Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements

Abstract

The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.

Figure 1

Artemisinin biosynthetic pathway. ADS, amorphadiene synthase; Aldh1, aldehyde dehydrogenase 1; CYP, CYP71AV1; DBR2, double bond reductase 2; DMAPP, dimethylallyl diphosphate; DXS, 1-deoxyxylulose 5- phosphate synthase; DXR, 1-deoxyxylulouse 5-phosphate reductoisomerase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; MEP, methyl erythritol phosphate pathway; MVA, mevalonic acid pathway.

Figure 2

(A) Changes in HMGR and FPS in A. annua seedlings grown 14 days in glucose, fructose, or sucrose relative to day zero. (B) Relative response of HMGR and FPS in A. annua seedlings grown in glucose or fructose and compared to growth in sucrose. Bars are ± SD.

Figure 3

(A) Changes in DXS and DXR in A. annua seedlings grown 14 days in glucose, fructose, or sucrose relative to day zero. (B) Relative response of DXS and DXR in A. annua seedlings grown in glucose or fructose and compared to growth in sucrose. Bars are ± SD.

Figure 4

(A) Changes in ADS and CYP in A. annua seedlings grown 14 days in glucose, fructose, or sucrose relative to day zero. (B) Relative response of ADS and CYP in A. annua seedlings grown in glucose or fructose and compared to growth in sucrose. Bars are ± SD.

Figure 5

AN and DHAA levels in A. annua seedlings grown 14 days in glucose, fructose, or sucrose. Bars are SE.

Figure 6

AA and AB levels in A. annua seedlings grown 14 days in glucose, fructose, or sucrose. Bars are SE.

Figure 7

Root growth of A. annua seedlings grown in different concentrations of artemisinin. Letters indicate statistical differences at p ≤ 0.05(via ANOVA/Tukey HSD).