Transcriptional regulation of artemisinin biosynthesis in Artemisia annua L.

Sci. Bull. (2016) 61(1):18–25 DOI 10.1007/s11434-015-0983-9

www.scibull.com www.springer.com/scp

Review

Life & Medical Sciences

Transcriptional regulation of artemisinin biosynthesis in Artemisia annua L. Qian Shen • Tingxiang Yan • Xueqing Fu Kexuan Tang

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Received: 20 November 2015 / Revised: 7 December 2015 / Accepted: 8 December 2015 / Published online: 8 January 2016 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2016

Abstract Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives. Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin. In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research. Keywords Artemisia annua L.  Artemisinin  Transcription factor  Biosynthesis

1 Introduction Artemisia annua L. (Chinese wormwood herb, Asteraceae) synthesizes and accumulates artemisinin, a unique sesquiterpene endoperoxide lactone. A. annua and artemisinin have obtained much attention due to the antimalarial properties of artemisinin and its derivatives against chloroquine-resistant strains of Plasmodium falciparum SPECIAL TOPIC: Advances in Artemisinin Study Q. Shen  T. Yan  X. Fu  K. Tang (&) Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, Plant Biotechnology Research Center, Fudan-SJTUNottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China e-mail: [email protected]

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[1]. Malaria, one of the most serious health problems in human history, is responsible for more than 600,000 deaths last year [2]. Artemisinin-based combination therapies (ACTs) are recommended by WHO to be the best choice for acute malaria [1–3]. It has saved millions of lives in Africa countries. The Chinese pharmacologist Youyou Tu is best known for her contribution to the isolation of artemisinin, and Professor Tu received the 2011 Lasker Award in clinical medicine and the 2015 Nobel Prize in Physiology or Medicine. Besides the antimalarial activities, artemisinin and its derivatives have also been reported to have antiviral [4], anticancer [5, 6], and antischistosomal activities [7]. Therefore, artemisinin has been considered to be a promising natural product with multifunctions. Although semisynthesis of artemisinin via artemisinic acid, which can be obtained from genetically modified yeast, is feasible at present [8, 9], plant of A. annua is still the main commercial source of artemisinin. Unfortunately, the supply is restricted by the relatively low amounts of artemisinin at a range of 0.1 %–1 % dry leaf weight of A. annua, which results in a high cost of this effective product that most of the poorer population of malarial victims in Africa could not afford. Therefore, numerous attempts have been made to improve the artemisinin yield in plant during the last two decades. Not surprisingly, at the beginning, investigation of artemisinin product is largely focused on elucidating the biosynthetic pathway. These studies have progressed from isolating the biosynthetic pathway relevant enzymes to genetically modifying plants by over-expressing or silencing these enzyme coding genes. But regulatory mechanisms of artemisinin biosynthesis in plant are still poorly understood. In this review, we summarize current knowledge of the transcriptional regulation of artemisinin production in A. annua and hope to give insight into further improvement in artemisinin biosynthesis studies.

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2 Biosynthetic pathway of artemisinin biosynthesis As a sesquiterpenoid, the biosynthetic pathway for the common precursor isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) of artemisinin is mostly the same. In plants, IPP and DMAPP, which are the basic C5 precursors for terpenoid biosynthesis, are produced via two distinct pathways: the mevalonate (MVA) pathway in cytosol and the methylerythritol phosphate (MEP) pathway in plastid [10]. Generally, MVAderived isoprene units for farnesyl pyrophosphate (FPP) are mainly used for the production of sesquiterpenes, triterpenes, and polyisoprenoids. And the MEP-derived IPP and DMAPP precursors are responsible for the biosynthesis of geranyl pyrophosphate (GPP) and geranylgeranyl pyrophosphate (GGPP) for the production of monoterpenes, diterpenes, and tetraterpenes [10, 11]. However, a 13 CO2 study showed that the FPP used for artemisinin biosynthesis was arose from one MEP-derived IPP unit, one MVA-derived IPP unit, and one MVA-derived DMAPP unit [12]. The first committed step in the artemisinin-specific biosynthetic pathway is the conversion of FPP to amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) [13, 14]. Subsequently, amorpha-4,11-diene is hydroxylated to artemisinic alcohol, which is oxidized to artemisinic aldehyde and subsequently to artemisinic acid in three steps that are catalyzed by the multifunction cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) as the native redox partner [8, 15]. Recently, an additional alcohol dehydrogenase (ADH1) was proved to be involved in helping the oxidation of artemisinic alcohol to artemisinic aldehyde acid in genetically modified yeast [9]. The artemisinic aldehyde D11(13) reductase (DBR2), a double-bond reductase, catalyzes the formation of dihydroartemisinic aldehyde [16], and then converted into dihydroartemisinic acid by aldehyde dehydrogenase 1 (ALDH1) [17]. Dihydroartemisinic acid is regarded as the direct precursor of artemisinin. The conversion of dihydroartemisinic acid to artemisinin occurs in the cell-free cuticular space, and it appears to be non-enzymatic photooxidation process [18, 19]. Similarly, artemisinic acid is converted into arteannuin B also via a photooxidative reaction [18, 19]. A simplified biosynthetic pathway of artemisinin is shown in Fig. 1.

