TrichomeLess Regulator 3 is required for trichome initial and cuticle biosynthesis in Artemisia annua.

Artemisinin is primarily synthesized and stored in the subepidermal space of the glandular trichomes of Artemisia annua. The augmentation of trichome density has been demonstrated to enhance artemisinin yield. However, existing literature lacks insights into the correlation between the stratum corneum and trichomes. This study aims to unravel the involvement of TrichomeLess Regulator 3 (TLR3), which encodes the transcription factor, in artemisinin biosynthesis and its potential association with the stratum corneum. TLR3 was identified as a candidate gene through transcriptome analysis. The role of TLR3 in trichome development and morphology was investigated using yeast two-hybrid, pull-down analysis, and RNA electrophoresis mobility assay. Our research revealed that TLR3 negatively regulates trichome development. It modulates the morphology of Arabidopsis thaliana trichomes by inhibiting branching and inducing the formation of abnormal trichomes in Artemisia annua. Overexpression of the TLR3 gene disrupts the arrangement of the stratum corneum and reduces artemisinin content. Simultaneously, TLR3 possesses the capacity to regulate stratum corneum development and trichome follicle morphology by interacting with TRICHOME AND ARTEMISININ REGULATOR 1, and CycTL. Consequently, our findings underscore the pivotal role of TLR3 in the development of glandular trichomes and stratum corneum biosynthesis, thereby influencing the morphology of Artemisia annua trichomes.

Feedback regulation of plant secondary metabolism: Applications and challenges.

Plant secondary metabolites offer resistance to invasion by herbivorous organisms, and are also useful in the chemical, pharmaceutical, cosmetic, and fragrance industries. There are numerous approaches to enhancing secondary metabolite yields. However, a growing number of studies has indicated that feedback regulation may be critical in regulating secondary metabolite biosynthesis. Here, we review examples of feedback regulation in secondary metabolite biosynthesis pathways, phytohormone signal transduction, and complex deposition sites associated with secondary metabolite biosynthesis. We propose a new strategy to enhance secondary metabolite production based on plant feedback regulation. We also discuss challenges in feedback regulation that must be overcome before its application to enhancing secondary metabolite yields. This review discusses recent advances in the field and highlights a strategy to overcome feedback regulation-related obstacles and obtain high secondary metabolite yields.

Corrigendum: SbWRKY75- and SbWRKY41- mediated jasmonic acid signaling regulates baicalin biosynthesis.

[This corrects the article DOI: 10.3389/fpls.2023.1213662.].

Phenotyping of Salvia miltiorrhiza Roots Reveals Associations between Root Traits and Bioactive Components.

Plant phenomics aims to perform high-throughput, rapid, and accurate measurement of plant traits, facilitating the identification of desirable traits and optimal genotypes for crop breeding. Salvia miltiorrhiza (Danshen) roots possess remarkable therapeutic effect on cardiovascular diseases, with huge market demands. Although great advances have been made in metabolic studies of the bioactive metabolites, investigation for S. miltiorrhiza roots on other physiological aspects is poor. Here, we developed a framework that utilizes image feature extraction software for in-depth phenotyping of S. miltiorrhiza roots. By employing multiple software programs, S. miltiorrhiza roots were described from 3 aspects: agronomic traits, anatomy traits, and root system architecture. Through K-means clustering based on the diameter ranges of each root branch, all roots were categorized into 3 groups, with primary root-associated key traits. As a proof of concept, we examined the phenotypic components in a series of randomly collected S. miltiorrhiza roots, demonstrating that the total surface of root was the best parameter for the biomass prediction with high linear regression correlation (R2 = 0.8312), which was sufficient for subsequently estimating the production of bioactive metabolites without content determination. This study provides an important approach for further grading of medicinal materials and breeding practices.

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

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

SbWRKY75- and SbWRKY41-mediated jasmonic acid signaling regulates baicalin biosynthesis.

