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The 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.

Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

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.

The present study was undertaken to understand the epigenetic regulation of flavonoid biosynthesis under the influence of ultraviolet-B radiation using Artemisia annua L. as an experimental model. In-vitro propagated and acclimatized plantlets were treated with UV-B radiation (2.8 W m⁻²; 3 h), which resulted in enhanced accumulation of total flavonoid and phenolics content as well as eleven individual flavonoids measured through HPLC-DAC. Expression of eight genes (phenylanaline ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase; chalcone synthase, chalcone isomerase, cinnamoyl reductase, flavonoid-3'-hydroxylase, and flavones synthase) from upstream and downstream flavonoid biosynthetic pathways was measured through RT-PCR and RT-Q-PCR and all were variably induced under UV-B irradiation. Among them, AaPAL1 transcript and its protein were most significantly upregulated. Global DNA methylation analysis revealed hypomethylation of genomic DNA in A. annua. Further epigenetic characterization of promoter region of AaPAL1 revealed cytosine demethylation at five sites, which in turn caused epigenetic activation of six transcription factor-binding sites including QELEMENT, EBOXBNNAPA/MYCCONSENSUSAT, MYBCORE, MYBCOREATCYCB1, and GCCCORE. MYB transcription factors are positive regulators of flavonoid biosynthesis. Epigenetic activation of transcription-enhancing cis-regulatory elements in AaPAL1 promoter and subsequent overexpression of AaMYB1 and AaMYC and AaWRKY transcription factors under UV-B irradiation may probably be the reason for higher AaPAL1 expression and hence greater biosynthesis of flavonoids in A. annua L. The present study is the first report that provides mechanistic evidence of epigenetic regulation of flavonoid biosynthesis under UV-B radiation in A. annua L.

Artemisinin, which has been isolated from Artemisia annua L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in A. annua is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in A. annua, but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich A. annua.

The 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 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.

Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by  Artemisia annua ( A. annua ) .  The lower  in planta  content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of  A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in  A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular.

Artemisinin, the key antimalarial drug, is synthesized in Artemisia annua glandular secretory trichomes (GSTs), yet their development and artemisinin’s precise cellular origins are unclear. Utilizing single-nucleus RNA sequencing and spatial transcriptomics, we construct a high-resolution cellular atlas mapping metabolic dynamics across GST development. We define three developmental states: the initiation phase, transcriptional activation of core metabolic pathways establishes fundamental cellular machinery; the intermediate phase, marked lipid metabolism activation with coordinated fatty acid and wax biosynthesis, accompanied by active photosynthetic activity; the terminal differentiation phase, metabolite specialized through spatial compartmentalization of terpenoid and lipid biosynthetic pathways. Notably, we discover that six specific secretory cells within the 10-cell GSTs constitute the primary site for artemisinin production. We identify hundreds of hub genes potentially contributing to trichome development or artemisinin biosynthesis. Overall, this study systematically elucidates GST development and artemisinin biosynthesis, revealing its spatial production mechanism and providing essential cellular and genetic foundations for metabolic engineering and fundamental trichome biology. This study provides a cellular atlas of A. annua glandular trichomes using single-nucleus and spatial transcriptomics. It defines three developmental states and identifies hundreds of hub genes for trichome morphogenesis and artemisinin synthesis.

Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants.