Cloning and Characterization of AabHLH1, a bHLH Transcription Factor that Positively Regulates Artemisinin Biosynthesis in Artemisia annua

Abstract

Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. 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 possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (β-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.

The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula

“…In A. annua, two JA-responsive AP2 family TFs and a MYC-type bHLH TF have been identified as activators of the expression of genes encoding the sesquiterpene synthase and P450 essential for the production of the antimalaria compound artemisinin (Yu et al, 2012;Ji et al, 2014). In Arabidopsis, it has been observed that the bHLH TF MYC2 directly binds sesquiterpene synthase gene promoters, resulting in an elevated release of volatile sesquiterpenes (Hong et al, 2012).…”

“…In fact, only a few TFs specifically modulating plant terpene biosynthesis have been identified in general. The basic helix-loop-helix (bHLH) TF MYC2, also known as a primary player in the JA signaling cascade (Kazan and Manners, 2013), and its homologs have been shown to play a role in the regulation of the biosynthesis of sesquiterpenes in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), and Artemisia annua (Hong et al, 2012;Ji et al, 2014;Spyropoulou et al, 2014). Very recently, two other bHLH TFs, Bl (bitter leaf) and Bt (bitter fruit), not related to MYC2, were found to regulate the accumulation of cucurbitacin triterpenes in cucumber (Cucumis sativus; Shang et al, 2014).…”

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

“…The basic helix-loop-helix (bHLH) TF MYC2 has emerged as a central regulator in JA signaling cascades, including those leading to the biosynthesis of several classes of specialized metabolites (9). MYC2-type TFs have been shown to be involved in the regulation of terpene biosynthesis genes in Arabidopsis thaliana, Artemisia annua (sweet wormwood), and Solanum lycopersicum (tomato) (10)(11)(12). In C. roseus, MYC2 regulates the expression of the ethylene response factor (ERF) Octadecanoid derivativeResponsive Catharanthus APETALA2-domain 3 (ORCA3) (13), the JA-inducible regulatory TF that modulates the JA-induced expression of the genes of the indole branch of the pathway, strictosidine synthase (STR), and several steps downstream of strictosidine, thereby controlling part of the JA-responsive production of MIAs (14,15).…”

JA-mediated transcriptional regulation of secondary metabolism in medicinal plants

“…To date, A. annua is the only natural source of artemisinin. The relatively low yield (0.1 %-0.8 % by dry weight) of artemisinin in A. annua is a major limitation to the commercialization of the drug, so the enhanced production of artemisinin is highly desirable [70]. Caretto et al [71] have recognized that the plant A. annua are able to produce high content of artemisinin in respond to the elicitor effect of JAs.…”