Introduction: Scutellaria baicalensis Georgi is a traditional Chinese medicinal plant with broad pharmacological activities whose main active ingredient is the flavonoid baicalin. Given its medicinal value and increasing market demand, it is essential to improve the plant's baicalin content. Flavonoid biosynthesis is regulated by several phytohormones, primarily jasmonic acid (JA). Methods: In this study, we conducted transcriptome deep sequencing analysis of S. baicalensis roots treated with methyl jasmonate for different durations (1, 3, or 7 hours). Leveraging weighted gene co-expression network analysis and transcriptome data, we identified candidate transcription factor genes involved in the regulation of baicalin biosynthesis. To validate the regulatory interactions, we performed functional assays such as yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Results: Our findings demonstrated that SbWRKY75 directly regulates the expression of the flavonoid biosynthetic gene SbCLL-7, whereas SbWRKY41 directly regulates the expression of two other flavonoid biosynthetic genes, SbF6H and SbUGT, thus regulating baicalin biosynthesis. We also obtained transgenic S.baicalensis plants by somatic embryo induction and determined that overexpressing SbWRKY75 increased baicalin content by 14%, while RNAi reduced it by 22%. Notably, SbWRKY41 indirectly regulated baicalin biosynthesis by modulating the expression of SbMYC2.1, SbJAZ3 and SbWRKY75. Discussion: This study provides valuable insights into the molecular mechanisms underlying JA-mediated baicalin biosynthesis in S. baicalensis. Our results highlight the specific roles of transcription factors, namely SbWRKY75 and SbWRKY41, in the regulation of key biosynthetic genes. Understanding these regulatory mechanisms holds significant potential for developing targeted strategies to enhance baicalin content in S. baicalensis through genetic interventions.

AaWRKY6 contributes to artemisinin accumulation during growth in Artemisia annua.

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

The transcription factors SbMYB45 and SbMYB86.1 regulate flavone biosynthesis in Scutellaria baicalensis.

Scutellaria baicalensis Georgi is an important Chinese medicinal plant that is rich in the flavones baicalin, wogonoside, and wogonin, providing it with anti-cancer, anti-inflammatory, and antibacterial properties. However, although the biosynthetic pathways of baicalin and its derivates have been elucidated, the regulation of flavone biosynthesis in S. baicalensis is poorly understood. Here, we found that the contents of baicalin and its derivates increased and that baicalin biosynthetic pathway genes were induced in response to light, and baicalin and baicalein are not exclusively produced in the roots of S. baicalensis. Based on the fact that MYB transcription factors are known to play important roles in flavone biosynthesis, we identified SbMYB45 and SbMYB86.1 in S. baicalensis and determined that they bind to the promoter of the flavone biosynthesis gene SbCHI to enhance its transcription. Moreover, overexpressing SbMYB45 and SbMYB86.1 enhanced the accumulation of baicalin in S. baicalensis leaves. We demonstrate that SbMYB45 and SbMYB86.1 bind to the cis-acting element MBSII in the promoter of CHI to redundantly induce its expression upon light exposure. These findings indicate that SbMYB45 and SbMYB86.1 transcriptionally activate SbCHI in response to light and enhance flavone contents in S. baicalensis.

Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an essential traditional Chinese herb that is commonly used to treat colds, fever, and influenza. Indole alkaloids, such as indigo and indirubin, are the primary active constituents of B. cusia. The indole-producing reaction is crucial for regulating the flow of indole alkaloids metabolites along the pathways and coordinating primary and secondary product biosynthesis in plants. The tryptophan synthase alpha-subunit (TSA) can catalyse a process that produces indole, which is free to enter secondary metabolite pathways; however, the underlying potential mechanism of regulating indigo alkaloids synthesis remains unknown. Here, a BcTSA was cloned from the transcriptome of B. cusia. The BcTSA has a significant degree of similarity with other plant TSAs according to bioinformatics and phylogenetic analyses. Quantitative real-time PCR (RT-qPCR) research showed that BcTSA was dramatically enhanced in response to treatment with methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA), and was predominantly expressed in the stems as opposed to the leaves and rhizomes. Subcellular localization revealed that BcTSA is localized in chloroplasts, which is compatible with the fact that the conversion of indole-3-glycerol phosphate (IGP) to indole occurs in chloroplasts. The complementation assay results showed that BcTSA was functional, demonstrating that it was capable of catalyzing the conversion of IGP to indole. BcTSA was shown to stimulate the manufacture of indigo alkaloids including isatin, indigo, and indirubin when the gene was overexpressed in the hairy roots of Isatis indigotica. In conclusion, our research provides novel perspectives that might be applied to manipulating the indole alkaloid composition of B. cusia.

