Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes

Abstract

Artemisia annua 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) A. annua 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 in planta, AaCAD 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.

Overexpression of Pear (Pyrus pyrifolia) CAD2 in Tomato Affects Lignin Content

“…CAD genes were demonstrated to control lignification in plants [39,40,41]. In agreement with our results, Brachypodium distachyon CAD gene BdCAD1 mutants displayed a reduced CAD activity and lower lignin content [39], suggesting that the CAD activity is positively correlated with the lignin content.…”

“…In Brassica chinensis , the induced expression of BcCAD1-1 and BcCAD2 could increase the lignification of stems [40]. AaCAD has also been reported to positively enhance lignin formation in Artemisia annua [41]. In summary, these results support that PpCAD2 positively regulates lignin biosynthesis by increasing the CAD activity in the stems, fruit pericarp and leaves of transgenic tomato plants.…”

“…The explants were placed back onto the co-cultivation medium and incubated in the dark for 1.5 d under 28 °C. The explants were transferred to a callus induction/selection medium containing MS salts, 3% sucrose, 1.5 mg·L −1 zeatin, 50 mg·L −1 kanamycin and 500 mg·L −1 Cef, and 0.7% agar at pH 5.8 [41]. The explants were transferred onto a fresh medium every 2 weeks.…”

Flavonoid Versus Artemisinin Anti-malarial Activity in Artemisia annua Whole-Leaf Extracts

“…The reduction of artemisinin levels in chi1-1 might be explained by lower DHAA levels (Supplementary Table S1), which could be due to either decreased DHAA synthesis or enhanced DHAA degradation, but the connection to the chi1-1 mutation is unclear. The crosstalk between phenylpropanoid and terpenoid metabolism is further highlighted by the report that overexpression of the A. annua CINNAMYL ALCOHOL DEHYDROGENASE results in an increase in lignin and coumarin and a reduction in artemisinin and other sesquiterpenes (Ma et al, 2018).…”

Pluronic F-68 Improves Callus Proliferation of Recalcitrant Rice Cultivar via Enhanced Carbon and Nitrogen Metabolism and Nutrients Uptake

“…In this study, cinnamyl alcohol dehydrogenase (CAD) protein was found to be exclusive in a callus treated with high PF-68 concentration as compared with optimum PF-68 concentration ( Supplementary Table 4 ). This CAD has been widely characterized, and it is known to play a role in lignin biosynthesis via the conversion of phenylpropenyl aldehydes to alcohols (Ma et al, 2018 ). Suppression of CAD was demonstrated to reduce the lignin content and alter the lignin composition in switchgrass (Fu et al, 2011 ).…”