Chemotype-dependent Metabolic Response to Methyl Jasmonate Elicitation in Artemisia annua

Abstract

Considerable difference in artemisinin and its direct precursors, artemisinic acid and dihydroartemisinic acid, was detected between two chemotypes within the species Artemisia annua (A. annua). These two chemotypes showed differential metabolic response to methyl jasmonate (MeJA) elicitation. Exogenous application of MeJA resulted in an accumulation of dihydroartemisinic acid and artemisinin in Type I plants. In Type II plants, however, artemisinic acid and artemisinin level decreased dramatically under MeJA elicitation. Squalene and other sesquiterpenes, (e.g., caryophyllene, germacrene D), were stimulated by MeJA in both chemotypes. The effect of MeJA elicitation was also studied at the transcription level. Real time RT-PCR analysis showed a coordinated activation of most artemisinin pathway genes by MeJA in Type I plants. The lack of change in cytochrome P450 reductase (CPR) transcript in Type I plants indicates that the rate-limiting enzymes in artemisinin biosynthesis have yet to be identified. Other chemotype-specific electron donor proteins likely exist in A. annua to meet the demand for P450-mediated reactions in MeJA-mediated cellular processes. In Type II plants, mRNA expression patterns of most pathway genes were consistent with the reduced artemisinin level. Intriguingly, the mRNA transcript of aldehyde dehydrogenase1 (ADHL1), an enzyme which catalyzes the oxidation of artemisinic and dihydroartemisinic aldehydes, was upregulated by MeJA. The differential metabolic response to MeJA suggests a chemotype-dependent metabolic flux control towards artemisinin and sterol production in the species A. annua.

Key words

Artemisia annua L. - Asteraceae - methyl jasmonate - chemotype - GC-MS - real time RT-PCR

References

• 1 Xiao S. Development of antischistosomal drugs in China, with particular consideration to praziquantel and the artemisinins.  Acta Trop. 2005;  96 153-167
• 2 Romero M R, Efferth T, Serrano M A, Castaño B, Macias R I R, Briz O, Marin J J G. Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an “in vitro” replicative system.  Antiviral Res. 2005;  68 75-83
• 3 Efferth T. Molecular pharmacology and pharmacogenomics of artemisinin and its derivatives in cancer cells.  Curr Drug Targets. 2006;  7 407-421
• 4 Abdin M Z, Israr M, Rehman R U, Jain S K. Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production.  Planta Med. 2003;  69 289-299
• 5 Towler M J, Weathers P J. Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways.  Plant Cell Rep. 2007;  26 2129-2136
• 6 Ro D, Ehlting J, Douglas C J. Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar.  Plant Physiol. 2002;  130 1837-1851
• 7 Teoh K H, Polichuk D R, Reed D W, Nowak G, Covello P S. 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 1411-1416
• 8 Brown G D, Sy L. In vivo transformations of dihydroartemisinic acid in Artemisia annua plants.  Tetrahedron. 2004;  60 1139-1159
• 9 Smith C A, Want E J, O'Maille G, Abagyan R, Siuzdak G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification.  Anal Chem. 2006;  78 779-787
• 10 Bertea C M, Freije J R, van der Woude H, Verstappen F W, Perk L, Marquez V, De Kraker J W, Posthumus M A, Jansen B J, de Groot A, Franssen M C, Bouwmeester H J. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.  Planta Med. 2005;  71 40-47
• 11 Zhang Y, Teoh K H, Read D W, Maes L, Goosens A, Olson D J H, Ross A R, Covello P S. The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua.  J Biol Chem. 2008;  283 21501-21508
• 12 Teoh K H, Polichuk D R, Reed D W, Covello P S. Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua.  Botany. 2009;  87 635-642
• 13 Koda Y. The role of jasmonic acid and related compounds in the regulation of plant development.  Int Rev Cytol. 1992;  135 155-199
• 14 Creelman R A, Mullet J E. Biosynthesis and action of jasmonates in plants.  Plant Mol Biol. 1997;  48 355-381
• 15 Sy L, Brown G D. The mechanism of the spontaneous autoxidation of dihydroartemisinic acid.  Tetrahedron. 2002;  58 897-908
• 16 Mueller M J, Brodschelm W, Spannagl E, Zenk M H. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid.  Proc Natl Acad Sci USA. 1993;  90 7490-7494
• 17 Gundlach H, Müller M J, Kutchan T M, Zenk M H. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures.  Proc Natl Acad Sci USA. 1992;  89 2389-2393
• 18 Baldi A, Dixit V. Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua.  Bioresour Technol. 2008;  99 4609-4614
• 19 De Vos R C, Moco S, Lommen A, Keurentjes J J B, Bino R J, Hall R D. Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry.  Nat Protoc. 2007;  2 778-791
• 20 Sasaki Y, Asamizu E, Shibata D, Nakamura Y, Kaneko T, Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T, Shimada H, Takamiya K, Ohta K, Tabata S. Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways.  DNA Res. 2001;  8 153-161
• 21 Wallaart T E, Pras N, Beekman A C, Quax W J. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes.  Planta Med. 2000;  66 57-62
• 22 Cheng A, Xiang C Y, Li J X, Yang C Q, Hu W L, Wang L J, Lou Y G, Chen X Y. The rice (E)-beta-caryophyllene synthase (OsTPS3) accounts for the major inducible volatile sesquiterpenes.  Phytochemistry. 2007;  68 1632-1641
• 23 Aguilera Y, Dorado M E, Prada F A, Martínez J J, Quesada A, Ruiz-Gutiérrez V. The protective role of squalene in alcohol damage in the chick embryo retina.  Exp Eye Res. 2005;  80 535-543
• 24 Ro D, Paradise E M, Ouellet M, Fisher K J, Newman K L, Ndungu J M, Ho K A, Eachus R A, Ham T S, Kirby J, Chang M C Y, Withers S T, Shiba Y, Sarpong R, Keasling J D. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.  Nature. 2006;  440 940-943
• 25 Koopmann E, Hahlbrock K. Differentially regulated NADPH: cytochrome P450 oxidoreductases in parsley.  Proc Natl Acad Sci USA. 1997;  94 14954-14959
• 26 Mizutani M, Ohta D. Two isoforms of NADPH : cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, heterologous expression in insect cells, and differential regulation.  Plant Physiol. 1998;  116 357-367
• 27 Kirch H H, Dorothea B, Wei Y, Schnable P S, Wood A J. The ALDH gene superfamily of Arabidopsis.  Trends Plant Sci. 2004;  9 371-377