Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L.

Abstract

This study aims to elucidate the underlying molecular mechanisms of artemisinin accumulation induced by cadmium (Cd). The effects of different Cd concentrations (0, 20, 60, and 120 μmol/L) on the biosynthesis of Artemisia annua L. were examined. Intermediate and end products were quantified by HPLC-ESI-MS/MS analysis. The expression of key biosynthesis enzymes was also determined by qRT-PCR. The results showed that the application of treatment with 60 and 120 μmol/L Cd for 3 days significantly improved the biosynthesis of artemisinic acid, arteannuin B, and artemisinin. The concentrations of artemisinic acid, arteannuin B, and artemisinin in the 120 μmol/L Cd-treated group were 2.26, 102.08, and 33.63 times higher than those in the control group, respectively. The concentrations of arteannuin B and artemisinin in 60 μmol/L Cd-treated leaves were 61.10 and 26.40 times higher than those in the control group, respectively. The relative expression levels of HMGR, FPS, ADS, CYP71AV1, DBR2, ALDH1, and DXR were up-regulated in the 120 μmol/L Cd-treated group because of increased contents of artemisinic metabolites after 3 days of treatment. Hence, appropriate doses of Cd can increase the concentrations of artemisinic metabolites at a certain time point by up-regulating the relative expression levels of key enzyme genes involved in artemisinin biosynthesis.

Keywords

Cd / secondary metabolites / gene expressions / Artemisia annua L.

References

[1] Dixon RA. Natural products and plant disease resistance. Nature 2001; 411(6839): 843–847

[2] Wink M. Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 2003; 64(1): 3–19

[3] Huang CY, Bazzaz FA, Vanderhoef LN. The inhibition of soybean metabolism by cadmium and lead. Plant Physiol 1974; 54(1): 122–124

[4] Larbi A, Morales F, Abadía A, Gogorcena Y, Lucena JJ, Abadía J. Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition. Funct Plant Biol 2002; 29(12): 1453–1464

[5] Ekmekçi Y, Tanyolaç D, Ayhan B. Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars. J Plant Physiol 2008; 165(6): 600–611

[6] Uraguchi S, Watanabe I, Yoshitomi A, Kiyono M, Kuno K. Characteristics of cadmium accumulation and tolerance in novel Cd-accumulating crops, Avena strigosa and Crotalaria juncea. J Exp Bot 2006; 57(12): 2955–2965

[7] Shi GX, Xu QS, Xie KB, Xu N, Zhang XL, Zeng XM, Zhou HW, Zhu L. Physiology and ultrastructure of Azolla imbricata as affected by Hg²⁺ and Cd²⁺ toxicity. Acta Botanica Sinica (Zhi Wu Xue Bao) 2003; 45: 437–444 (in Chinese)

[8] Vitti A, Nuzzaci M, Scopa A, Tataranni G, Remans T, Vangronsveld J, Sofo A. Auxin and cytokinin metabolism and root morphological modifications in Arabidopsis thaliana seedlings infected with Cucumber mosaic virus (CMV) or exposed to cadmium. Int J Mol Sci 2013; 14(4): 6889–6902

[9] Daud MK, Ali S, Variath MT, Zhu SJ. Differential physiological, ultramorphological and metabolic responses of cotton cultivars under cadmium stress. Chemosphere 2013; 93(10): 2593–2602

[10] Li X, Zhao M, Guo L, Huang L. Effect of cadmium on photosynthetic pigments, lipid peroxidation, antioxidants, and artemisinin in hydroponically grown Artemisia annua. J Environ Sci (China) 2012; 24(8): 1511–1518

[11] Zheng Z, Wu M. Cadmium treatment enhances the production of alkaloid secondary metabolites in Catharanthus roseus. Plant Sci 2004; 166(2): 507–514

[12] Liu D, Zhang L, Li C, Yang K, Wang Y, Sun X, Tang K. Effect of wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua. Russ J Plant Physiol 2010; 57(6): 882–886

[13] Yadav RK, Sangwan RS, Sabir F, Srivastava AK, Sangwan NS. Effect of prolonged water stress on specialized secondary metabolites, peltate glandular trichomes, and pathway gene expression in Artemisia annua L. Plant Physiol Biochem 2014; 74: 70–83

[14] Lei CY, Ma DM, Pu GB, Qi XF, Du ZG, Wang H, Li GF, Ye HC, Liu BY. Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L. Ind Crops Prod 2011; 33(1): 176–182

[15] Arsenault PR, Vail DR, Wobbe KK, Weathers PJ. Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements. Molecules 2010; 15(4): 2302–2318

