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Microalgae are the preferred species for producing astaxanthin because they pose a low toxicity risk than chemical synthesis. Astaxanthin has multiple health benefits and is being used in: medicines, nutraceuticals, cosmetics, and functional foods. Haematococcus pluvialis is a model microalga for astaxanthin biosynthesis; however, its natural astaxanthin content is low. Therefore, it is necessary to develop methods to improve the biosynthesis of astaxanthin to meet industrial demands, making its commercialization cost-effective. Several strategies related to cultivation conditions are employed to enhance the biosynthesis of astaxanthin in H. pluvialis. However, the mechanism of its regulation by transcription factors is unknown. For the first time, this study critically reviewed the studies on identifying transcription factors, progress in H. pluvialis genetic transformation, and use of phytohormones that increase the gene expression related to astaxanthin biosynthesis. In addition, we propose future approaches, including (i) Cloning and characterization of transcription factors, (ii) Transcriptional engineering through overexpression of positive regulators or downregulation/silencing of negative regulators, (iii) Gene editing for enrichment or deletion of transcription factors binding sites, (iv) Hormonal modulation of transcription factors. This review provides considerable knowledge about the molecular regulation of astaxanthin biosynthesis and the existing research gap. Besides, it provides the basis for transcription factors mediated metabolic engineering of astaxanthin biosynthesis in H. pluvialis. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Summary: Plant glandular secretory trichomes (GSTs) produce various specialized metabolites. Increasing GST density represents a strategy to enhance the yield of these chemicals; however, the gene regulatory network that controls GST initiation remains unclear.In a previous study of Artemisiaannua L., we found that a HD‐ZIP IV transcription factor, AaHD1, promotes GST initiation by directly regulating AaGSW2. Here, we identified two AaHD1‐interacting transcription factors, namely AaMIXTA‐like 2 (AaMYB16) and AaMYB5.Through the generation and characterization of transgenic plants, we found that AaMYB16 is a positive regulator of GST initiation, whereas AaMYB5 has the opposite effect. Notably, neither of them regulates GST formation independently. Rather, they act competitively, by interacting and modulating AaHD1 promoter binding activity. Additionally, the phytohormone jasmonic acid (JA) was shown to be associated with the AaHD1‐AaMYB16/AaMYB5 regulatory network through transcriptional regulation via a JASMONATE‐ZIM DOMAIN (JAZ) protein repressor.These results bring new insights into the mechanism of GST initiation through regulatory complexes, which appear to have similar functions in a range of vascular plant taxa. [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]

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua , AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx , in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint. [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]

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]

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 Aa ORA 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., Aa ORA 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 Aa ORA is targeted to the nuclei and the cytoplasm., Overexpression and RNA interference ( RNAi) of Aa ORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71 AV1, DBR2 and Aa ERF1. 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 Aa ORA is a positive regulator in the biosynthesis of artemisinin., Overexpression of Aa ORA 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 Aa ORA overexpression in A. thaliana and Aa ORA RNAi in A. annua demonstrate that Aa ORA is a positive regulator of disease resistance to B. cinerea. [ABSTRACT FROM AUTHOR]