3 Transcription factors (TFs) regulating artemisinin biosynthesis

Fig. 1 (Color online) Biosynthetic pathway and a transcriptional regulation network of artemisinin in A. annua. Phytohormones such as JA and ABA induce the expression levels of transcription factors; subsequently, these phytohormone-responsive TFs bind the promoters of artemisinin biosynthetic pathway genes such as ADS, CYP71AV1, and DBR2. The activation of gene expression results an increase in artemisinin content in plant. JA, jasmonate acid; ABA, abscisic acid; IDI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthase; ADS, amorpha-4,11-diene synthase; CYP71AV1, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase; ADH1, alcohol dehydrogenase 1; DBR2, artemisinic aldehyde D11(13) reductase; ALDH1, aldehyde dehydrogenase 1. Blue arrow lines represent transcription factors induced by phytohormones. Red arrow lines represent transcription factors bind to the promoter of genes. Black bend arrow lines represent TFs active target genes expressing. Dotted arrow lines represent possible interactions between two TFs

In plants, the synthesis and accumulation of secondary metabolites are properly controlled in a spatial and temporal manner. And this spatial–temporal regulation is

usually controlled by a complex network containing regulatory proteins known as TFs [20]. TFs are sequencespecific DNA-binding proteins that recognize specific cis-

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regulatory sequences in the promoters of target genes. In response to developmental or environmental signaling, they activate or repress their target genes expression, thus controlling the specific accumulation of secondary metabolites [21]. Recent investigations in plant exhibited that plant transcription factors often regulate a series of genes in one specific pathway and overexpression of these factors has been proposed as a promising approach for more efficiently regulating plant secondary metabolism [22–24]. 3.1 WRKY family WRKY proteins family is one of the largest families of TFs found in plant. Like most of TFs, the defining feature of WRKY transcription factors is their DNA-binding domain which is called the WRKY domain [25]. The WRKY domain contains about 60 residues in length and has two components. There are invariant WRKY amino acid sequences at the N-terminal and a zinc finger structure at the C-terminal [25]. The conservation of the WRKY domain is marked as the W-box (TTGACC/T) cis-elementbinding protein. Almost all WRKY TFs bind preferentially to W-boxes, and the binding to W-boxes is a feature of both biotic and abiotic stress responses [26]. AaWRKY1, the first A. annua transcription factor isolated and characterized, regulates artemisinin biosynthesis. In A. annua, AaWRKY1 gene is highly expressed in glandular trichome cells and is strongly induced by methyl jasmonate treatment. In vivo and in vitro analyses revealed that AaWRKY1 transcription factor had the binding ability with the W-box in ADS promoter and activates ADS gene expression in transgenic tobacco and transient expression A. annua leaf system [27]. Then, another group studied the functions of transcription factor AaWRKY1 in stably transformed A. annua plants by overexpressing with CaMV35S promoter or trichome-specific CYP71AV1 promoter. Both of the approaches can elevate the expression of CYP71AV1. But over-expressing AaWRKY1 with the trichome specific promoter improves the transcription level of CYP71AV1 more effectively. However, the transcription levels of ADS and DBR2 did not change significantly in transgenic plants. As a result, the HPLC analysis of metabolites showed that the artemisinin content in transgenic lines reached 19 mg/g (DW), which is 1.9-fold times compared to the control lines [28]. It indicated that WRKY family TFs play important roles in mediating biosynthesis of plant secondary metabolites. 3.2 AP2/ERF family The APETALA2/ethylene-responsive factor (AP2/ERF) family is conservatively widespread in the plants. AP2/

