Isolation and characterization of AaAER, a novel double bond reductase from Artemisia annua

  • Yu-Kun Wei
  • Jian-Xu Li
  • Wen-Li Hu
  • Chang-Qing Yang
  • Ling-Jian Wang
Original Article


We isolated a 2-alkenal reductase (AaAER) gene from Artemisia annua L. cDNA library through homologous cloning strategy. Enzymatic properties of AaAER, including substrate selectivity, enzyme kinetics, and key factors affecting enzyme activity, were characterized in vitro. We found that AaAER mainly functions in catalyzing the reduction of adjacent straight chain “C = C” double bond in the carbonyl group. This suggests the possibility of its participation in downstream events of the HPL (hydroperoxide lyase) pathway and contribution to cell detoxification in plants. Although, AaAER shares some substrates with AaDbr 1 and AaDbr 2, which are involved in artemisinin biosynthesis, AaAER was unable to reduce the adjacent double bond of carbonyl group in artemisinic aldehyde. We also analyzed the possible causes for highly homologous reductases with different types of substrates and non-homologous enzymes sharing the same substrate. The identification of a novel double bond reductase in A. annua provides an opportunity to explore the mechanism underlying the detoxification of aldehyde ketone molecules, which are generated by HPL downstream pathway and the redox process in the metabolic pathway of artemisinin synthesis.


Artemisia annuaDetoxification Double bond reductase Enzyme activity Reactive carbonyls 



Methyl jasmonate


Salicylic acid


Rapid amplification of cDNA ends


2-alkenal reductase of Artemisia annua


Double bond reductase 1


Artemisinic aldehyde Δ11(13) reductase


Molecular evolutionary genetics analysis

Supplementary material

13562_2014_278_MOESM1_ESM.doc (32 kb)
Table S1 The primers used in this study (DOC 32 kb)
13562_2014_278_Fig3_ESM.gif (190 kb)
Fig. S1

The alignment of amino acid sequences of AaAER and its homologs. The boxed AXXGXXG is NADP + binding site. Three glycines indicated with arrowheads are key sites of the active enzyme with high-level structure. (GIF 189 kb)

13562_2014_278_MOESM2_ESM.tif (256 kb)
High resolution image (TIFF 256 kb)
13562_2014_278_Fig4_ESM.gif (91 kb)
Fig. S2

Inducible expression and purification of AaAER. (a) In vitro Inducible expression and purification of AaAER protein. Lane 1, in vitro total AaAER protein; lane 2, empty vector; lane 3, 10 mM imidazol elution; lane 4–5, 20 mM imidazol elution; lane 6–8 250 mM imidazol elution; Lane 9, protein marker. (b) Western blotting data further identified target proteins. Data showed the protein size of the target is around 55 kD. Lane 1, protein target; lane 2, protein marker. (GIF 91 kb)

13562_2014_278_MOESM3_ESM.tif (1.7 mb)
High resolution image (TIFF 1722 kb)
13562_2014_278_Fig5_ESM.gif (220 kb)
Fig. S3

GC-MS analyses of the reaction between AaAER and 2-nonenal. Reduction activity of AaAER was identified in reaction systems with inactive proteins and without NADPH. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (2-nonanal). b, Chromatogram of standard (2-nonanal). (GIF 219 kb)

13562_2014_278_MOESM4_ESM.tif (3 mb)
High resolution image (TIFF 3063 kb)
13562_2014_278_Fig6_ESM.gif (162 kb)
Fig. S4

GC-MS analyses of the reaction between AaAER and 3-nonen-2-one. Reduction activity of AaAER was identified in reaction systems with inactived proteins. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (2-nonanone). b, Chromatogram of standard (2-nonanone). (GIF 161 kb)

13562_2014_278_MOESM5_ESM.tif (3.3 mb)
High resolution image (TIFF 3333 kb)
13562_2014_278_Fig7_ESM.gif (172 kb)
Fig. S5

GC-MS analyses of the reaction between AaAER and cinnamaldehyde. Reduction activity of AaAER was identified in reaction systems with inactivate proteins. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (hydrocinnamaldehyde). b, Chromatogram of standard (hydrocinnamaldehyde). (GIF 172 kb)

13562_2014_278_MOESM6_ESM.tif (3.4 mb)
High resolution image (TIFF 3517 kb)
13562_2014_278_Fig8_ESM.gif (198 kb)
Fig. S6

GC-MS analyses of the reaction between AaAER and trans-2, cis-6-nonadienal. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (6-nonenal). b, Chromatogram of standard (6-nonenal). (GIF 198 kb)

13562_2014_278_MOESM7_ESM.tif (3.2 mb)
High resolution image (TIFF 3249 kb)
13562_2014_278_Fig9_ESM.gif (14 kb)
Fig. S7

Effect of temperature on AaAER activity. Reactions with 3-nonen-2-one as a substrate were carried out in HEPES buffer at pH 6.0. The curve of reaction rate against the temperature. (GIF 13 kb)

13562_2014_278_MOESM8_ESM.tif (16.5 mb)
High resolution image (TIFF 16899 kb)
13562_2014_278_Fig10_ESM.gif (12 kb)
Fig. S8

Effects of pH on AaAER activity. Reactions were carried out in HEPES buffer at 37 °C. The curve of reaction rate against pH. (GIF 12 kb)

13562_2014_278_MOESM9_ESM.tif (16.1 mb)
High resolution image (TIFF 16437 kb)
13562_2014_278_Fig11_ESM.gif (28 kb)
Fig. S9

