a positive regulator of artemisinin biosynthesis in A. annua. AaMYB108 promotes artemisinin biosynthesis by interacting with a previously characterized positive regulator of artemisinin, AaGSW1. Then, we found that AaMYB108 interacted with AaCOP1 and AaJAZ8, respectively. The function of AaMYB108 was influenced by AaCOP1 and AaJAZ8. Through the treatment of AaMYB108 transgenic plants with light and JA, it was found that the promotion of artemisinin by light and JA depends on the presence of AaMYB108. Taken together, our results reveal the molecular mechanism of JA regulating artemisinin biosynthesis depending on light in A. annua. This study provides new insights into the integration of light and phytohormone signaling to regulate terpene biosynthesis in plants., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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.)

Background: A. annua (also named Artemisia annua, sweet wormwood) is the main source of the anti-malarial drug artemisinin, which is synthesised and stored in its trichomes. Members of the basic Helix-Loop-Helix (bHLH) family of transcription factors (TFs) have been implicated in artemisinin biosynthesis in A. annua and in trichome development in other plant species., Results: Here, we have systematically identified and characterised 226 putative bHLH TFs in A. annua. All of the proteins contain a HLH domain, 213 of which also contain the basic motif that mediates DNA binding of HLH dimers. Of these, 22 also contained a Myc domain that permits dimerisation with other families of TFs; only two proteins lacking the basic motif contained a Myc domain. Highly conserved GO annotations reflected the transcriptional regulatory role of the identified TFs, and suggested conserved roles in biological processes such as iron homeostasis, and guard cell and endosperm development. Expression analysis revealed that three genes (AabHLH80, AabHLH96, and AaMyc-bHLH3) exhibited spatiotemporal expression patterns similar to genes encoding key enzymes in artemisinin synthesis., Conclusions: This comprehensive analysis of bHLH TFs provides a new resource to direct further analysis into key molecular mechanisms underlying and regulating artemisinin biosynthesis and trichome development, as well as other biological processes, in the key medicinal plant A. annua., (© 2023. The Author(s).)

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.)

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 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.)

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.

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.)

Background: The Artemisia species are widely distributed around the world, and have found important usage in traditional medicinal practice. This study was designed to investigate the metabolites of Tibetan Artemisia species and understand the metabolic pathways., Methods: The metabolites from three Artemisia species in Tibet, were analyzed using LC-MS/MS. The differential metabolites were classified and analyzed by principal component analysis (PCA), partial least squares analysis and hierarchical clustering. KEGG Pathway enrichment analysis was used to identify the key metabolic pathways involved in the differential metabolites of three Artemisia species., Result: The metabolites of three Artemisia species were analyzed. Under the positive ion mode in LC-MS/MS, 262 distinct metabolites were differentially detected from Artemisia sieversiana and Artemisia annua, 312 differential metabolites were detected from Artemisia wellbyi and Artemisia sieversiana, 306 differential metabolites were screened from Artemisia wellbyi and Artemisia annua. With the negative ion mode, 106 differential metabolites were identified from Artemisia sieversiana and Artemisia annua, 131 differential metabolites were identified from Artemisia wellbyi and Artemisia sieversiana,133 differential metabolites were differentially detected from Artemisia wellbyi and Artemisia annua. The selected differential metabolites were mainly organic acids and their derivatives, ketones, phenols, alcohols and coumarins. Among these natural compounds, artemisinin, has the highest relative content in Artemisia annua., Conclusions: This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet., (© 2022. The Author(s).)

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.)

