Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), Chongqing Key Laboratory of Plant Ecology and Resources Research in Three Gorges Reservoir Region, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing, China.
College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China.
J Exp Bot. 2018 Feb 23;69(5):1109-1123. doi: 10.1093/jxb/erx444.
The plant Artemisia annua produces the anti-malarial compound artemisinin. Although the transcriptional regulation of artemisinin biosynthesis has been extensively studied, its post-translational regulatory mechanisms, especially that of protein phosphorylation, remain unknown. Here, we report that an ABA-responsive kinase (AaAPK1), a member of the SnRK2 family, is involved in regulating artemisinin biosynthesis. The physical interaction of AaAPK1 with AabZIP1 was confirmed by multiple assays, including yeast two-hybrid, bimolecular fluorescence complementation, and pull-down. AaAPK1, mainly expressed in flower buds and leaves, could be induced by ABA, drought, and NaCl treatments. Phos-tag mobility shift assays indicated that AaAPK1 phosphorylated both itself and AabZIP1. As a result, the phosphorylated AaAPK1 significantly enhanced the transactivational activity of AabZIP1 on the artemisinin biosynthesis genes. Substituting the Ser37 with Ala37 of AabZIP1 significantly suppressed its phosphorylation, which inhibited the transactivational activity of AabZIP1. Consistent overexpression of AaAPK1 significantly increased the production of artemisinin, as well as the expression levels of the artemisinin biosynthesis genes. Our study opens a window into the regulatory network underlying artemisinin biosynthesis at the post-translational level. Importantly, and for the first time, we provide evidence for why the kinase gene AaAPK1 is a key candidate for the metabolic engineering of artemisinin biosynthesis.
青蒿植物产生抗疟化合物青蒿素。尽管青蒿素生物合成的转录调控已经得到了广泛的研究，但它的翻译后调控机制，特别是蛋白质磷酸化的调控机制，仍然未知。在这里，我们报告说一个 ABA 响应激酶（AaAPK1），属于 SnRK2 家族的一员，参与调节青蒿素生物合成。通过多种测定方法，包括酵母双杂交、双分子荧光互补和下拉实验，证实了 AaAPK1 与 AabZIP1 的物理相互作用。AaAPK1 主要在花蕾和叶片中表达，可被 ABA、干旱和 NaCl 处理诱导。Phos-tag 迁移率变动分析表明，AaAPK1 自身和 AabZIP1 都能被磷酸化。结果，磷酸化的 AaAPK1 显著增强了 AabZIP1 对青蒿素生物合成基因的转录激活活性。用 Ala37 取代 AabZIP1 的 Ser37 可显著抑制其磷酸化，从而抑制 AabZIP1 的转录激活活性。过表达 AaAPK1 可显著增加青蒿素的产量以及青蒿素生物合成基因的表达水平。我们的研究为青蒿素生物合成的翻译后调控网络打开了一扇窗。重要的是，这也是首次提供了为什么激酶基因 AaAPK1 是青蒿素生物合成代谢工程的关键候选基因的证据。