[16] Pu GB, Ma DM, Chen JL, Ma LQ, Wang H, Li GF, Ye HC, Liu BY. Salicylic acid activates artemisinin biosynthesis in Artemisia annua L. Plant Cell Rep 2009; 28(7): 1127–1135

[17] Caretto S, Quarta A, Durante M, Nisi R, De Paolis A, Blando F, Mita G. Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua suspension cultures. Plant Biol (Stuttg) 2011; 13(1): 51–58

[18] Duke SO, Paul RN. Development and fine structure of the glandular trichomes of Artemisia annua L. Int J Plant Sci 1993; 154(1): 107–118

[19] Akhila A, Thakur RS, Popli SP. Biosynthesis of artemisinin in Artemisia annua. Phytochemistry 1987; 26(7): 1927–1930

[20] Towler MJ, Weathers PJ. Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways. Plant Cell Rep 2007; 26(12): 2129–2136

[21] Bouwmeester HJ, Wallaart TE, Janssen MH, van Loo B, Jansen BJ, Posthumus MA, Schmidt CO, De Kraker JW, König WA, Franssen MC. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 1999; 52(5): 843–854

[22] Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006; 440(7086): 940–943

[23] Teoh KH, Polichuk DR, Reed DW, Nowak G, Covello PS. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett 2006; 580(5): 1411–1416

[24] Zhang Y, Teoh KH, Reed DW, Maes L, Goossens A, Olson DJ, Ross AR, Covello PS. The molecular cloning of artemisinic aldehyde D11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem 2008; 283(31): 21501–21508

[25] Teoh KH, Polichuk DR, Reed DW, Covello PS. Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany 2009; 87(6): 635–642

[26] Wallaart TE, Bouwmeester HJ, Hille J, Poppinga L, Maijers NC. Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 2001; 212(3): 460–465

[27] Wallaart TE, Lubberink HG, Woerdenbag HJ, Pras N, Quax WJ, Quax WJ. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. J Nat Prod 1999; 62(3): 430–433

[28] Han XL, Huang LQ, Guo LP, Li MJ, Liu XH, Zhang XB. Accumulation and translocation of cadmium in soil and plant and its effects on growth of Artemisia annua and artemisinin content. China J Chin Materia Medica (Zhongguo Zhong Yao Za Zhi) 2010; 35(13): 1655–1659 (in Chinese)

[29] Jessing KK, Juhler RK, Strobel BW. Monitoring of artemisinin, dihydroartemisinin, and artemether in environmental matrices using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). J Agric Food Chem 2011; 59(21): 11735–11743

[30] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3(6): 1101–1108

[31] Cheng Q, Su P, Hu Y, He Y, Gao W, Huang L. RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS. Biotechnol Lett 2014; 36(2): 363–369

[32] Gu XC, Chen JF, Xiao Y, Di P, Xuan HJ, Zhou X, Zhang L, Chen WS. Overexpression of allene oxide cyclase promoted tanshinone/phenolic acid production in Salvia miltiorrhiza. Plant Cell Rep 2012; 31(12): 2247–2259

[33] Shen Q, Chen YF, Wang T, Wu SY, Lu X, Zhang L, Zhang FY, Jiang WM, Wang GF, Tang KX. Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae). Genet Mol Res 2012; 11(3): 3298–3309

[34] Xiang L, Zeng LX, Yuan Y, Chen M, Wang F, Liu XQ, Zeng LJ, Lan XZ, Liao ZH. Enhancement of artemisinin biosynthesis by overexpressing dxr, cyp71av1 and cpr in the plants of Artemisia annua L. Plant Omics 2012; 5: 503–507

[35] Wang HH, Ma CF, Li ZQ, Ma LQ, Wang H, Ye HC, Xu GW, Liu BY. Effects of exogenous methyl jasmonate on artemisinin biosynthesis and secondary metabolites in Artemisia annua L. Ind Crops Prod 2010; 31(2): 214–218

[36] Wang H, Ma C, Ma L, Du Z, Wang H, Ye H, Li G, Liu B, Xu G. Secondary metabolic profiling and artemisinin biosynthesis of two genotypes of Artemisia annua. Planta Med 2009; 75(15): 1625–1633

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Nos. 81130070, 81325023, and 81473307), Natural Key Technologies R&D Program of China (Nos. 2012BAI29B02 and 2012BAI28B02), and the Innovative Funding for PhD Students at China Academy of Chinese Medical Sciences (No. CX201608).

Compliance with ethics guidelines

Liangyun Zhou, Guang Yang, Haifeng Sun, Jinfu Tang, Jian Yang, Yizhan Wang, Thomas Avery Garran, and Lanping Guo declare no conflict of interest. This article does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee.

RIGHTS & PERMISSIONS

2016 Higher Education Press and Springer-Verlag Berlin Heidelberg