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ERF proteins contain at least one DNA-binding AP2 domain, and there are three separate subfamilies, namely the ERF, AP2, and RAV families [29]. These transcription factors are involved in the regulation of plant primary and secondary metabolism, growth and developmental programs, and response to environmental stimuli [30]. The well-known transcription factor ORCA3 of Catharanthus roseus is a member of AP2/ERF family, which controls the expression of multiple genes involved in the terpenoid indole alkaloids (TIAs) biosynthetic pathway in C. roseus [23, 31]. Recently, several AP2/ERF genes were reported to participate in artemisinin biosynthesis, too. Our laboratory screened a cDNA library of A. annua and retrieved six AP2/ERF transcription factors, and then six AP2/ERF TFs were characterized. Comparative and bioinformatic analyses revealed that one of them named as AaORA had the closest evolutionary relationship to ORCA3 and ORCA2 of C. roseus. Moreover, promoter GUS-staining analysis of AaORA promoter showed that AaORA was a trichomespecific transcription factor, which was only highly expressed in both glandular and filamentous trichomes of A. annua [32]. Subsequently, transgenic plants overexpressing AaORA were generated for further analysis. Compared with the control plant leaves, the expression of AaORA was increased, whereas the expression of ADS, CYP71AV1, and DBR2 was also promoted in AaORAoverexpressing transgenic plants. As a consequence, there was an increase in artemisinin and dihydroartemisinic acid contents by 40 %–53 % and 22 %–35 %, respectively, in overexpressing transgenic plants [32]. These results demonstrated that AaORA was a positive regulator in artemisinin biosynthesis. Another two JA-response AP2/ERF family TFs were retrieved from A. annua glandular trichome cDNA library which were named AaERF1 and AaERF2 [33]. Six groups (B1–B6) were further divided into ERF subfamily based on the conserved amino acid residues and the existence of other motifs [34]. Sequence comparison and phylogenetic analysis exhibited both AaERF1 and AaERF2 belong to the B3 group, and B3 group was consisted of transcription activators with positive functions in JA-dependent responses and plant defense [35]. Different tissues expression and phytohormones induction patterns analyzation of AaERF1 and AaERF2 displayed that both of them exhibited a similar spatial expression pattern to that of ADS and CYP71AV1, with the highest level of transcripts in flowers [33]. The strong expression of AaERF1 and AaERF2 genes in inflorescence correlates with a relatively high glandular trichome density in these organs. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that the two ERF factors were able to bind to both the CBF2 and the RAA motifs which exist

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in both ADS and CYP71AV1 promoters. Overexpression of the AaERF1 or AaERF2 genes in A. annua plants led to a significant increase in artemisinin and artemisinic acid contents. These data demonstrate clearly that AaERF1 and AaERF2 were positive regulators of ADS and CYP71AV1 and contribute to the production of artemisinin and artemisinic acid in plants [33]. Artemisinin is synthesized and stored in epidermal appendages, which are called glandular trichomes, on the aerial parts of the plant leaves, stems, and flowers [36, 37]. Recently, studies of trichome development (both glandular and filamentous trichome) in Arabidopsis and tomato plants demonstrated that the biosynthesis of wax or cuticle was involved in this process [38–40]. In Arabidopsis, AP2/ ERF TFs WAX INDUCER1/SHINE1 triggered the wax production and modulated the amount and composition of cutin, when overexpressed in A. thaliana in transgenic plants, and then affected trichomes development [41–43]. An A. annua AP2/ERF which seemed to be an orthologous gene of AtWIN1 in Arabidopsis was retrieved from National Center for Biotechnology Information (NCBI) and named TRICHOME AND ARTEMISININ REGULATOR 1 (TAR1) [44]. Sequence comparison indicated that TAR1 shared 67 % and 58 % overall identity with AtWIN1 and AtSHINE3, respectively. Gene expression pattern of TAR1 analyzed by real-time Q-PCR and promoter GUS-staining showed that TAR1 was expressed highly in inflorescence and young leaves but not trichome specific, which is similar with AaERF1 and AaERF2 [33, 44]. Silencing TAR1 expression by RNAi method in A. annua revealed that the adaxial side leaf of transgenic plants was covered with abnormal wax deposition and displayed higher cuticle permeability while compared with the wild-type plants. As a result, the morphology of both glandular and filamentous trichomes was altered and became abnormal [44]. Moreover, EMSA and Agrobacterium-mediated transient expression assay exhibited that TAR1 could bind to CBF2 and RAA motifs existing in ADS and CYP71AV1 promoters and activate their expression just as AaERF1 and AaERF2. As expected, overexpressing TAR1 gene in A. annua increased the content of artemisinin, dihydroartemisinic acid, and artemisinic acid in leaves and flower buds compared with the wild-type plants [44]. These results demonstrate that TAR1 was an important regulator in the biosynthesis of artemisinin and was crucial for the development of trichomes in A. annua plants. 3.3 bZIP family In plants, the basic leucine zipper (bZIP) transcription factor family is another one of the largest and most diverse families. Similarly, the bZIP TFs are also named by their

