Isolation and characterization of AaZFP1, a C2H2 zinc finger protein that regulates the AaIPPI1 gene involved in artemisinin biosynthesis in Artemisia annua

AaZFP1, a C2H2-type transcription factor, was found to bind the AGT-N1-10-AGT box of AaIPPI1pro and activate the expression of AaIPPI1 involved in artemisinin biosynthesis. Artemisinin, an endoperoxide sesquiterpene lactone, is a widely used antimalarial drug isolated from Artemisia annua L. Isopentenyl pyrophosphate isomerase (AaIPPI1) catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate and is the key gene involved in the biosynthesis of artemisinin. However, the AaIPPI1 gene regulation network remains largely unknown. Here, we isolated the AaIPPI1 promoter (AaIPPI1pro) and predicted that it contains cis-elements involved in stress responses, including the TGACG motif (a methyl jasmonate-responsive element), GARE motif (a gibberellin-responsive element), ABRE (an abscisic acid-responsive element), TC-rich repeats (a stress-responsive element), and the AGT-N1-10-AGT box, which is the binding site of Cys/His2 zinc finger protein (C2H2 ZFP). The C2H2 ZFP gene AaZFP1 was discovered by screening a cDNA library using AaIPPI1pro as bait in yeast. AaZFP1 contains two conserved C2H2 regions, a nuclear localization domain (B box), a Leu-rich domain (L box), and a conserved DLN sequence (DLN box) close to its C terminus. A subcellular localization assay indicated that AaZFP1 protein is localized in the nucleus and cytoplasm. An electrophoretic mobility shift assay demonstrated that AaZFP1 binds to the AGT-N1-10-AGT box of AaIPPI1pro. A dual-luciferase assay indicated that AaZFP1 enhanced the promoter activity of AaIPPI1 in vivo. Transient overexpression of AaZFP1 in A. annua increased the expression of AaIPPI1 and the content of artemisinin. Our data demonstrated that AaZFP1 functions as a transcriptional activator that regulates the expression of AaIPPI1 by directly binding to its promoter. The present study provides insights into the transcriptional regulation of genes involved in artemisinin biosynthesis in A. annua.