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Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis

Plant Cell Reports volume 31, pages 1759–1768 (2012)Cite this article

Abstract

Agarwood is a high-valued woody material for medicine, perfume, and incense production in Asia, Middle East, and Europe. The wild resources of agarwood-producing tree species, e.g., Aquilaria sinensis have been greatly threatened. The formation of agarwood is considered to be associated with the plant stress and defensive responses, thus it would be urgent and significant to investigate the molecular mechanism of these species responding to a variety of stresses. This is the first report regarding the reference gene selection of Aquilaria species for studying the molecular mechanism of stress-related agarwood production. Candidate reference genes were selected according to previous reports and the sequences were obtained from the 454 EST library of A. sinensis. To obtain the robust genes, we applied three independent programs depending on distinct assumptions and combined these results by a rank aggregation algorithm. The result supports tubulin, ribosomal protein, and glyceraldehyde-3-phosphate dehydrogenase to be the most stable reference genes for quantification of target gene expression in the overall samples examined. Validation of these genes through normalizing the expression of a terpene synthase demonstrated that these three genes are reliable. The selective usage of three algorithms based on their characteristics was underlined. However, more robust genes could be identified if the results of all algorithms were combined by a proper method such as the rank aggregation algorithm.

Key message Reference genes which are critical in gene expression studies are recommended for future molecular studies of stress response and agarwood production in the endangered Aquilaria and other tree species.

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References

  • Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64(15):5245–5250

    PubMed  Article  CAS  Google Scholar 

  • Banerjee AK, Laya MS, Poon PS (2006) Sesquiterpenes classified as phytoalexins. In: Atta-ur R (ed) Studies in natural products chemistry, vol 33, part 13. Elsevier, Amsterdam, pp 193–237

  • Bustin S (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29(1):23–39

    PubMed  Article  CAS  Google Scholar 

  • Bustin SA, Nolan T (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15(3):155–166

    PubMed  Google Scholar 

  • Chen L, Zhong HY, Kuang JF, Li JG, Lu WJ, Chen JY (2011) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234(2):377–390

    PubMed  Article  CAS  Google Scholar 

  • Cordoba EM, Die JV, Gonzalez-Verdejo CI, Nadal S, Roman B (2011) Selection of reference genes in Hedysarum coronarium under various stresses and stages of development. Anal Biochem 409(2):236–243

    PubMed  Article  CAS  Google Scholar 

  • Die JV, Roman B, Nadal S, Gonzalez-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232(1):145–153

    PubMed  Article  CAS  Google Scholar 

  • Exposito-Rodriguez M, Borges AA, Borges-Perez A, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131

    PubMed  Article  Google Scholar 

  • Feng J, Gan B, Meng H, Wei J, Yang Y, Zhang Z (2010) Introducing eaglewood on Aquilaria sinensis tree involves using transfusion method, creating holes on trunk of Aquilaria sinensis tree, dripping eaglewood aromatizing agent slowly into holes and then sealing holes. CN101755629-A

  • Gershenzon J, Dudareva N (2007) The function of terpene natural products in the natural world. Nat Chem Biol 3(7):408–414

    PubMed  Article  CAS  Google Scholar 

  • Gonçalves S, Cairney J, Maroco J, Oliveira M, Miguel C (2005) Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta 222(3):556–563

    PubMed  Article  Google Scholar 

  • Hacquard S, Veneault-Fourrey C, Delaruelle C, Frey P, Martin F, Duplessis S (2011) Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves. Physiol Mol Plant Pathol 75(3):106–112

    Article  CAS  Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6(10):986–994

    PubMed  Article  CAS  Google Scholar 

  • Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112

    PubMed  Article  Google Scholar 

  • Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6(4):279–284

    PubMed  Article  CAS  Google Scholar 

  • Ishihara M, Tsuneya T, Uneyama K (1993) Sesquiterpene constituents in agarwood. 4. Fragrant sesquiterpenes from agarwood. Phytochemistry 33(5):1147–1155

    Article  CAS  Google Scholar 

  • Iskandar HM, Simpson RS, Casu RE, Bonnett GD, Maclean DJ, Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression. Plant Mol Biol Rep 22(4):325–337

    Article  CAS  Google Scholar 

  • Ito M, Honda G (2008) Agarwood—its sedative effect on mice and current state in the production sites. Aromather Res 9(2):122–127

    CAS  Google Scholar 

  • Ito M, Okimoto K, Yagura T, Honda G, Kiuchi F, Shimada Y (2005) Induction of sesquiterpenoid production by methyl jasmonate in Aquilaria sinensis cell suspension culture. J Essent Oil Res 17(2):175–180

    Article  Google Scholar 

  • Itoh T, Tabata Y, Widjaja E, Mulyaningsih T, Wiriadinata H, Parman, Mandang YI (2002) Structure and artificial induction of aloe wood. IAWA J 23(4):466–467

    Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345(2):646–651

    PubMed  Article  CAS  Google Scholar 

  • Kakino M, Tazawa S, Maruyama H, Tsuruma K, Araki Y, Shimazawa M, Hara H (2010) Laxative effects of agarwood on low-fiber diet-induced constipation in rats. BMC Complement Altern Med 10:68

    PubMed  Article  Google Scholar 

  • Kumeta Y, Ito M (2010) Characterization of delta-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood. Plant Physiol 154(4):1998–2007

    PubMed  Article  CAS  Google Scholar 

  • Maldonado-Bonilla L, Betancourt-Jiménez M, Lozoya-Gloria E (2008) Local and systemic gene expression of sesquiterpene phytoalexin biosynthetic enzymes in plant leaves. Eur J Plant Pathol 121(4):439–449

