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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. [ABSTRACT FROM AUTHOR]

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua. [ABSTRACT FROM AUTHOR]

Plants regulate the synthesis of specialized compounds through the actions of individual transcription factors (TFs) or sets of TFs. One such compound, artemisinin from Artemisia annua, is widely used as a pharmacological product in the first-line treatment of malaria. However, the emergence of resistance to artemisinin in Plasmodium species, as well as its low production rates, have required innovative treatments such as exploiting the synergistic effects of flavonoids with artemisinin. We overview current knowledge about flavonoid and artemisinin transcriptional regulation in A. annua , and review the dual action of TFs and structural genes that can regulate both pathways simultaneously. Understanding the concerted action of these TFs and their associated structural genes can guide the development of strategies to further improve flavonoid and artemisinin production. It is increasingly recognized that different TFs act conjointly to regulate the production of plant metabolites. However, their dual functionality in regulating distinct pathways of synthesis has not been well studied. The identification of cis- regulatory elements within the promoter regions of genes encoding different pathway enzymes enables the identification of TF families that regulate their expression. Parallel regulation of the enzyme-coding genes for distinct pathways can simplify overexpression strategies. This provides an excellent starting point to unravel how individual TFs act in a coordinated manner to simultaneously regulate different pathways of specialized metabolism. [ABSTRACT FROM AUTHOR]

Biological synthesis of green nanomaterials holds great promise for sustainable catalysis with versatile applications. Herein, Shewanella oneidensis MR‐1 is used to develop a green method to synthesize Pd/reductive graphene oxide (rGO) nanocomposites without the addition of toxic reducing agents. It was found that S. oneidensis MR‐1 cells could efficiently reduce graphene oxide to rGO and synthesize Pd nanoparticles that uniformly dispersed on the rGO nanosheets. More strikingly, the addition of GO greatly increases the recovery rate of Pd from 37.8 % to 90.4 %. After carbonization, the biosynthesized Pd/rGO nanocomposites exhibits promising catalytic activities toward oxygen reduction reaction with an onset potential of 0.92 V, a half‐wave potential of 0.81 V, and a limiting current density of 5.2 mA cm−2 in 0.1 M KOH electrolyte. This work provides a simple, "green", and cost‐effective method for the preparation of graphene supported nanocomposites with extended applications. [ABSTRACT FROM AUTHOR]

Artemisinin, a sesquiterpene lactone obtained from Artemisia annua , is an essential therapeutic against malaria. YABBY family transcription factor AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450–dependent hydroxylase) and AaDBR2 (double-bond reductase 2); however, the protein–protein interactions of AaYABBY5, as well as the mechanism of its regulation, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome–specific WRKY1) and AaDBR2 (double-bond reductase 2). In this study, YABBY–WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Toward the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1 and AaDBR2 promoters, respectively. In AaYABBY5 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti- AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY–WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

2-methyl-6-phytyl-1, 4-benzoquinol methyltransferase (MPBQ-MT) is a vital enzyme catalyzing a key methylation step in both α/γ-tocopherol and plastoquinone biosynthetic pathway. In this study, the gene encoding MPBQ-MT was isolated from lettuce (Lactuca sativa) by rapid amplification of cDNA ends (RACE), named LsMT. Overexpression of LsMT in lettuce brought about a significant increase of α- and γ-tocopherol contents with a reduction of phylloquinone (vitamin K1) content, suggesting a competition for a common substrate phytyl diphosphate (PDP) between the two biosynthetic pathways. Besides, overexpression of LsMT significantly increased plastoquinone (PQ) level. The increase of tocopherol and plastoquinone levels by LsMT overexpression conduced to the improvement of plants’ tolerance and photosynthesis under high light stress, by directing excessive light energy toward photosynthetic production rather than toward generation of more photooxidative damage. These findings suggest that the role and function of MPBQ-MT can be further explored for enhancing vitamin E value, strengthening photosynthesis and phototolerance under high light in plants. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA) was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase), CYP71AV1 (cytochrome P450 dependent hydroxylase) and DBR2 (double bond reductase 2) were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A.annua. [ABSTRACT FROM AUTHOR]

