DOI QR코드

DOI QR Code

Enhanced biosynthesis of artemisinin by environmental stresses in Artemisia annua

환경스트레스 처리에 의한 개똥쑥 artemisinin 생합성 증진

  • Received : 2022.11.09
  • Accepted : 2022.11.25
  • Published : 2022.12.31

Abstract

Artemisinin is a secondary metabolite of Artemisia annua that shows potent anti-malarial, anti-bacterial, antiviral, and anti-tumor effects. The supply of artemisinin depends on its content in Artemisia annua, in which various environmental factors can affect the plant's biosynthetic yield. In this study, the effects of different light-emitting diode (LED)-irradiation conditions were tested to optimize the germination and growth of Artemisia annua for the enhanced production of artemisinin. Specifically, the ratio between the red and blue lights in the irradiating LED was varied for investigation as follows: [Red : Blue] = [6 : 4], [7 : 3], and [8 : 2]. Furthermore, additional stress factors like UV-B-irradiation (1,395 ㎼/cm2), low temperature (4℃), and dehydration were also explored to induce hormetic expressions of ADS, CYP, and ALDH1, which are essential genes for the biosynthesis of artemisinin. Quantitative polymerase chain reaction (qPCR) was used to analyze the expression levels of the respective genes and their correlation with the specified conditions. [8 : 2] LED-irradiation was the most optimal among the tested conditions for the cultivation of Artemisia annua in terms of both fresh and dry weights post-harvest. For the production of artemisinin, however, [7 : 3] LED-irradiation with dehydration for six hours pre-harvest was the most optimal condition by inducing around twofold enhancement in the biosynthetic yield of artemisinin. As expected, a correlation was observed between the expression levels of the genes and the contents of artemisinin accumulated.

개똥쑥은 말라리아 등 다양한 질병의 치료물질인 artemisinin 제공하나, 식물체 내 농도가 낮고, 생산이 불안정하여 국제적 수요에 대응하지 못하고 있다. 재배환경을 인공적으로 제어하는 식물공장 시스템은 계절이나 장소에 제한 없이 약용식물의 공장식 생산체계가 가능하다(Kim 2010). 본 연구에서는 식물공장에서 개똥쑥의 artemisinin의 대량생산이 가능한 최적의 조건을 찾기 위하여 파종부터 수확까지 적색광(R)과 청색광(B)을 혼합한 3종류의 LED (R : B = 6 : 4, 7 : 3, 8 : 2)에서 생장 및 물질생산에 적합한 광조건을 탐색하였다. 개똥쑥의 수확 전, 1,395 ㎼/cm2의 UV-B, 4℃의 저온, 그리고 건조 처리로 식물에 hormesis를 유도하여 artemisinin의 생산 증가를 확인하였다. Artemisinin 생합성에 관여하는 효소들 중에서 ADS, CYP, ALDH1의 발현량을 qPCR로 측정하였고, artemisinin 정량을 통해 전사체와 대사물질의 연관성을 확인하고, artemisinin 생산에 적합한 재배 광조건과 hormesis 처리 조건을 탐색하였다. 3종의 LED 비율 중 8 : 2에서 높은 생체중 및 건물중을 생산했으며, hormesis를 유도하기 위한 3종의 물리 처리에서 이를 통해 7 : 3 식물을 수확전 6시간 건조처리했을 때 artemisinin 함량이 약 2배 증가하였다.

Keywords

Acknowledgement

본 연구는 IPET ARC 지원사업(단국대 천연물 신의약소재산업화 연구센터)의 일부로 수행되었습니다.

