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Terpenoid metabolic profiling analysis of transgenic Artemisia annua L. by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

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Abstract

Amorpha-4,11-diene synthase (ADS) is a very important enzyme which catalyzes the committed step of artemisinin biosynthesis. In this work, two lines of transgenic Artemisia annua L. which ADS was over-expressed (line A9) and suppressed (line Amsi), respectively, were utilized. And the transgenic line GUS with β-Glucuronidase gene was regarded as the control. Their terpenoid metabolic profiling was investigated by using GC × GC–TOFMS. The metabolic profiling method established included simple extraction, two-dimension separation and multivariate analysis. Partial least squares discriminant analysis (PLS-DA) was used to classify two transgenic lines and the control line. Eleven important compounds in classification were identified. Most of them were sesquiterpenoids including monoterpenoid, diterpenoid and four bioprecursors of artemsisnin. Compared with the control, artemisinin and bioprecursors in the line A9 increased as a result of over-expressing ADS. Borneol and phytol also increased in the line A9, but (E)-β-farnesene and germacrene D were reversely altered. The result indicated that over-expression of the ADS affected not only artemisinin biosynthesis, but also the whole metabolic network of terpenoid. Compared with the line A9, no opposite change of artemisinin and related derivatives was observed in the line Amsi, the ADS inhibition had no significant effect on artemisinin biosynthesis in the line Amsi.

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References

  • Abdin, M. Z., Israr, M., Rehman, R. U., & Jain, S. K. (2003). Artemisinin, a novel antimalarial drug: Biochemical and molecular approaches for enhanced production. Planta Medica, 69, 289–299.

    Article  CAS  PubMed  Google Scholar 

  • Adahchour, M., Beens, J., & Brinkman, U. A. T. (2008). Recent developments in the application of comprehensive two-dimensional gas chromatography. Journal of Chromatography. A, 1186, 67–108.

    Article  CAS  PubMed  Google Scholar 

  • Bertea, C. M., Freije, J. R., van der Woude, H., Verstappen, F. W., Perk, L., Marquez, V., et al. (2005). Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Medica, 71, 40–47.

    Article  CAS  PubMed  Google Scholar 

  • Bhandari, P., Gupta, A. P., Singh, B., & Kaul, V. K. (2005). Simultaneous densitometric determination of artemisinin, artemisinic acid and arteannuin-B in Artemisia annua using reversed-phase thin layer chromatography. Journal of Separation Science, 28, 2288–2292.

    Article  CAS  PubMed  Google Scholar 

  • Bijlsma, S., Bobeldijk, L., Verheij, E. R., Ramaker, R., Kochhar, S., Macdonald, I. A., et al. (2006). Large-scale human metabolomics studies: A strategy for data (pre-) processing and validation. Analytical Chemistry, 78, 567–574.

    Article  CAS  PubMed  Google Scholar 

  • Bouwmeester, H. J., Wallaart, T. E., Janssen, M. H. A., van Loo, B., Jansen, B. J. M., Posthumus, M. A., et al. (1999). Amorpha-4, 11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry, 52, 843–854.

    Article  CAS  PubMed  Google Scholar 

  • Brown, G. D., & Sy, L. K. (2004). In vivo transformations of dihydroartemisinic acid in Artemisia annua plants. Tetrahedron, 60, 1139–1159.

    Article  CAS  Google Scholar 

  • Brown, G. D., & Sy, L. K. (2007). In vivo transformations of artemisinic acid in Artemisia annua plants. Tetrahedron, 63, 9548–9566.

    Article  CAS  Google Scholar 

  • Chen, H. L., Wang, K. T., Pu, Q. S., Chen, X. G., & Hu, Z. D. (2002). On-line conversion and determination of artemisinin using a flow-injection capillary electrophoresis system. Electrophoresis, 23, 2865–2871.

    Article  PubMed  Google Scholar 

  • deVries, P. J., & Dien, T. K. (1996). Clinical pharmacology and therapeutic potential of artemisinin and its derivatives in the treatment of malaria. Drugs, 52, 818–836.

    Article  CAS  Google Scholar 

  • Eriksson, L., Johansson, E., Kettaneh-Wold, N., Trygg, J., Wikström, C., & Wold, S. (2006). Mult-and megavariate data analysis—Basic principles and applications. Umeå: Umetrics AB.

