Presentation Information
[3Open-01]AaHY5 ChIP-seq based on transient expression system reveals the role of AaWRKY14 in artemisinin biosynthesis gene regulation
○Dianjing GUO1, Limeng Zhou1, Yingzhang Huang1 (1. The Chinese University of Hong Kong (Hong Kong))
Keywords:
AaHY5,AaWRKY14,Artemisia annua;,Artemisinin biosynthesis,ChIP-seq
Artemisinin, produced from the medicinal herb Artemisia annua, is identified as the fastest-acting anti-Plasmodium falciparum malaria drug. Artemisinin combination therapy (ACT) has been the standard method for the treatment of Plasmodium falciparum malaria worldwide since 2008. Despite its importance, the artemisinin abundance in Artemisia annua is too low (about 0.1–1.0% dry weight) to meet the increasing demands. A comprehensive understanding of artemisinin biosynthesis regulation is thus of great significance.
Artemisinin is mainly biosynthesized in the glandular trichome of Artemisia annua, involving a sophisticated network incorporating regulatory transcription factors (TFs) and multiple genes. Key artemisinin biosynthesis enzymes e.g. ADS, CYP71AV1, DBR2, and ALDH1 that are specifically expressed in glandular trichome have been identified in the past decades. Light is an important environmental cue regulating artemisinin biosynthesis and artemisinin production under high light intensity is almost two times higher than that under low light intensity. The bZIP transcription factor AaHY5 is an important regulator involved in light-mediated artemisinin biosynthesis via inducing critical downstream transcription factor AaGSW1 and AaWRKY9, which then directly activate the expression of artemisinin biosynthetic genes.
ChIP-seq (Chromatin immunoprecipitation with sequencing) is the gold standard for determining genome-wide in vivo transcription factor binding sites, the first step for targets prediction and network construction. For non-model plants, it is challenging to perform ChIP-seq due to the difficulty in generating stable transgenic plants. AaHY5 is a positive regulator in artemisinin biosynthesis, whose detailed mode of action remains elusive.
Here we established a protoplast transformation procedure for Artemisia annua by optimizing different conditions in protoplast isolation and transfection. We then performed AaHY5 ChIP-seq based on the established transient expression system. Combining RNA-seq data for various tissues, we identified four transcription factors (one MYB and three WRKY family members) in AaHY5 targets that potentially regulated artemisinin biosynthesis. The three WRKY transcription factors could be induced by light and the overexpression of AaHY5 and upregulate two artemisinin biosynthetic genes, ADS and CYP71AV1. Furthermore, AaWRKY14 showed transcriptional and activity on artemisinin biosynthetic gene CYP71AV1, and was identified as a potential transcription factor linking AaHY5 and the artemisinin biosynthesis gene regulation.
Artemisinin is mainly biosynthesized in the glandular trichome of Artemisia annua, involving a sophisticated network incorporating regulatory transcription factors (TFs) and multiple genes. Key artemisinin biosynthesis enzymes e.g. ADS, CYP71AV1, DBR2, and ALDH1 that are specifically expressed in glandular trichome have been identified in the past decades. Light is an important environmental cue regulating artemisinin biosynthesis and artemisinin production under high light intensity is almost two times higher than that under low light intensity. The bZIP transcription factor AaHY5 is an important regulator involved in light-mediated artemisinin biosynthesis via inducing critical downstream transcription factor AaGSW1 and AaWRKY9, which then directly activate the expression of artemisinin biosynthetic genes.
ChIP-seq (Chromatin immunoprecipitation with sequencing) is the gold standard for determining genome-wide in vivo transcription factor binding sites, the first step for targets prediction and network construction. For non-model plants, it is challenging to perform ChIP-seq due to the difficulty in generating stable transgenic plants. AaHY5 is a positive regulator in artemisinin biosynthesis, whose detailed mode of action remains elusive.
Here we established a protoplast transformation procedure for Artemisia annua by optimizing different conditions in protoplast isolation and transfection. We then performed AaHY5 ChIP-seq based on the established transient expression system. Combining RNA-seq data for various tissues, we identified four transcription factors (one MYB and three WRKY family members) in AaHY5 targets that potentially regulated artemisinin biosynthesis. The three WRKY transcription factors could be induced by light and the overexpression of AaHY5 and upregulate two artemisinin biosynthetic genes, ADS and CYP71AV1. Furthermore, AaWRKY14 showed transcriptional and activity on artemisinin biosynthetic gene CYP71AV1, and was identified as a potential transcription factor linking AaHY5 and the artemisinin biosynthesis gene regulation.
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