References

  1. Alam P, Abdin MZ (2011) Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content. Plant Cell Rep 30:1919-1928 
  2. Arsenault PR, Vail DR, Wobbe KK, Weathers PJ (2010) Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements. Molecules 15:2302-2318  https://doi.org/10.3390/molecules15042302
  3. Bharati A, Sabat SC (2010) A spectrophotometric assay for quatification of artemisinin. Talanta 82:1033-1037  https://doi.org/10.1016/j.talanta.2010.06.015
  4. Chen Y, Shen Q, Wang Y, Wang T, Wu S, Zhang L, Lu X, Zhang F, Jiang W, Qiu B, Gao E, Sun X, Tang K (2012) The stacked overexresssion of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L. Plant Biotech Rep 7:287-295 
  5. Choi MK, Back GY, Kwan SJ, Yoon YC, Kim HT (2014) Effect of LED light wavelength on growth, vitamin C and anthocyanin contents. Protect Horti and Plant Factory 23(1):19-25  https://doi.org/10.12791/KSBEC.2014.23.1.019
  6. Erum D, Cusido RM, Palazon J, Estrada KR, Bonfill M, Mirza B (2015) Enhanced artemisinin yield by expression of rol genes in Artemisia annua. Malaria J 14:424 
  7. Ferreira JF (2007) Nutrient deficiency in the production of artemisinin, dihydroartemisinic acid, and artemisinic acid in Artemisia annua L. J Agric Food Chem 55(5):1686-1694  https://doi.org/10.1021/jf063017v
  8. Ferreira JFS, Laughlin JC, Delabays N, Magalhaes PM (2005) Cultivation and genetics of Artemisia annua for increased production of the anti-malarial artemisinin. Plant Gen Res 3:206-229  https://doi.org/10.1079/PGR200585
  9. Geldre EV, Vergauwe A, Eekhout EV (1997) State of the art of production of antimalarial compound artemisinin in plants. Plant Mol Biol 33:199-209  https://doi.org/10.1023/A:1005716600612
  10. Kim JH (2010) Trend and direction for plant factory system. J Plant Biotech 37:442-455 
  11. Liu C, Zhao Y, Wang Y (2006) Artemisinin: current state and perspectives for biotechnological production of an antimalarial drug. Appl Microbiol Biotechnol 72(1):11-20  https://doi.org/10.1007/s00253-006-0452-0
  12. Lulu Y, Chang Z, Ying H, Ruiyi Y, Qingqing Z (2008) Abiotic stress-induced expression of artemisinin biosynthesis genes in Artemisia annua L. Chinese J of App and Env Biol 14(1):1-5 
  13. Lv Z, Zhang F, Pan Q, Fu X, Jiang W, Shen Q, Yan T, Shi P, Lu X, Sun X, Tang K (2016) Branch pathway blocking in Artemisia annua is a useful method for obtaining high yield artemisinin Plant & Cell Physiology 57(3):588-602  https://doi.org/10.1093/pcp/pcw014
  14. Marchese JA, Ferreira JFS, Rehder VLG, Rodrigues O (2010) Water deficit effect on the accumulation of biomass and artemisinin in annual wormwood (Artemisia annua L., Asteraceae). Braz J Plant Physiol 22(1):1-9  https://doi.org/10.1590/S1677-04202010000100001
  15. Massa GD, Kim H-H, Wheeler RM, Mitchell CA (2008) Plant productivity in response to LED lighting. Hort Sci 43(7):1951-1956 
  16. Moon BC, Lee YM, Ji Y, Choi G, Chun JM, Kim HK (2013) Molecular authentication and phylogenetic analysis of plant species for Breeae and Cirsii herba based DNA barcodes. Kor J Herbology 28:75-84 
  17. Olofsson L, Lundgren A, Brodelius PE (2012) Trichome isolation with without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L. Plant Science 183:9-13  https://doi.org/10.1016/j.plantsci.2011.10.019
  18. Pandey N, Pandey-Rai S (2015) Updates on artemisinin: an insight to mode of actions and strategies for enhanced global production. Protoplasma 253:15-30  https://doi.org/10.1007/s00709-015-0805-6
  19. Pasternak T, Rudas V, Potteers G, Jansen MAK (2005) Morphogenic effects of abiotic stress: reorientation of growth in Arabidopsis thaliana seedlings. Environ and Exp Botany 53:299-314  https://doi.org/10.1016/j.envexpbot.2004.04.009
  20. Qureshi MI, Israr M, Abdin MZ, Iqbal M (2005) Responses of Artemisia annua L. to lead and salt-induced oxidative stress. Environ Exp Bot 53:185-193  https://doi.org/10.1016/j.envexpbot.2004.03.014
  21. Rai R, Meena RP, Smita SS, Shukla A, Rai SK, Pandey-Rai S (2011) UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis in Artemisia annua L.-an antimalarial plant. J of Photochem and Photobiol B: biol 105(3):216-225  https://doi.org/10.1016/j.jphotobiol.2011.09.004
  22. Vargas-Hernandez M, Macias-Bobadilla I, Guevara-Gonzalez RG, Romero-Gomez S de J, Rico-Garcia E, Ocampo-Verazquez RV, Alvarez-Arquieta L de L, Torres-Pacheco I (2017) Plant hormesis management with biostimulants of biotic origin in agriculture. Front in Plant Sci 8:1762 
  23. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Meth and Appl 18:315-322
  24. Yang R-Y, Zeng X-M, Lu Y-Y, Lu W-J, Feng L-L, Yang X-Q, Zeng Q-P (2010) Senescent leaves of Artemisia annua are one of the most active organs for overexpression of artemisinin biosynthesis responsible genes upon burst of singlet oxygen. Planta Med 2010 76:734-742  https://doi.org/10.1055/s-0029-1240620