    Google Scholar 

  • Han, J. L., Liu, B. Y., Ye, H. C., Wang, H., Li, Z. Q., & Li, G. F. (2006). Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L. Journal of Integrative Plant Biology, 48, 482–487.

    Article  CAS  Google Scholar 

  • Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). Gus fusions-beta-glucuronidase as a sensitive and versatile gene fusion marker in higher-plants. EMBO Journal, 6, 3901–3907.

    CAS  PubMed  Google Scholar 

  • Klayman, D. L. (1985). Qinghaosu (artemisinin): An antimalarial drug from China. Science, 228, 1049–1055.

    Article  CAS  PubMed  Google Scholar 

  • Lindahl, A. L., Olsson, M. E., Mercke, P., Tollbom, O., Schelin, J., Brodelius, M., et al. (2006). Production of the artemisinin precursor amorpha-4, 11-diene by engineered Saccharomyces cerevisiae. Biotechnological Letters, 28, 571–580.

    Article  CAS  Google Scholar 

  • Liu, H. J., Yeh, W. L., & Chew, S. Y. (1993). A total synthesis of the antimalarial natural product (+)-qinghaosu. Tetrahedron Letters, 34, 4435–4438.

    Article  CAS  Google Scholar 

  • Ma, C. F., Wang, H. H., Lu, X., Xu, G. W., & Liu, B. Y. (2008). Metabolic fingerprinting investigation of Artemisia annua L. in different stages of development by gas chromatography and gas chromatography–mass spectrometry. Journal of Chromatography. A, 1186, 412–419.

    Article  CAS  PubMed  Google Scholar 

  • Marriott, P., & Shellie, R. (2002). Principles and applications of comprehensive two-dimensional gas chromatography. TrAC-Trends in Analytical Chemistry, 21, 573–583.

    Article  CAS  Google Scholar 

  • Mohamed, S. S., Khalid, S. A., Ward, S. A., Wan, T. S. M., Tang, H. P. O., Zheng, M., et al. (1999). Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography-mass spectrometry-selected ion monitoring. Journal of Chromatography. B, 731, 251–260.

    Article  CAS  Google Scholar 

  • Mohler, R. E., Dombek, K. M., Hoggard, J. C., Young, E. T., & Synovec, R. E. (2006). Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry analysis of metabolites in fermenting and respiring yeast cells. Analytical Chemistry, 78, 2700–2709.

    Article  CAS  PubMed  Google Scholar 

  • Mutabingwa, T. K. (2005). Artemisinin-based combination therapies (ACTS): Best hope for malaria treatment but inaccessible to the needy!. Acta Tropica, 95, 305–315.

    Article  CAS  PubMed  Google Scholar 

  • Peng, C. Y. A., Ferreira, J. F. S., & Wood, A. J. (2006). Direct analysis of artemisinin from Artemisia annua L. using high-performance liquid chromatography with evaporative light scattering detector, and gas chromatography with flame ionization detector. Journal of Chromatography. A, 1133, 254–258.

    Article  CAS  PubMed  Google Scholar 

  • Ro, D. K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 440, 940–943.

    Article  CAS  PubMed  Google Scholar 

  • Rocha, S. M., Coelho, E., Zrostlikova, J., Delgadillo, I., & Coimbra, M. A. (2007). Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry of monoterpenoids as a powerful tool for grape origin traceability. Journal of Chromatography. A, 1161, 292–299.

    Article  CAS  PubMed  Google Scholar 

  • Shellie, R. A., Welthagen, W., Zrostlikova, J., Spranger, J., Ristow, M., Fiehn, O., et al. (2005). Statistical methods for comparing comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry results: Metabolomic analysis of mouse tissue extracts. Journal of Chromatography. A, 1086, 83–90.

    Article  CAS  PubMed  Google Scholar 

  • Sy, L. K., & Brown, G. D. (1998). Three sesquiterpenes from Artemisia annua. Phytochemistry, 48, 1207–1211.

    Article  CAS  Google Scholar 

  • Sy, L. K., & Brown, G. D. (2001). Deoxyarteannuin B, dihydro-deoxyarteannuin B and tans-5-hydroxy-2-isopropenyl-5-methylhex-3-en-1-ol from Artemisia anuua. Phytochemistry, 58, 1159–1166.

