Ma Dongming, Xu Chong, Alejos-Gonzalez Fatima, Wang Hong, Yang Jinfen, Judd Rika, Xie De-Yu
Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, China.
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, NC, United States.
Front Plant Sci. 2018 Jun 19;9:828. doi: 10.3389/fpls.2018.00828. eCollection 2018.
is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function , was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.
是唯一能产生用于疟疾治疗的青蒿素的药用作物。在此，我们描述了从一个自花授粉（SP）自交系品种中克隆肉桂醇脱氢酶（AaCAD）及其对木质素和青蒿素产量的影响。通过异源表达纯化了重组AaCAD。酶活性测定表明，重组AaCAD将对香豆醛、松柏醛和芥子醛转化为其相应的醇，这些醇是木质素生物合成中的关键中间体。计算了所有三种底物的Km、Vmax和Vmax/Km值。为了表征其功能，在SP植物中过表达。使用乙酰溴（AcBr）进行定量分析表明，与野生型（WT）植物相比，转基因植物中的木质素含量显著更高。此外，基于气相色谱-质谱联用（GC-MS）的分析表明，转基因植物中的香豆素含量显著增加。相比之下，高效液相色谱-质谱联用（HPLC-MS）分析表明，与WT植物相比，转基因植物中的青蒿素含量显著降低。此外，气相色谱-质谱分析表明，转基因植物中青蒿琥酯B和其他六种倍半萜的含量降低。共聚焦显微镜分析显示AaCAD定位于细胞质。这些数据表明，AaCAD在细胞质中发挥双重途径功能，其中它正向增强木质素形成，但负向控制青蒿素形成。基于这些数据，讨论了由AaCAD催化介导的这两条途径之间的相互作用，以了解植物中青蒿素生物合成的代谢控制以实现高产。