Effects of metal ions on AaAER activity. Comparison of reaction rate with different metal ions. HEPES buffer without metal ions was used as a control. Reactions with 3-nonen-2-one as a substrate were carried out at 37 °C and pH 6.0. The difference in the average reaction rate of different metal ions was evaluated by Dunnett’s Multiple Comparison Test. ** and *** indicate different degrees of difference compared to controls. (GIF 28 kb)

13562_2014_278_MOESM10_ESM.tif (15.2 mb)
High resolution image (TIFF 15612 kb)


  1. Chehab EW, Perea JV, Gopalan B, Theg S, Dehesh K (2007) Oxylipin pathway in rice and Arabidopsis. J Integr Plant Biol 49:43–51CrossRefGoogle Scholar
  2. Dick RA, Kwak M, Sutter TR, Kensler TW (2001) Antioxidative function and substrate specificity of NAD(P)H dependent alkenal/one oxidoreductase. J Biol Chem 276:40803–40810PubMedCrossRefGoogle Scholar
  3. Farmer EE, Ryan C (1990) A Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci U S A 87:7713–7716PubMedCentralPubMedCrossRefGoogle Scholar
  4. Hernandez JA, Corpas FJ, Gomez M, del Río LA, Sevilla F (1993) Salt induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria. Physiol Plant 89:103–110CrossRefGoogle Scholar
  5. Hirata T, Tamura Y, Yokobatake N, Shimoda K, Ashida Y (2000) A 38 kDa allylic alcohol dehydrogenase from the cultured cells of Nicotiana tabacum. Phytochemistry 55:297–303PubMedCrossRefGoogle Scholar
  6. Mano J, Torii Y, Hayashi S, Takimoto K, Matsui K, Nakamura K, Inze D, Babiychuk E, Kushnir S, Asada K (2002) The NADPH: quinone oxidoreductase P1-ζ-crystallin in Arabidopsis catalyzes the α, β-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes. Plant Cell Physiol 43:1445–1455PubMedCrossRefGoogle Scholar
  7. Mano J, Belles-Boix E, Babiychuk E, Inze D, Torii Y, Hiraoka E, Takimoto K, Slooten L, Asada K, Kushnir S (2005) Protection against photooxidative injury of tobacco leaves by 2-alkenal reductase. detoxication of lipid peroxide-derived reactive carbonyls. Plant Physiol 139:1773–1783PubMedCentralPubMedCrossRefGoogle Scholar
  8. Moran JF, Becana M, Iturbe-Ormaetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P (1994) Drought induces oxidative stress in pea plants. Planta 194:346–352CrossRefGoogle Scholar
  9. Nordling E, Jornvall H, Persson B (2002) Medium-chain dehydrogenases/reductases (MDR) Family characterizations including genome comparisons and active site modelling. Eur J Biochem 269:4267–4276PubMedCrossRefGoogle Scholar
  10. Prasad TK, Anderson MD, Martin BA, Stewart CR (1994) Evidence for chilling induced oxidative stress in maize seedlings and regulatory role of hydrogen peroxide. Plant Cell 6:65–74PubMedCentralPubMedCrossRefGoogle Scholar
  11. Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr Opin Plant Biol 1:404–411PubMedCrossRefGoogle Scholar
  12. Ringer KL, McConkey ME, Davis EM, Rushing GW, Croteau R (2003) Monoterpene double-bond reductases of the (−)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (−)-isopiperitenone reductase and (+)-pulegone reductase of peppermint. Arch Biochem Biophys 418:80–92PubMedCrossRefGoogle Scholar
  13. Riveros-Rosas H, Julian-Sanchez A, Villalobos-Molina R, Pardo JP, Pina E (2003) Diversity, taxonomy and evolution of medium-chain dehydrogenase/reductase superfamily. Eur J Biochem 270:3309–3334PubMedCrossRefGoogle Scholar
  14. Strassner J, Schaller F, Frick UB, Howe GA, Weiler EW, Amrhein N, Macheroux P, Schaller A (2002) Characterization and cDNA-microarray expression analysis of 12-oxophytodienoate reductases reveals differential roles for octadecanoid biosynthesis in the local versus the systemic wound response. Plant J 32:585–601PubMedCrossRefGoogle Scholar
  15. Takeuchi Y, Fukumoto R, Kasahara H, Sasaki T, Mitsutoshi K (1995) Peroxidation of lipids and growth inhibition induced by UV-B irradiation. Plant Cell Rep 14:566–570PubMedCrossRefGoogle Scholar
  16. Uchida K (2003) 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 42:318–343PubMedCrossRefGoogle Scholar
  17. Youn B, Kim S, Moinuddin SGA, Lee C, Bedgar DL, Harper AR, Davin LB, Lewis NG, Kang CH (2006) Mechanistic and structural studies of apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase At5g16970. J Biol Chem 281:40076–40088PubMedCrossRefGoogle Scholar
  18. Zhang Y, Teoh KH, Reed DW, Maes L, Goossens A, Olson DJH, Ross ARS, Covello PS (2008) The molecular cloning of artemisinic aldehyde 11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem 283:21501–21508PubMedCrossRefGoogle Scholar
  19. Zhang Y, Teoh KH, Reed DW, Covello PS (2009) Molecular cloning and characterization of Dbr1, a 2-alkenal reductase from Artemisia annua. Botany 87:643–649CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2014

Authors and Affiliations

  • Yu-Kun Wei
    • 1
  • Jian-Xu Li
    • 2
  • Wen-Li Hu
    • 2
  • Chang-Qing Yang
    • 2
  • Ling-Jian Wang
    • 2
  1. 1.Shanghai Chenshan Plant Science Research Center, Chinese Academy of SciencesShanghai Chenshan Botanical GardenShanghaiPeople’s Republic of China
  2. 2.Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiPeople’s Republic of China

Personalised recommendations