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

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

Strategic nanoparticle-mediated plant disease resistance.

Nanotechnology is a promising means for development of sustainable agriculture while the study of nanoparticle-mediated plant disease resistance is still in its primary stage. Nanotechnology has shown great promise in regulating: the content of secondary metabolites, inducing disease resistance genes, delivering hormones, delivering biomolecules (such as: nucleotides, proteins, and activators), and obtaining transgenic plants to resist plant diseases. In this review, we conclude its versatility and applicability in disease management strategies and diagnostics and as molecular tools. With the advent of new biotechnologies (e.g. de novo regeneration, CRISPR/Cas9, and GRF4-GIF1 fusion protein), we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering. In order to ensure the safe use and social acceptance of plant nanoparticle technology, its adverse effects are discussed, including the risk of transferring nanoparticles through the food chain.

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

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

Phytohormone-Based Regulation of Trichome Development.

Phytohormones affect plant growth and development. Many phytohormones are involved in the initiation of trichome development, which can help prevent damage from UV radiation and insect bites and produce fragrance, flavors, and compounds used as pharmaceuticals. Phytohormones promote the participation of transcription factors in the initiation of trichome development; for example, the transcription factors HDZIP, bHLH and MYB interact and form transcriptional complexes to regulate trichome development. Jasmonic acid (JA) mediates the progression of the endoreduplication cycle to increase the number of multicellular trichomes or trichome size. Moreover, there is crosstalk between phytohormones, and some phytohormones interact with each other to affect trichome development. Several new techniques, such as the CRISPR-Cas9 system and single-cell transcriptomics, are available for investigating gene function, determining the trajectory of individual trichome cells and elucidating the regulatory network underlying trichome cell lineages. This review discusses recent advances in the modulation of trichome development by phytohormones, emphasizes the differences and similarities between phytohormones initially present in trichomes and provides suggestions for future research.

Structure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases.

Pinoresinol-lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8' lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. ß4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.

Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

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.

Isatis indigotica: from (ethno) botany, biochemistry to synthetic biology.

Isatis indigotica Fort. (Chinese woad) is a species with an ancient and well-documented history as an indigo dye and medicinal plant. It is often confused with Isatis tinctoria L. (European woad), a medicinal plant in Europe. Here, the differences between I. indigotica and I. tinctoria are systematically described. The usage development history, clinical applications and pharmacological activities, and chemical components of I. indigotica are also summarized. Lignans, indole alkaloids, and their corresponding derivatives have been identified as the major active ingredients of I. indigotica and are associated with anti-viral, anti-inflammatory, anti-cancer, and other health-promoting activities. Notable progress has been made in understanding the biosynthetic pathway and regulation mechanism of lignans and indole alkaloids in I. indigotica, the results from which should facilitate the process of targeted metabolic engineering or synthetic biology. Moreover, multiple biotechnology methods such as polyploid breeding and genetic engineering have been used with I. indigotica to result in, for example, greater yields, higher levels of bioactive component accumulation, and enhanced stress tolerance to salt, drought, and insects. Some issues require additional analyses, and suggestions for future research on I. indigotica are also discussed.

SmMYC2b Enhances Tanshinone Accumulation in Salvia miltiorrhiza by Activating Pathway Genes and Promoting Lateral Root Development.