emisinin is currently the best therapeutic against both drug-resistant and cerebral-malaria-causing strains of Plasmodium falciparum. Allene oxide cyclase (AOC) is the key enzyme in the biosynthetic pathway of jasmonates. In this study, a full-length cDNA of AOC gene (named as AaAOC) was cloned from Artemisia annua L. AaAOC was 1,007 bp, containing an open reading frame (750 bp) encoding 249 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of AaAOC was highly homologous to AOC from other plant species. Phylogenetic analysis indicated that AaAOC was clustered in a closely related subgroup with AOC of Camptotheca acuminata. Southern blot analysis revealed that AaAOC was a multicopy gene. Reverse transcriptase-polymerase chain reaction (RT-PCR) and QPCR analysis showed that AaAOC mRNA accumulated most abundantly in alabastrums, in which the content of artemisinin was previously proven to be the highest. RT-PCR analysis revealed that MeJA, ABA, and ethylene treatments significantly enhanced AaAOC transcript expression. And the results of HPLC showed that the contents of artemisinin were greatly increased after the treatments of ABA and MeJA. [ABSTRACT FROM AUTHOR]

Summary: Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1 , thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90–120% and decreased by 20–60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production. [ABSTRACT FROM AUTHOR]

Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions. [ABSTRACT FROM AUTHOR]

Artemisia annua is the only natural source of the sesquiterpenoid artemisinin, which is widely used to treat malaria. The phytohormone jasmonic acid (JA) can significantly promote artemisinin biosynthesis in A. annua. AabHLH1 can bind and activate artemisinin biosynthetic genes, such as AaADS and AaCYP71AV1. In this study, we proved that AabHLH1 was responsive to MeJA treatment and highly expressed in glandular trichome‐enriched tissues, and that its expression profile was similar to that of AaADS. Yeast two‐hybrid assays showed that AabHLH1 interacted with all nine AaJAZ proteins in A. annua. Functional analysis with transgenic plants showed that several artemisinin biosynthetic genes were upregulated in AabHLH1‐OE transgenic A. annua lines and downregulated in AabHLH1‐EAR lines; furthermore, the artemisinin content was increased in the AabHLH1‐OE lines and decreased in the AabHLH1‐EAR lines. These results demonstrate that the JA‐induced AabHLH1 positively regulates artemisinin biosynthesis by regulating the biosynthetic genes, and thus provide new insight into the regulatory mechanism of JA‐induced artemisinin biosynthesis in A. annua. [ABSTRACT FROM AUTHOR]

The MYB transcription factor (TF) family is among the largest TF families and plays an important role in plant growth and stress response. However, few studies have investigated the role of the MYB gene in drought resistance in cotton. In this study, we analysed the drought transcriptomic data of cotton and identified that the GhMYB102 gene was significantly upregulated in upland cotton during the early stages of drought stress. Bioinformatics analysis showed that the amino acid sequence encoded by GhMYB102 contained two highly conserved MYB binding domains belonging to R2R3-MYB TFs. GhMYB102 was most closely related to AtMYB102. GhMYB102 is mainly expressed in roots and is induced by abiotic stresses and abscisic acid (ABA); it is localised in the nucleus and has transcriptional activation activity. Silencing of GhMYB102 decreased plant drought resistance. In addition, dual-luciferase assays and yeast single hybridisation analysis showed that GhMYB102 could directly bind the MYB motif elements in the promoter regions of GhNCED1 and GhZAT10. These results indicate that GhMYB102 plays a positive role in drought tolerance by regulating the expression of GhNCED1 and GhZAT10. Thus, GhMYB102 enhances drought resistance by participating in ABA biosynthesis or regulating the expression of drought-responsive genes. • Drought resistance transcription factors were screened from drought transcriptome. • Functional validation of MYB transcription factors under drought stress in plants. • MYB transcription factors regulates plant drought tolerance by regulating ABA synthesis. • Drought-responsive genes regulated by MYB transcription factors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway. [ABSTRACT FROM AUTHOR]