title: 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.
authors: University of the Chinese Academy of Sciences, Beijing 100049, China These authors contributed equally to this work.
authors: Yunpeng
authors: Y
authors: Ji
authors: National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China These authors contributed equally to this work.
authors: Jingwei
authors: J
authors: Xiao
authors: University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Yalin
authors: Y
authors: Shen
authors: Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
authors: Dongming
authors: D
authors: Ma
authors: Hebei University, Baoding 071002, China.
authors: Zhenqiu
authors: Z
authors: Li
authors: Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
authors: Gaobin
authors: G
authors: Pu
authors: University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Xing
authors: X
authors: Li
authors: Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
authors: Lili
authors: L
authors: Huang
authors: Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
authors: Benye
authors: B
authors: Liu
authors: Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China.
authors: Hechun
authors: H
authors: Ye
authors: University of the Chinese Academy of Sciences, Beijing 100049, China hwang@ucas.ac.cn.
authors: Hong
authors: H
authors: Wang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D031172:Artemisia annua / Q000737:chemistry / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D037621:Artemisinins / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D019883:Alkyl and Aryl Transferases; D037621:Artemisinins; D051792:Basic Helix-Loop-Helix Proteins; D003577:Cytochrome P-450 Enzyme System; D010940:Plant Proteins; C031327:artemisinin; C403714:amorpha-4,11-diene synthase
keywords: Amorpha-4,11-diene synthase (ADS); Artemisia annua L; Artemisinin biosynthesis; Cyt P450 monooxygenase (CYP71AV1); bHLH transcription factor