Salvia miltiorrhiza Bunge (Lamiaceae) is an economically important medicinal plant as well as an emerging model plant. Our previous studies indicate that SmMYC2b is a positive transcription factor that can affect the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza. Moreover, MYC2s are well known to induce the development of lateral roots. As tanshinones are mainly distributed in the periderm, the promotion of lateral root development probably leads to increased accumulation of tanshinones. In this paper, we firstly discovered that SmMYC2b played a dual regulatory role in effectively enhancing the tanshinone accumulation by activating tanshinone biosynthetic pathway and promoting lateral root development. The expression levels of the previously studied pathway genes SmCPS1, SmKSL1, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 dramatically increased. In addition, SmMYC2b was proved to exhibit a similar function as other homologs in promoting lateral root development, which increased the tanshinone produced tissue and further enhanced the biosynthesis of tanshinones. RNA-seq assays revealed that SmMYC2b-regulated genes comprised 30.6% (1,901 of 6,210) of JA-responsive genes, confirming that SmMYC2b played a crucial role in transcriptional regulation of JA-regulated genes. Overall, we concluded that SmMYC2b could enhance tanshinone accumulation by activating the tanshinone biosynthetic pathway and promoting lateral root development. Our study provides an effective approach to enhance the production of desired tanshinones and enriches our knowledge of the related regulatory network.

Nanoparticle-mediated gene transformation strategies for plant genetic engineering.

Plant genetic engineering, a recent technological advancement in the field of plant science, is an important tool used to improve crop quality and yield, to enhance secondary metabolite content in medicinal plants or to develop crops for sustainable agriculture. A new approach based on nanoparticle-mediated gene transformation can overcome the obstacle of the plant cell wall and accurately transfer DNA or RNA into plants to produce transient or stable transformation. In this review, several nanoparticle-based approaches are discussed, taking into account recent advances and challenges to hint at potential applications of these approaches in transgenic plant improvement programs. This review also highlights challenges in implementing the nanoparticle-based approaches used in plant genetic engineering. A new technology that improves gene transformation efficiency and overcomes difficulties in plant regeneration has been established and will be used for the de novo production of transgenic plants, and CRISPR/Cas9 genome editing has accelerated crop improvement. Therefore, we outline future perspectives based on combinations of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies to accelerate crop improvement. The information provided here will assist an effective exploration of the technological advances in plant genetic engineering to support plant breeding and important crop improvement programs.

Interaction of bZIP transcription factor TGA6 with salicylic acid signaling modulates artemisinin biosynthesis in Artemisia annua.

Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1, thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90-120% and decreased by 20-60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production.

The SPB-Box Transcription Factor AaSPL2 Positively Regulates Artemisinin Biosynthesis in Artemisia annua L.

Artemisinin, an important compound produced by Artemisia annua, is the active ingredient in the treatment of malaria. Jasmonic acid, one of the phytohormones, is an important elicitor of artemisinin biosynthesis by enhancing transcription levels of transcription factors. SPL transcription factors are plant-specific transcription factors of plant growth, development, and secondary metabolism regulation. However, to date, the SPL transcription factors that regulate artemisinin biosynthesis is currently unclear. Here, we show that an SPL transcription factor can positively regulate artemisinin biosynthesis by binding to the promoter of artemisinin biosynthetic pathway genes. We screened AaSPL2 by gene expression profiles analysis in 14 SPL transcription factors. We demonstrated that AaSPL2 can activate the promoter of DBR2 by dual-LUC assy. Moreover, in the AaSPL2 overexpression plants, the artemisinin content was increased by 33-86%, and in the AaSPL2 -RNAi transgenic plants, artemisinin content was decreased by 33-65%. These data suggest that AaSPL2 and DBR2 interact with a "GTAC" cis-element in the DBR2 promoter, mediating the transcriptional activation of DBR2 in response to JA and resulting in the improvement on artemisinin content.