Transient expression of transcription factors belonging to WRKY and MYB family and two enzymes DBR2 and OPR3 in two Artemisia annua chemotypes (ANAMED and Iranian wild type) in order to increase the artemisinin

Document Type : Research Paper


1 Post Graduate Student, University College of Agriculture & Natural Resources, University of Tehran, Karaj and Assistant Professor, Department of Plant Breeding and Biotechnology, Tarbiat Modares University, Tehran, Iran

2 Professor, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran

3 Assistant Professor, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran

4 School of Natural Sciences, Linnaeus University, Kalmar, Sweden


Artemisia annua is an annual herb native to Asia most probably China produce one of the most important sesquiterpene namely artemisinin which has antimalarial and anticancer activity. Three transcription factors belonging to WRKY family, two transcription factors belonging to MYB family and two enzymes DBR2 and OPR3 were isolated from A.  annua leaves and first inserted into pJET1.2 vector then sub cloned into cowpea mosaic virus (CPMV) based viral vector (pEAQ-HT). Thegenes were transiently expressed in A.  annua young leaves of two different chemotypes ANAMED and Iranian wild type under the control of a constitutive cauliflower mosaic virus (CaMV) 35S promoter as vacuum infiltration. An agroinfitration method was carried out for transient expression of different genes. The analysis of metabolites by GC-MS method showed that the transcription factors which have expressed transiently, could effect on the rate of DHAA and AA metabolites. The results show that the TFs (WRKY and MYB) are involved in artemisinin biosynthesis pathway.


