Tingxiang Yan

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.

Molecular Cloning and Characterization of a Trichome-Specific Promoter of Artemisinic Aldehyde Δ11(13) Reductase (DBR2) in Artemisia annua

Artemisinin is widely used as an antimalarial drug around the world. Artemisinic aldehyde Δ11(13) reductase (DBR2) is a key enzyme which reduces artemisinic aldehyde to dihydroartemisinic aldehyde in the biosynthesis of artemisinin. In this study, two fragments encompassing a putative promoter of DBR2, designated as DBR2pro1 and DBR2pro2, were isolated using genomic DNA walking. The transcription start site and the putative cis-elements of each version of promoter were predicted using bioinformatic analysis. In order to study the function of the cloned promoter, Artemisia annua was transformed with β-glucuronidase (GUS) reporter gene driven by DBR2pro1 and DBR2pro2, respectively. GUS staining results demonstrated that both DBR2pro1 and DBR2pro2 were strongly expressed in glandular secretory trichomes (GSTs) of leaf primordia and flower buds, but were not obviously expressed in roots, stems, old leaves, and fully developed flowers, thus indicating that the two versions of promoter were functional and specifically expressed in GSTs.

Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin.

There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at branching points and the artemisinin biosynthetic pathway, β-caryophyllene, β-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of β-caryophyllene synthase (CPS), β-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by β-caryophyllene, β-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of β-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while β-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.

Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua

Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future.

Characterization of the Promoter of Artemisia annua Amorpha-4,11-diene Synthase (ADS) Gene Using Homologous and Heterologous Expression as well as Deletion Analysis

Amorpha-4,11-diene synthase (ADS) is the first key enzyme of artemisinin biosynthetic pathway in Artemisia annua L. In this study, the promoter region of the ADS gene has been cloned and used to demonstrate the expression of GUS reporter gene in both glandular trichomes of A. annua and non-glandular trichomes of Arabidopsis thaliana following homologous and heterologous expression of ADS promoter–GUS fusion. Subsequently, 5′ sequential deletion analysis of the ADS promoter revealed that a short sequence, −350 upstream of the transcription start site, was sufficient for trichome-specific expression in A. thaliana and that the region from −350 to −300 contained essential elements for this observed specificity. However, frequencies of transgenic A. thaliana plants displaying trichome-specific expressions varied between different lines, and all the lines with deleted fragments of the ADS promoter showed lower frequencies than the line with full-length ADS promoter. Most lines with deleted ADS promoter–GUS fusions showed GUS expressions in the guard cells of stomata as well, which was not observed in A. thaliana plants transformed with the full-length ADS promoter. GUS activities varied among different transgenic lines as well, both in transiently transformed Nicotiana benthamiana and stably transformed A. thaliana, with promoter–deletion lines exhibiting higher GUS activities than the full-length ADS promoter line.

Transcriptome Analysis of Genes Associated with the Artemisinin Biosynthesis by Jasmonic Acid Treatment under the Light in Artemisia annua

Artemisinin is a sesquiterpene lactone endoperoxide extracted from a traditional Chinese medicinal plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended as the best treatment of malaria by the World Health Organization (WHO). Both the phytohormone jasmonic acid (JA) and light promote artemisinin biosynthesis in A. annua. Interestingly, we found that the increase of artemisinin biosynthesis by JA was dependent on light. However, the relationship between the two signal pathways mediated by JA and light remains unclear. Here, we collected the A. annua seedlings of 24 h continuous light (Light), 24 h dark treatment (Dark), 4 h MeJA treatment under the continuous light conditions (Light-MeJA-4h) and 4 h MeJA treatment under the dark conditions (Dark-MeJA-4h) and performed the transcriptome sequencing using Illumina HiSeq 4000 System. A total of 266.7 million clean data were produced and assembled into 185,653 unigenes, with an average length of 537 bp. Among them, 59,490 unigenes were annotated and classified based on the public information. Differential expression analyses were performed between Light and Dark, Light and Light-MeJA-4h, Dark and Dark-MeJA-4h, Light-MeJA-4h, and Dark-MeJA-4h, respectively. Furthermore, transcription factor (TF) analysis revealed that 1588 TFs were identified and divided into 55 TF families, with 284 TFs down-regulated in the Dark relative to Light and 96 TFs up-regulated in the Light-MeJA-4h relative to Light. 8 TFs were selected as candidates for regulating the artemisinin biosynthesis and one of them was validated to be involved in artemisinin transcriptional regulation by Dual-Luciferase (Dual-LUC) assay. The transcriptome data shown in our study offered a comprehensive transcriptional expression pattern influenced by the MeJA and light in A. annua seedling, which will serve as a valuable resource for further studies on transcriptional regulation mechanisms underlying artemisinin biosynthesis.

