AaCycTL Regulates Cuticle and Trichome Development in Arabidopsis and Artemisia annua L.

Artemisinin is an important drug for resistance against malaria. Artemisinin is derived from the glandular trichome of leaves, stems, or buds of the Chinese traditional herb Artemisia annua. Increasing the trichome density may enhance the artemisinin content of A. annua. It has been proven that cyclins are involved in the development of trichomes in tomato, Arabidopsis, and tobacco, but it is unclear whether the cyclins in A. annua influence trichome development. In this study, we showed that AaCycTL may regulate trichome development and affect the content of artemisinin. We cloned AaCycTL and found that it has the same expression files as the artemisinin biosynthesis pathway gene. We overexpressed AaCycTL in Arabidopsis, and the results indicated that AaCycTL changed the wax coverage on the surface of Arabidopsis leaves. The trichome density decreased as well. Using yeast two-hybrid and BiFC assays, we show that AaCycTL can interact with AaTAR1. Moreover, we overexpressed AaCycTL in A. annua and found that the expression of AaCycTL was increased to 82–195%. Changes in wax coverage on the surface of transgenic A. annua leaves or stems were found as well. We identified the expression of the artemisinin biosynthesis pathway genes ADS, CYP71AV1, and ALDH1 has decreased to 88–98%, 76–97%, and 82–97% in the AaCycTL-overexpressing A. annua lines, respectively. Furthermore, we found reduced the content of artemisinin. In agreement, overexpression of AaCycTL in A. annua or Arabidopsis may alter waxy loading, change the initiation of trichomes and downregulate trichome density. Altogether, AaCycTL mediates trichome development in A. annua and thus may serve to regulate trichome density and be used for artemisinin biosynthesis.

AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua

Artemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.

Amorpha-4,11-diene synthase: a key enzyme in artemisinin biosynthesis and engineering

Amorpha-4,11-diene synthase (ADS) catalyzes the first committed step in the artemisinin biosynthetic pathway, which is the first catalytic reaction enzymatically and genetically characterized in artemisinin biosynthesis. The advent of ADS in Artemisia annua is considered crucial for the emergence of the specialized artemisinin biosynthetic pathway in the species. Microbial production of amorpha-4,11-diene is a breakthrough in metabolic engineering and synthetic biology. Recently, numerous new techniques have been used in ADS engineering; for example, assessing the substrate promiscuity of ADS to chemoenzymatically produce artemisinin. In this review, we discuss the discovery and catalytic mechanism of ADS, its application in metabolic engineering and synthetic biology, as well as the role of sesquiterpene synthases in the evolutionary origin of artemisinin.

Heterologous Biosynthesis of Artemisinin in Chrysanthemum morifolium Ramat

Artemisinin-based drugs are the most effective medicine against multidrug-resistant Plasmodium spp., the parasite that causes malaria. To this day, wormwood A. annua L. is the sole commercial source of artemisinin, where it is produced in minor amounts. The artemisinin yield depends on numerous poorly regulated agricultural factors and the genetic variability of this non-domesticated plant. This has aroused significant interest in the development of heterologous expression platforms for artemisinin production. Previously, we obtained lines of Chrysanthemum morifolium Ramat. (C. morifolium Ramat.), cvs. White Snowdon and Egyptianka, transformed with artemisinin biosynthesis genes. Here, we report the results of an analysis of artemisinin production in transgenic chrysanthemums. Transcription of heterologous amorpha-4,11-diene monooxygenase and cytochrome P450 reductase genes in transgenic lines was confirmed using high-resolution melting analysis. Artemisinin accumulation was detected using GC-MS in White Snowdon plants, but not in Egyptianka ones, thereby demonstrating the possibility of transplanting active artemisinin biosynthetic pathway into chrysanthemum. Ways of increasing its content in producer plants are discussed.

Chromatin Accessibility Is Associated with Artemisinin Biosynthesis Regulation in Artemisia annua

Glandular trichome (GT) is the dominant site for artemisinin production in Artemisia annua. Several critical genes involved in artemisinin biosynthesis are specifically expressed in GT. However, the molecular mechanism of differential gene expression between GT and other tissue types remains elusive. Chromatin accessibility, defined as the degree to which nuclear molecules are able to interact with chromatin DNA, reflects gene expression capacity to a certain extent. Here, we investigated and compared the landscape of chromatin accessibility in Artemisia annua leaf and GT using the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) technique. We identified 5413 GT high accessible and 4045 GT low accessible regions, and these GT high accessible regions may contribute to GT-specific biological functions. Several GT-specific artemisinin biosynthetic genes, such as DBR2 and CYP71AV1, showed higher accessible regions in GT compared to that in leaf, implying that they might be regulated by chromatin accessibility. In addition, transcription factor binding motifs for MYB, bZIP, C2H2, and AP2 were overrepresented in the highly accessible chromatin regions associated with artemisinin biosynthetic genes in glandular trichomes. Finally, we proposed a working model illustrating the chromatin accessibility dynamics in regulating artemisinin biosynthetic gene expression. This work provided new insights into epigenetic regulation of gene expression in GT.