  1. Borrone, J.W., Meerow, A.W., Kuhn, D.N., Whitlock, B.A. & Schnell, R.J. (2007). The potential of the WRKY gene family for phylogenetic reconstruction: An example from the Malvaceae. Molecular Phylogenetics and Evolution, 44, 1141-1154.
  2. Bouwmeester, H.J., Wallaart, T.E., Janssen, M.H., van Loo, B., Jansen, B.J., Posthumus, M.A., Broun, P., Liu, Y., Queen, E.,Schwarz, Y., Lleva, M. & Leibman, A.M. (2006).  Importance of transcription factors in the regulation of plant secondary metabolism and their relevance to the control of terpenoid accumulation. Phytochem Rev, 5, 27-38.
  3. Bouwmeester, H.J., Wallaart, T.E., Janssen, M.H., van Loo, B., Jansen, B.J., Posthumus, M.A., Schmidt, C.O., De Kraker, J.W., Konig, W.A. & Franssen, M.C. (1999). Amorpha-4, 11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry, 52, 843-854.
  4. Capell, T. & Christou, P. (2004). Progress in plant metabolic engineering. Current Opinion in Biotechnology, 15, 148-154.
  5. Covello, P.S., Teoh, K.H., Polichuk, D.R., Reed, D.R. & Nowak, G. (2007). Functional genomics and the biosynthesis of artemisinin. Phytochemistry, 68, 1864-1871.
  6. Iwase, A., Matsui, K & Ohme-Takagi, M. (2009). Manipulation of plant metabolic pathways by transcription factors. Plant Biotechnology, 26, 29-38.
  7. Kim,  C.Y., Ahn,  Y.O., Kim, S.H., Kim, Y.H.., Lee, H.S.,  Catanach, A.S., Jacobs, J.M., Conner, A.J. & Kwak S.S. (2010). The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system. Physiologia Plantarum, 139, 229-240.
  8. Lommen, W.J., Schenk, E., Bouwmeester, H.J. & Verstappen, F.W. (2006). Trichome dynamics and artemisininaccumulation during development and senescence of Artemisia annua leaves. Planta Med., 72, 336-345.
  9. Ma, Dongming., Gaobin, Pu., Caiyan, Lei., Lanqing, Ma., Huahong, W., Yanwu, G., Jianlin, C., Zhigao, D., Hong, W., Guofeng, L., Hechun, Y.  & Benye, L. (2009). Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis. Plant Cell Physiology, 50, 2146-2161.
  10. Mahjoub, A., Hernould, M., Joube, J., Decendit, A., Mars, M., Barrieu, F., Hamdi, S., Delrot, S. (2009). Overexpression of a grapevine R2R3-MYB factor in tomato affects vegetative development, flower morphology and flavonoid and terpenoid metabolism. Plant Physiology and Biochemistry, 47, 551-561.
  11. Olofsson, L., Engström, A., Lundgren, A. & Brodelius, P.E. (2011). Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L, BMC Plant Biology, 45, 1-12.
  12. Olsson, M.E., Olofsson, L.M., Lindahl, A.L., Lundgren, A., Brodelius, M. & Brodelius, P. E. (2009). Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L. Phytochemistry, 70, 1123–1128.
  13. Petroni, K., Falasca, G., Calvenzani, V., Allegra, D., Stolfi, C., Fabrizi, L., Altamura, M.M. & Tonelli, C. (2008).  The AtMYB11 gene from Arabidopsis is expressed in meristematic cells and modulates growth in planta and organogenesis in vitro. Journal of Experimental Botany, 59, 1201-1213.
  14. Ranjabar, M., Naghavi, M.R., Alizadeh, H. (2014). Comparative analysis of ADS gene promoter in seven Artemisia species. Journal of Genetics, 93. (in Press).
  15. Rashidi Monfared, S., Naghavi, M.R., Yazdi samadi, B., Alizadeh, H. & Soltanlou, H. (2011). Study of expression regulation of the key gene ADS of artemisinin biosynthesis pathway in different Iranian Artemisia annua genotypes. Iranian Journal of Field Crop Science, 42(2), 409-419.
  16. Sainsbury, F., Thuenemann, E.C., Lomonossoff, G.P. (2009). pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. J Plant Biotechnol, 7, 682-693.
  17. Teoh, K.H., Polichuk, D.R., Reed, D.W. & Covello, P.S. (2009). Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemsia annua. Botany, 87, 635-642.
  18. Vannini, C., Iriti, M., Bracale, M., Locatelli, F., Faoro, F., Croce, P., Pirona, R., Di Maro, A., Coraggio, I. & Genga, A. (2006). The ectopic expression of the rice Osmyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic stresses. Physiology and Molecular Plant Pathology, 69, 26-42.
  19. Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J., 33, 949-956.
  20. Wallaart, T.E., Bouwmeester, H.J., Hille, J., Poppinga, L. & Maijers, N.C. (2001). Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta, 212, 460-465.
  21. Wang,  J.W., Zheng, L.P., Zhang, B. & Zou, T. (2009). Stimulation of artemisinin synthesis by combined  cerebroside and nitric oxide elicitation in Artemisia annua hairy roots. Appl Microbiol Biotechnol, 85, 285-292.
  22. Weathers, P.J., Bunk, G. & Mccoy, M.C. (1997).  Effects of gibberellic acid on hairy root cultures of Artemisia annua: growth and artemisinin production. In Vitro Cellular & Developmental Biology – Plant, 41, 47-53.
  23. White, N.J. (2004). Anti-malarial drug resistance. The Journal of Clinical Investigation, 113, 1084-1092.
  24. Wray, G., Matthew, A., Hahn, W. Abouheif, E., Balhoff, J.P., Pizer M., Rockman, M.V. & Romano, L.A. (2003).  The Evolution of Transcriptional Regulation in Eukaryotes. Molecular Biology Evolution, 20, 1377-1419.
  25. Yanhui, C., Xiaoyuan, Y., Kun, H., Meihua, L., Jigang, L., Zhaofeng, G., Zhiqiang, L., Yunfei, Z., Xiaoxiao, W., Xiaoming, Q., Yunping, S., Li, Z., Xiaohui, D., Jingchu, L., Xing-Wang, Chen, D., Gu Hongya, Z. & Li-Jia, Q. (2006) .The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Molecular Biology, 60, 107-124.
Volume 46, Issue 3 - Serial Number 3
October 2015
Pages 339-349
  • Receive Date: 15 September 2014
  • Revise Date: 21 December 2015
  • Accept Date: 14 April 2015
  • Publish Date: 23 September 2015