    Article  CAS  PubMed  Google Scholar 

  • Teoh, K. H., Polichuk, D. R., Reed, D. W., Nowak, G., & Covello, P. S. (2006). 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 Letters, 580, 1411–1416.

    Article  CAS  PubMed  Google Scholar 

  • Towler, M. J., & Weathers, P. J. (2007). Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways. Plant Cell Reports, 26, 2129–2136.

    Article  CAS  PubMed  Google Scholar 

  • van Agtmael, M. A., Eggelte, T. A., & van Boxtel, C. J. (1999). Artemisinin drugs in the treatment of malaria: From medicinal herb to registered medication. Trends in Pharmacological Sciences, 20, 199–205.

    Article  PubMed  Google Scholar 

  • Van der Kooy, F., Verpoorte, R., & Meyer, J. J. M. (2008). Metabolomic quality control of claimed anti-malarial Artemisia afra herbal remedy and A. afra and A. annua plant extracts. South African Journal of Botany, 74, 186–189.

    Article  CAS  Google Scholar 

  • VanGeldre, E., Vergauwe, A., & VandenEeckhout, E. (1997). State of the art of the production of the antimalarial compound artemisinin in plants. Plant Molecular Biology, 33, 199–209.

    Article  CAS  Google Scholar 

  • Wallaart, T. E., Pras, N., Beekman, A. C., & Quax, W. J. (2000). Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: Proof for the existence of chemotypes. Planta Medica, 66, 57–62.

    Article  CAS  PubMed  Google Scholar 

  • Wallaart, T. E., van Uden, W., Lubberink, H. G. M., Woerdenbag, H. J., Pras, N., & Quax, W. J. (1999). Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. Journal of Natural Products, 62, 430–433.

    Article  CAS  PubMed  Google Scholar 

  • Wiklund, S., Johansson, E., Sjostrom, L., Mellerowicz, E. J., Edlund, U., Shockcor, J. P., et al. (2008). Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models. Analytical Chemistry, 80, 115–122.

    Article  CAS  PubMed  Google Scholar 

  • Yang, R. Y., Feng, L. L., Yang, S. Q., Yin, L. L., Xu, X. L., & Zeng, Q. P. (2008). Quantitative Transcript profiling reveals down-regulation of a sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants. Planta Medica, 74, 1510–1516.

    Article  CAS  PubMed  Google Scholar 

  • Yang, J., Zhao, X. J., Liu, X. L., Wang, C., Gao, P., Wang, J. S., et al. (2006). High Performance liquid chromatography–mass spectrometry for metabonomics: Potential biomarkers for acute deterioration of liver function in chronic hepatitis B. Journal of Proteome Research, 5(3), 554–561.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, Y., Teoh, K. H., Reed, D. W., Maes, L., Goossens, A., Olson, D. J. H., et al. (2008). The molecular cloning of artemisinic aldehyde delta 11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. Journal of Biological Chemistry, 283, 21501–21508.

    Article  CAS  PubMed  Google Scholar 

  • Zhao, Y. J., Ye, H. C., Li, G. F., Chen, D. H., & Liu, Y. (2003). Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of Artemisia annua L. Chinese Science Bulletin, 48, 63–67.

    Article  CAS  Google Scholar 

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Acknowledgements

This study has been supported by National Basic Research Program of China (2007CB109201), State Key Project for Cultivating New Type of Transgenic Organisms (2008ZX08012-002) and the projects (No. 20775078, 60773164) of National Natural Science Foundation of China.

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Authors and Affiliations

  1. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, 116023, China

    Chenfei Ma, Xin Lu & Guowang Xu

  2. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, 100093, China

    Huahong Wang, Hong Wang & Benye Liu

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  1. Chenfei Ma
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  2. Huahong Wang
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  3. Xin Lu
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  4. Hong Wang
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  5. Guowang Xu
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  6. Benye Liu
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Corresponding authors

Correspondence to Guowang Xu or Benye Liu.

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Chenfei Ma and Huahong Wang contributed equally to this work.

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Ma, C., Wang, H., Lu, X. et al. Terpenoid metabolic profiling analysis of transgenic Artemisia annua L. by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. Metabolomics 5, 497–506 (2009). https://doi.org/10.1007/s11306-009-0170-6

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  • DOI: https://doi.org/10.1007/s11306-009-0170-6

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