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conserved DNA-binding domain, the basic region that binds DNA and a leucine zipper dimerization motif [45– 47]. Investigations show that bZIP TFs have involved in various biological processes during growth, such as plant development, organ and tissue differentiation [48], plant energy metabolism [49]. On the other hand, a lot of data show that bZIP TFs also function as important regulators in response to various biotic/abiotic stresses and signaling. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, and using common domains, the AtbZIP family can be subdivided into 10 groups. In group A, the bZIP TFs suggested playing roles in abscisic acid (ABA) or stress signaling [50–52]. Group A bZIP TFs induce gene expression through binding to ACGTG-containing cis-elements that include the ABA response element (ABRE), which are known as ABA-responsive elements (ABREs) [53]. Only a few bZIP TFs have been reported to play roles in regulating plant secondary metabolism. Since group A bZIP TFs revealed important roles in ABA signaling and exogenous ABA promoting artemisinin biosynthesis [54], the A. annua AabZIP1 was reported to regulate artemisinin biosynthesis. Based on global expression and phylogenetic analyses as well as dual-LUC screening, AabZIP1 which showed a similar expression profile of ADS and CYP71AV1 was identified from more than 100 bZIP TFs in A. annua. The results gained from yeast one-hybrid assay, dual-LUC analysis, and EMSA revealed that AabZIP1 activated ADS and CYP71AV1 gene expression levels by binding to the ABRE motifs existed in the promoters [55]. As expected, overexpression of AabZIP1 increased the expression levels of ADS and CYP71AV1 and thus enhanced artemisinin biosynthesis in transgenic A. annua. Moreover, the AabZIP1-overexpressed plants exhibited ABA sensitivity and exogenous ABA treatment could further induce artemisinin accumulation [55]. 3.4 bHLH family The basic helix–loop–helix (bHLH) proteins are from one of the largest transcription factor families in plant genome and found throughout the three eukaryotic kingdoms [56, 57]. Members of this family share the bHLH conserved domain, which consists of around 60 amino acids with two distinct regions. The basic region at the N-terminus functions as a DNA-binding motif [58, 59], and the HLH region contains two amphiphilic a helices with a linking loop allowing the formation of homodimers or heterodimers [57, 60]. The core DNA sequence motif recognized by the bHLH proteins is a consensus hexanucleotide sequence known as the E-box (50 -CANNTG-30 ) [58, 59]. The bHLH TFs are found to be involved in important developmental and physiological processes, such as stomata development

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[61], light signaling [62], and various biotic/abiotic stresses [63–65]. Interestingly, more and more bHLH transcription factors were reported to regulate plant secondary metabolisms, such as alkaloid biosynthesis in C. roseus [24, 66], nicotine biosynthesis in Nicotiana tabacum [67], anthocyanin biosynthesis in A. thaliana [68], and sesquiterpene biosynthesis in A. thaliana [69]. In consideration of the promoters of ADS and CYP71AV1 containing E-box elements, which were putative binding sites for bHLH transcription factors, it was interesting to find out whether there were bHLH TFs involved in regulating the biosynthesis of artemisinin. A bHLH TF, named AabHLH1, which is cloned from A. annua glandular trichome cDNA library, was reported to regulate artemisinin biosynthesis. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and strongly activated the expression of ADS and CYP71AV1. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin [70]. Recently, our laboratory also cloned and identified a JA-response bHLH transcription factor named AaMYC2, which could bind to the G-box-like cis-elements that present in both CYP71AV1 and DBR2 promoters, and then strongly activated the expression of CYP71AV1 and DBR2 in overexpressed transgenic A. annua plants. In consequence, there was an increase in artemisinin content in transgenic plants compared with the wild type [71]. Based on all information discussed above, a summary of transcriptional regulation network of artemisinin biosynthetic pathway is shown in Fig. 1.