    Article  CAS  Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    PubMed  Article  Google Scholar 

  • Mascia T, Santovito E, Gallitelli D, Cillo F (2010) Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Mol Plant Pathol 11(6):805–816

    PubMed  CAS  Google Scholar 

  • Nakanishi T, Yamagata E, Yoneda K, Miura I (1981) Jinkohol, a prezizane sesquiterpene alcohol from agarwood. Phytochemistry 20(7):1597–1599

    Article  CAS  Google Scholar 

  • Ng LT, Chang YS, Kadir AA (1997) A review on agar (gaharu) producing Aquilaria species. J Trop For Prod 2(2):272–285

    Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56(421):2907–2914

    PubMed  Article  CAS  Google Scholar 

  • Nobuchi T, Siripatanadilok S (1991) Preliminary observation of Aquilaria crassna wood associated with the formation of aloeswood. Bull Kyoto Univ For 63:226–235

    Google Scholar 

  • Okudera Y, Ito M (2009) Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures. Plant Biotechnol 26(3):307–315

    Article  CAS  Google Scholar 

  • Oldfield S, Lusty C, MacKinven A (1998) The world list of threatened trees. World conservation press, Cambridge

    Google Scholar 

  • Persoon GA (2008) Growing ‘the wood of the gods’: agarwood production in Southeast Asia. In: Smallholder tree growing for rural development and environmental services: lessons from Asia, vol 5, pp 245–262

  • Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515

    PubMed  Article  CAS  Google Scholar 

  • Pihur V, Datta S, Datta S (2009) RankAggreg, an R package for weighted rank aggregation. BMC Bioinforma 10(1):62

    Article  Google Scholar 

  • Pojanagaroon S, Kaewrak C (2005) Mechanical methods to stimulate aloes wood formation in Aquilaria crassna Pierre ex H.Lec. (Kritsana) trees. In: WOCMAP III: conservation, cultivation and sustainable use of MAPs (676), pp 161–166

  • Qi SY, He ML, Lin LD, Zhang CH, Hu LJ, Zhang HZ (2005) Production of 2-(2-phenylethyl) chromones in cell suspension cultures of Aquilaria sinensis. Plant Cell Tissue Organ Cult 83(2):217–221

    Article  CAS  Google Scholar 

  • Rao KR, Dayal R (1992) The secondary xylem of Aquilaria agallocha (Thymelaeaceae) and the formation of agar. Iawa Bull 13(2):163–172

    Google Scholar 

  • Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27

    PubMed  Article  Google Scholar 

  • Stolf-Moreira R, Lemos EGDM, Abdelnoor RV, Beneventi MA, Rolla AAP, Pereira SD, de Oliveira MCN, Nepomuceno AL, Marcelino-Guimaraes FC (2011) Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean. Pesquisa Agropecuaria Brasileira 46(1):58–65

    Article  Google Scholar 

  • Takemoto H, Ito M, Shiraki T, Yagura T, Honda G (2008) Sedative effects of vapor inhalation of agarwood oil and spikenard extract and identification of their active components. J Nat Med 62(1):41–46

    PubMed  Article  CAS  Google Scholar 

  • Tong Z, Gao Z, Wang F, Zhou J, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10:71

    PubMed  Article  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):res34

    Google Scholar 

  • VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44(5):619–626

    PubMed  Article  CAS  Google Scholar 

  • Wan HJ, Zhao ZG, Qian CT, Sui YH, Malik AA, Chen JF (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399(2):257–261

    PubMed  Article  CAS  Google Scholar 

  • Wijitphan P (2009) Stimulating Aquilaria resin formation by making wounds on Aquilaria trunk, comprises making wounds on stump of cut down Aquilaria for forming rectangular shaped wounds, and drilling holes within rectangular wound deep into heartwood. US7485309-B1

  • Yagura T, Ito M, Kiuchi F, Honda G, Shimada Y (2003) Four new 2-(2-phenylethyl)chromone derivatives from withered wood of Aquilaria sinensis. Chem Pharm Bull (Tokyo) 51(5):560–564

    Article  CAS  Google Scholar 

  • Yang Y, Shah J, Klessig DF (1997) Signal perception and transduction in plant defense responses. Gene Dev 11(13):1621–1639

    PubMed  Article  CAS  Google Scholar 

  • Zhong HY, Chen JW, Li CQ, Chen L, Wu JY, Chen JY, Lu WJ, Li JG (2011) Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions. Plant Cell Rep 30(4):641–653

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81001607) and the Program for New Century Excellent Talents in University Funded by the Ministry of Education of China (No. 2008).

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Affiliations

  1. Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medicinal College, Malianwabei Road, Beijing, 100193, China

    Zhi-Hui Gao, Jian-He Wei, Zheng Zhang & Wen-Ting Zhao

  2. Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Science, Wanning, 571533, China

    Jian-He Wei, Yun Yang & Zheng Zhang

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  1. Zhi-Hui Gao
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  2. Jian-He Wei
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  5. Wen-Ting Zhao
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Correspondence to Jian-He Wei.

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Communicated by C. Quiros.

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Gao, ZH., Wei, JH., Yang, Y. et al. Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis . Plant Cell Rep 31, 1759–1768 (2012). https://doi.org/10.1007/s00299-012-1289-x

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  • DOI: https://doi.org/10.1007/s00299-012-1289-x

Keywords

  • Quantitative gene expression
  • Reference gene
  • Agarwood
  • Aquilaria sinensis
  • Stress