AaPDR3, a PDR Transporter 3, Is Involved in Sesquiterpene β-Caryophyllene Transport in Artemisia annua

Artemisinin, a sesquiterpenoid endoperoxide, isolated from the plant Artemisia annua L., is widely used in the treatment of malaria. Another sesquiterpenoid, β-caryophyllene having antibiotic, antioxidant, anticarcinogenic and local anesthetic activities, is also presented in A. annua. The role played by sesquiterpene transporters in trichomes and accumulation of these metabolites is poorly understood in A. annua and in trichomes of other plant species. We identified AaPDR3, encoding a pleiotropic drug resistance (PDR) transporter located to the plasma membrane from A. annua. Expression of AaPDR3 is tissue-specifically and developmentally regulated in A. annua. GUS activity is primarily restricted to T-shaped trichomes of old leaves and roots of transgenic A. annua plants expressing proAaPDR3: GUS. The level of β-caryophyllene was decreased in transgenic A. annua plants expressing AaPDR3-RNAi while transgenic A. annua plants expressing increased levels of AaPDR3 accumulated higher levels of β-caryophyllene. When AaPDR3 was expressed in transformed yeast, yeasts expressing AaPDR3 accumulated more β-caryophyllene, rather than germacrene D and β-farnesene, compared to the non-expressing control.

T‐shaped trichome‐specific expression of monoterpene synthase ADH2 using promoter–β‐GUS fusion in transgenic Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (sweet wormwood), is extensively used in the treatment of malaria. In order to better understand the metabolism of terpenes in A. annua and the influence of terpene synthases on artemisinin yield, the expression pattern of a monoterpene alcohol dehydrogenase (ADH2) has been studied using transgenic plants expressing promoter–β‐glucuronidase (GUS) fusion. ADH2 played a major role in monoterpenoid biosynthesis including carveol, borneol, and artemisia ketone through in vitro biochemical analysis. In this study, the ADH2 promoter was cloned by the genome walking method. A number of putative cis‐acting elements were predicted in promoter region, suggesting that the ADH2 is driven by a complex regulation mechanism. ADH2 gene was highly expressed in old leaves, whereas the artemisinin biosynthetic genes were mainly expressed in bud and young leaves. The expression of ADH2 gene increased quickly during leaf development, revealed by qRT‐PCR. GUS expression analysis in different tissues of transgenic A. annua demonstrates that ADH2 expression is exclusively located to T‐shaped trichome, not glandular secretory trichome.

AaEIN3 mediates the downregulation of artemisinin biosynthesis by ethylene signaling through promoting leaf senescence in Artemisia annua

Artemisinin is an important drug for malaria treatment, which is exclusively produced in Artemisia annua. It’s important to dissect the regulatory mechanism of artemisinin biosynthesis by diverse plant hormones and transcription factors. Our study shows ethylene, a plant hormone which accelerates flower and leaf senescence and fruit ripening, suppressed the expression of genes encoding three key enzymes ADS, DBR2, CYP71AV1, and a positive regulator AaORA involved in artemisinin biosynthesis. Then we isolated the gene encoding ETHYLENE-INSENSITIVE3 (EIN3), a key transcription factor in ethylene signaling pathway, by screening the transcriptome and genome database from Artemisia annua, named AaEIN3. Overexpressing AaEIN3 suppressed artemisinin biosynthesis, while repressing its expression with RNAi enhanced artemisinin biosynthesis in Artemisia annua, indicating AaEIN3 negatively regulates artemisinin biosynthesis. Further study showed the downregulation of artemisinin biosynthesis by ethylene required the mediation of AaEIN3. AaEIN3 could accelerate leaf senescence, and leaf senescence attenuated the expression of ADS, DBR2, CYP71AV1, and AaORA that are involved in artemisinin biosynthesis. Collectively, our study demonstrated a negative correlation between ethylene signaling and artemisinin biosynthesis, which is ascribed to AaEIN3-induced senescence process of leaves. Our work provided novel knowledge on the regulatory network of plant hormones for artemisinin metabolic pathway.