AaHY5 ChIP-seq based on transient expression system reveals the role of AaWRKY14 in artemisinin biosynthetic gene regulation

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 activation activity on artemisinin biosynthetic gene CYP71AV1. Together, AaWRKY14 was identified as a potential transcription factor linking AaHY5 and the artemisinin biosynthetic gene regulation.