4 Perspectives Transcription factors play important roles in plant growth and development, including secondary metabolism. In recent years, a great number of studies have addressed the importance of the transcriptional regulation of target genes through transcription factors and elucidated their underlying mechanisms in regulating plant secondary metabolites in plant cells. Many secondary metabolites are highly valuable to humans, such as artemisinin, which saved millions of lives suffered from malaria. The biosynthetic pathways of secondary metabolites are usually complicated, and the accumulations are usually organ specific, for example, artemisinin was only synthesized and stored in glandular trichome, which resulted in low concentrations of those valuable products in plants [24, 31, 66]. Plant transcription factors have been proposed as a promising approach for more efficiently regulating plant secondary metabolic pathways due to their abilities to regulate specific pathways. As we reviewed in this paper, all of the

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transcription factors involved in artemisinin biosynthesis are likely activate more than one genes, either ADS and CYP71AV1 or CYP71AV1 and DBR2. Interestingly, almost all of the transcription factors reviewed here share a common feature of high expression in the glandular trichome cells and a similar expression pattern with ADS and CYP71AV1. It makes sense that since most of the genes in artemisinin biosynthetic pathway are glandular trichome preferential, trichome-specific transcription factors have preponderant influence in regulating those genes expression and contributing in artemisinin accumulation. Thus, RNA-seq of pure glandular trichome tissues collected by laser capture microdissection technology [36, 72, 73] will tremendously accelerate our speed in candidate genes screening and function characterizing. A few laser capture microdissection RNA-seq databases of A. annua glandular and filamentous trichomes are available now [73] and construct to advance the research on artemisinin biosynthesis and regulation. The available data have already proven useful for the identification of OSC2, a multifunctional oxidosqualene cyclase, and a cytochrome P450 enzyme CYP716A14v2. Together, these two filamentous trichome-specific enzymes catalyze the biosynthesis of triterpenoids for the cuticle of filamentous trichome of A. annua [74]. Since AaWRKY1, AaERF1, AaERF2, AaORA, AabZIP1, and AabHLH1 transcript levels are increased by abiotic stress, such as ABA and JA, it is interesting to expand the network on how abiotic stress stimulates transcriptional levels of biosynthetic genes. The phytohormone jasmonates play an important role in regulating plant secondary metabolism, especially in medicinal plants [11, 75], such as vinblastine biosynthesis in C. roseus [76], nicotine biosynthesis in N. tabacum [67], terpenoids biosynthesis in A. annua [77], Taxus chinensis [78], and Panax ginseng [75, 79]. Nowadays, more and more researches in A. annua show that JA is one of the most effective phytohormones in promoting artemisinin accumulation [77, 80–82]. It is worth finding new JA-responsive TFs that regulate artemisinin biosynthesis in the future. Meanwhile, transcription factors like bHLH often interact with other family proteins (such as MYB family, WD-repeat family) to form a complex and then regulate the downstream expression of target genes [83, 84]. Further elucidation of the interaction among transcription factors will help to elucidate the regulation of artemisinin biosynthesis and other plant secondary metabolisms. Although the artemisinin-based combination therapies have become the preferred norm in the fight against malaria, resistance to artemisinin has begun to emerge [2]. Recently, researches demonstrated the efficacy of the dried whole A. annua plant as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a mice malaria model. Moreover, using the whole

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plant can overcome parasite resistance and is actually more resilient to the evolution of parasite resistance [85, 86]. The mechanism underlying why whole plant treatment is better than pure artemisinin therapy is still unclear, and possible reasons may be attributable to other secondary metabolites accumulated in A. annua. Further intensive studies on artemisinin and other terpenoids or secondary metabolites will help to expand the value of A. annua plant.

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Acknowledgments This work was supported by National High Technology Research and Development Program (2011AA100605), Shanghai Key Discipline Cultivation and Construction Project (Horticulture, ZXDF150005), and Shanghai Jiao Tong University AgriEngineering Program (AF1500028).

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The authors declare that they have no conflict

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Conflict of interest of interest.

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