Alcohol dehydrogenase 1 identified from Artemisia annua (AaADH1) is a 40 kDa protein that predominately expressed in young leaves and buds, and catalyzes dehydrogenation of artemisinic alcohol to artemisinic aldehyde in artemisinin biosynthetic pathway. In this study, AaADH1 encoding gene was subcloned into vector pET-21a(+) and expressed in Escherichia coli. BL21(DE3), and purified by Co 2+ affinity chromatography. Anion exchange chromatography was performed until the protein purity reached more than 90%. Crystallization of AaADH1 was conducted for further investigation of the molecular mechanism of catalysis, and hanging-drop vapour diffusion method was used in experiments. The results showed that the apo AaADH1 crystal diffracted to 2.95 Å resolution, and belongs to space group P1, with unit-cell parameters, a = 77.53 Å, b = 78.49 Å, c = 102.44 Å, α = 71.88°, β = 74.02°, γ = 59.97°. The crystallization condition consists of 0.1 M Bis-Tris pH 6.0, 13% (w/v) PEG 8000 and 5% (v/v) glycerol., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Artemisinin, isolated from Artemisia annua, is recommended as the preferred drug to fight malaria. Previous research showed that jasmonate (JA)-mediated promotion of artemisinin accumulation depended on light. However, the mechanism underlying the interaction of light and JA in regulating artemisinin accumulation is still unknown. We identified a WRKY transcription factor, AaWRKY9, using transcriptome analysis. The glandular trichome-specific AaWRKY9 positively regulates artemisinin biosynthesis by directly binding to the promoters of AaDBR2 and AaGSW1. The key regulator in the light pathway AaHY5 activates the expression of AaWRKY9 by binding to its promoter. In addition, AaWRKY9 interacts with AaJAZ9, a repressor in the JA signalling pathway. AaJAZ9 represses the transcriptional activation activity of AaWRKY9 in the absence of methyl jasmonate. Notably, in the presence of methyl jasmonate, the transcriptional activation activity of AaWRKY9 is increased. Taken together, our results reveal a novel molecular mechanism underlying AaWRKY9 contributes to light-mediated and jasmonate-mediated to regulate the biosynthesis of artemisinin in A. annua. Our study provides new insights into integrating the two signalling pathways to regulate terpene biosynthesis in plants., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

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.)

Main Conclusion: This review analyses the most recent scientific research conducted for the purpose of enhancing artemisinin production. It may help to devise better artemisinin enhancement strategies, so that its production becomes cost effective and becomes available to masses. Malaria is a major threat to world population, particularly in South-East Asia and Africa, due to dearth of effective anti-malarial compounds, emergence of quinine resistant malarial strains, and lack of advanced healthcare facilities. Artemisinin, a sesquiterpene lactone obtained from Artemisia annua L., is the most potent drug against malaria and used in the formulation of artemisinin combination therapies (ACTs). Artemisinin is also effective against various types of cancers, many other microbes including viruses, parasites and bacteria. However, this specialty metabolite and its derivatives generally occur in low amounts in the source plant leading to its production scarcity. Considering the importance of this drug, researchers have been working worldwide to develop novel strategies to augment its production both in vivo and in vitro. Due to complex chemical structure, its chemical synthesis is quite expensive, so researchers need to devise synthetic protocols that are economically viable and also work on increasing the in-planta production of artemisinin by using various strategies like use of phytohormones, stress signals, bioinoculants, breeding and transgenic approaches. The focus of this review is to discuss these artemisinin enhancement strategies, understand mechanisms modulating its biosynthesis, and evaluate if roots play any role in artemisinin production. Furthermore, we also have a critical analysis of various assays used for artemisinin measurement. This may help to develop better artemisinin enhancement strategies which lead to decreased price of ACTs and increased profit to farmers., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual-responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA-induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA-AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA-responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1-AaTCP15/AaORA regulatory module to integrate JA and ABA-mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1-AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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.)

Plants synthesize diverse diterpenoids with numerous functions in organ development and stress resistance. However, the role of diterpenoids in glandular trichome (GT) development and GT-localized biosynthesis in plants remains unknown. Here, the identification of 10 diterpene synthases (diTPSs) revealed the diversity of diterpenoid biosynthesis in Artemisia annua. Protein-protein interactions (PPIs) between AaKSL1 and AaCPS2 in the plastids highlighted their potential functions in modulating metabolic flux to gibberellins (GAs) or ent-isopimara-7,15-diene-derived metabolites (IDMs) through metabolic engineering. A phenotypic analysis of transgenic plants suggested a complex repertoire of diterpenoids in Artemisia annua with important roles in GT formation, artemisinin accumulation and stress resilience. Metabolic engineering of diterpenoids simultaneously increased the artemisinin yield and stress resistance. Transcriptome and metabolic profiling suggested that bioactive GA 4 /GA 1 promote GT formation. Collectively, these results expand our knowledge of diterpenoids and show the potential of diterpenoids to simultaneously improve both the GT-localized metabolite yield and stress resistance, in planta., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

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.)

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.)

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua, AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx, in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)