AaWRKY4 upregulates artemisinin content through boosting the expressions of key enzymes in artemisinin biosynthetic pathway

Artemisinin is widely used as an antimalarial drug, and the regulation of artemisinin metabolism is at the forefront of artemisinin research. A WRKY transcription factor, named as AaWRKY4, was cloned from high artemisinin-yielding Artemisia annua, which has similar expression pattern with the key enzymes in artemisinin biosynthetic pathway. AaWRKY4 was preferentially expressed in glandular secretory trichomes (GSTs) of young leaves and flower buds, but weakly expressed in other tissues. To further study the function of AaWRKY4, plant expression vector pHB-AaWRKY4 containing AaWRKY4 driven by CaMV 35S promoter was constructed and introduced into A. annua via Agrobacterium tumafeciens-mediated transformation. Expression analysis showed that the expression of AaWRKY4 was increased in transgenic plants. Four independent transgenic plants overexpressing AaWRKY4 were selected for further analysis. The expression levels of artemisinin biosynthetic pathway genes ADS, CYP71AV1, DBR2 and ALDH1 were dramatically increased in AaWRKY4-overexpressing A. annua plants. Furthermore, the artemisinin yield was increased by 35–50% in AaWRKY4-overexpressing A. annua plants. These results indicate AaWRKY4 can upregulate artemisinin content through regulating artemisinin metabolism.