Artemisinin, a sesquiterpene lactone produced in Artemisia annua, is the most effective antimalarial compound, but its low natural abundance in A. annua remains a major bottleneck for large-scale production. Increasing its biosynthesis requires a better understanding of the regulatory networks controlling artemisinin biosynthesis genes. Several jasmonate (JA)-responsive transcription factors have been characterized, but the role of upstream kinases remains poorly understood. Here, we identify AaSnRK2.6, a SnRK2III-type protein kinase, as a novel positive regulator of artemisinin biosynthesis. AaSnRK2.6 expression was strongly induced by methyl jasmonate and abscisic acid, and the protein is localized to both the cytoplasm and nucleus. Protein interaction assays revealed that AaSnRK2.6 specifically interacts with the JA-responsive TF AaORA in the nucleus, and an in vitro kinase assay further showed that AaSnRK2.6 directly phosphorylates AaORA. Dual-luciferase assays showed that AaSnRK2.6 enhances AaORA-mediated activation of key artemisinin biosynthetic genes (amorpha-4,11-diene synthase, DBR2, and ALDH1). Overexpression of AaSnRK2.6 alone or AaORA alone significantly increased artemisinin and dihydroartemisinic acid accumulation, while co-overexpression of AaSnRK2.6 and AaORA in transgenic plants produced the stronger effect. By contrast, RNA interference knockdown of AaSnRK2.6 showed no significant effect on artemisinin levels, likely due to functional redundancy of kinases. Collectively, this study supports a positive kinase-TF regulatory module involved in hormone-responsive artemisinin biosynthesis in A. annua and provides new insights into its regulatory mechanism.

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