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Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1, an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The plant Artemisia annua produces the anti-malarial compound artemisinin. Although the transcriptional regulation of artemisinin biosynthesis has been extensively studied, its post-translational regulatory mechanisms, especially that of protein phosphorylation, remain unknown. Here, we report that an ABA-responsive kinase (AaAPK1), a member of the SnRK2 family, is involved in regulating artemisinin biosynthesis. The physical interaction of AaAPK1 with AabZIP1 was confirmed by multiple assays, including yeast two-hybrid, bimolecular fluorescence complementation, and pull-down. AaAPK1, mainly expressed in flower buds and leaves, could be induced by ABA, drought, and NaCl treatments. Phos-tag mobility shift assays indicated that AaAPK1 phosphorylated both itself and AabZIP1. As a result, the phosphorylated AaAPK1 significantly enhanced the transactivational activity of AabZIP1 on the artemisinin biosynthesis genes. Substituting the Ser37 with Ala37 of AabZIP1 significantly suppressed its phosphorylation, which inhibited the transactivational activity of AabZIP1. Consistent overexpression of AaAPK1 significantly increased the production of artemisinin, as well as the expression levels of the artemisinin biosynthesis genes. Our study opens a window into the regulatory network underlying artemisinin biosynthesis at the post-translational level. Importantly, and for the first time, we provide evidence for why the kinase gene AaAPK1 is a key candidate for the metabolic engineering of artemisinin biosynthesis.

In recent years, although several related genes had been cloned and characterized, the role of aldehyde dehydrogenase 1 (ALDH1), the newly cloned gene involved in artemisinin biosynthesis pathway, is still not clear. In this study, a 2,100-bp ALDH1 promoter region fused with GUS reporter gene was stably transferred into Arabidopsis thaliana. Histochemical staining showed the methyl jasmonate (MeJA) and wounding treatment induced the GUS gene expression specifically in the trichomes of transgenic A. thaliana, consistent with the results that the expression level of ALDH1 gene was increased in the A. annua under MeJA and wounding treatments. Two RAA motifs (AP2/ERF binding site) but no W box (WRKY binding site) motif were identified in the ALDH1 promoter by the analysis through PLACE and plantCARE. Through the dual luciferase reporter assay, we revealed that both AaORA and AaERF2, rather than AaWRKY1, could activate the expression of ALDH1 promoter. Our study shed light on the in-depth understanding of the role of ALDH1 in artemisinin biosynthesis., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Previous publications reported that the artemisinin level was increased in Artemisia annua following a night-frost period. However, the molecular mechanism was not clear. In this study, we found that exogenous jasmonate (JA) effectively enhanced the freezing tolerance of A. annua. The JA biosynthetic genes (LOX1, LOX2, allene oxide cyclase [AOC], and jasmonate resistant 1 [JAR1]) were induced by cold stress, leading to an increase in endogenous JA in cold-treated A. annua. Increased endogenous JA enhanced the expression of three JA-responsive transcription factors, ethylene response factor 1, ethylene response factor 2, and octadecanoid-responsive AP2/ERF, all of which were reported to transcriptionally activate the expression of artemisinin biosynthetic genes, such as amorpha-4,11-diene synthase (ADS), CYP71AV1, DBR2, and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the expression levels of the four artemisinin biosynthetic genes were also significantly increased under cold stress. Consequently, the levels of artemisinin and related secondary metabolites, such as dihydroartemisinic acid, artemisinin B, and artemisinic acid, were increased in A. annua under cold stress. Our study points to a molecular mechanism in which the production of artemisinin is regulated by cold stress in A. annua., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Artemisia annua is the main natural source of artemisinin production. In A. annua , extended drought stress severely reduces its biomass and artemisinin production while short-term water-withholding or abscisic acid (ABA) treatment can increase artemisinin biosynthesis. ABA-responsive transcription factor AabZIP1 and JA signaling AaMYC2 have been shown in separate studies to promote artemisinin production by targeting several artemisinin biosynthesis genes. Here, we found AabZIP1 promote the expression of multiple artemisinin biosynthesis genes including AaDBR2 and AaALDH1 , which AabZIP1 does not directly activate. Subsequently, it was found that AabZIP1 up-regulates AaMYC2 expression through direct binding to its promoter, and that AaMYC2 binds to the promoter of AaALDH1 to activate its transcription. In addition, AabZIP1 directly transactivates wax biosynthesis genes AaCER1 and AaCYP86A1. The biosynthesis of artemisinin and cuticular wax and the tolerance of drought stress were significantly increased by AabZIP1 overexpression, whereas they were significantly decreased in RNAi- AabZIP1 plants. Collectively, we have uncovered the AabZIP1-AaMYC2 transcriptional module as a point of cross-talk between ABA and JA signaling in artemisinin biosynthesis, which may have general implications. We have also identified AabZIP1 as a promising candidate gene for the development of A. annua plants with high artemisinin content and drought tolerance in metabolic engineering breeding. AabZIP1 regulates artemisinin biosynthesis via directly transactivating AaADS and AaCYP71AV1 , and indirectly up-regulating AaDBR2 and AaALDH1 via AaMYC2. Moreover, AabZIP1 improves drought tolerance by promoting wax biosynthesis in Artemisia annua. [Display omitted] [ABSTRACT FROM AUTHOR]

AaPIF3, a bHLH transcription factor phylogenetically associated with Arabidopsis PIF3, positively regulates artemisinin biosynthesis in Artemisia annua. Overexpression of AaPIF3 significantly promotes the production of artemisinin and its related metabolites. • AaPIF3 is a bHLH transcription factor that is phylogenetically related with Arabidopsis PIF3. • AaPIF3 is able to transactivate artemisinin biosynthesis genes, according to dual-luciferase assays. • Overexpression of AaPIF3 markedly increased the production of artemisinin in Artemisia annua. Artemisia annua is a Chinese traditional herbal plant that produces artemisinin, the potent anti-malarial drug. It is a common goal for the artemisinin industry to develop plants of A. annua with high yields of artemisinin. In this study, a bHLH transcription factor of A. annua (AaPIF3) was characterized and used to promote the production of artemisinin. AaPIF3 was highly expressed in flower buds and glandular secretory trichomes and its coding protein was localized to the nucleus. Dual-luciferase assays indicated that AaPIF3 was able to significantly enhance the promoter activities of artemisinin biosynthesis genes, including ADS , CYP71AV1 , DBR2 , and ALDH1 , suggesting that AaPIF3 positively regulated artemisinin biosynthesis. Overexpression of AaPIF3 markedly upregulated artemisinin biosynthesis genes at the transcriptional level, and consequently the production of artemisinin was significantly promoted in transgenic A. annua plants. On the contrary, suppression of AaPIF3 led to significantly decreased transcript levels of artemisinin biosynthesis genes, with overall production of artemisinin significantly reduced. In summary, AaPIF3 plays a positive role in regulating artemisinin biosynthesis and transgenic plants of A. annua with high yields of artemisinin have been successfully developed through overexpression of AaPIF3. [ABSTRACT FROM AUTHOR]