SummaryThe plant Artemisia annua is well known due to the production of artemisinin, a sesquiterpene lactone that is widely used in malaria treatment. Phytohormones play important roles in plant secondary metabolism, such as jasmonic acid (JA), which can induce artemisinin biosynthesis in A. annua. Nevertheless, the JA-inducing mechanism remains poorly understood.The expression of gene AaMYC2 was rapidly induced by JA and AaMYC2 binds the G-boxlike motifs within the promoters of gene CYP71AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway.Overexpression of AaMYC2 in A. annua significantly activated the transcript levels of CYP71AV1 and DBR2, which resulted in an increased artemisinin content. By contrast, artemisinin content was reduced in the RNAi transgenic A. annua plants in which the expression of AaMYC2 was suppressed. Meanwhile, the RNAi transgenic A. annua plants showed lower sensitivity to methyl jasmonate treatment than the wild-type plants.These results demonstrate that AaMYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production.

Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production.

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.

Glandular trichomes are generally considered biofactories that produce valuable chemicals. Increasing glandular trichome density is a very suitable way to improve the productivity of these valuable metabolites, but little is known about the regulation of glandular trichome formation. Phytohormone jasmonate (JA) promotes glandular trichome initiation in various plants, but its mechanism is also unknown. By searching transcription factors regulated by JA in Artemisia annua, we identified a novel homeodomain-leucine zipper transcription factor, HOMEODOMAIN PROTEIN 1 (AaHD1), which positively controls both glandular and nonglandular trichome initiations. Overexpression of AaHD1 in A. annua significantly increased glandular trichome density without harming plant growth. Consequently, the artemisinin content was improved. AaHD1 interacts with A. annua jasmonate ZIM-domain 8 (AaJAZ8), which is a repressor of JA, thereby resulting in decreased transcriptional activity. AaHD1 knockdown lines show decreased sensitivity to JA on glandular trichome initiation, which indicates that AaHD1 plays an important role in JA-mediated glandular trichome initiation. We identified a new transcription factor that promotes A. annua glandular trichome initiation and revealed a novel molecular mechanism by which a homeodomain protein transduces JA signal to promote glandular trichome initiation. Our results also suggested a connection between glandular and nonglandular trichome formations.

JA-induced JAZ8 degradation releases and activates the TCP14-ORA complex in enhancing artemisinin biosynthesis in Artemisia annua.

Artemisinin is a type of sesquiterpene lactone well known as an antimalarial drug, and is specifically produced in glandular trichomes of Artemisia annua. However, the regulatory network for the artemisinin biosynthetic pathway remains poorly understood. Exploration of trichome-specific transcription factors would facilitate the elucidation of regulatory mechanism of artemisinin biosynthesis. The WRKY transcription factor GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) was cloned and analysed in A. annua. AaGSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ERF transcription factor AaORA. A β-glucuronidase (GUS) staining assay further demonstrated that AaGSW1 is a glandular trichome-specific transcription factor. AaGSW1 positively regulates CYP71AV1 and AaORA expression by directly binding to the W-box motifs in their promoters. Overexpression of AaGSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, AaGSW1 can be directly regulated by AaMYC2 and AabZIP1, which are positive regulators of jasmonate (JA)- and abscisic acid (ABA)-mediated artemisinin biosynthetic pathways, respectively. These results demonstrate that AaGSW1 is a glandular trichome-specific WRKY transcription factor and a positive regulator in the artemisinin biosynthetic pathway. Moreover, we propose that two trifurcate feed-forward pathways involving AaGSW1, CYP71AV1 and AaMYC2/AabZIP1 function in the JA/ABA response in A. annua.

SummaryThe glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood.To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants.Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis.Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant.

Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as 'biofactories' for producing valuable natural products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive. Here we identify a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua. We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle-related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA-like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes. Our results reveal a molecular mechanism by which a novel HD-ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation.