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Functional characterization of a stilbene synthase gene using a transient expression system in planta

  • Genetic Transformation and Hybridization
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Abstract

The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.

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Abbreviations

RS :

Resveratrol synthase

CHS :

Chalcone synthase

STS :

Stilbene synthase

References

  • Austin MB, Noel JP (2003) The chalcone synthase superfamily of type III polyketide synthase. Nat Prod Rep 20:79–110

    Article  PubMed  CAS  Google Scholar 

  • Austin MB, Bowman ME, Ferrer JL, Schröder J, Noel JP (2004) An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chem Biol 11:1179–1194

    Article  PubMed  CAS  Google Scholar 

  • Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Courteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, De Cabo R, Sinclair DA (2006) Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444:337–342

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Burger C, Sabine R, Benveniste P, Schaller H (2003) Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14α-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L. J Exp Bot 54:1675–1683

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  PubMed  CAS  Google Scholar 

  • Hain R, Bieseler B, Kindl H, Schröder G, Stöcker R (1990) Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol. Plant Mol Biol 15:325–335

    Article  PubMed  CAS  Google Scholar 

  • Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA (2005) Transient expression vectors for functional genomics quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13

    Article  PubMed  Google Scholar 

  • Hipskind JD, Pavia NL (2000) Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phomo medicaginis. Mol Plant Microbe Interact 13:551–562

    Article  PubMed  CAS  Google Scholar 

  • Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    Article  PubMed  CAS  Google Scholar 

  • La Torre GL, Lagana G, Bellocco E, Vilasi F, Salvo F, Dugo G (2004) Improvement on enzymatic hydrolysis of resveratrol glucosides in wine. Food Chem 85:259–266

    Article  CAS  Google Scholar 

  • Lanz T, Schröder G, Schröder J (1990) Differential regulation of genes for resveratrol synthase in cell cultures of Arachis hypogaea. Planta 181:169–175

    Article  CAS  Google Scholar 

  • Lanz T, Tropf S, Marner FJ, Schröder J, Schröder G (1991) The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways. J Biol Chem 266:9971–9976

    PubMed  CAS  Google Scholar 

  • Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci USA 101:6852–6857

    Article  PubMed  CAS  Google Scholar 

  • Medina-Bolivar F, Cramer C (2004) Production of recombinant protein by hairy roots cultured in plastic sleeve bioreactor. In: Balbas P, Lorence A (eds) Recombinant gene expression: reviews and protocols, vol 267. Humana Press, Totowa, pp 351–363

    Google Scholar 

  • Medina-Bolivar F, Condori J, Rimando AM, Hubstenberger J, Shelton K, O’Keefe SF, Bennett S, Dolan MC (2007) Production and secretion of resveratrol in hairy root cultures of peanut. Phytochemistry 68:1992–2003

    Article  PubMed  CAS  Google Scholar 

  • Medrano G, Reidy MJ, Liu J, Ayala J, Dolan MC, Cramer CL (2009) Rapid system for evaluating bioproduction capacity of complex pharmaceutical proteins in plants. In: Faye L, Gomord V (eds) Recombinant proteins from plants: methods and protocols, vol 483. Humana Press, USA, pp 51–67

    Chapter  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36

    Article  PubMed  Google Scholar 

  • Püssa T, Floren J, Kuldkepp P, Raal A (2006) Survey of grapevine Vitis vinifera stem polyphenols by liquid chromatography–diode array detection–tandem mass spectrometry. J Agric Food Chem 54:7488–7494

    Article  PubMed  Google Scholar 

  • Roupe KA, Remsberg CM, Yañez JA, Davies NM (2006) Pharmacometrics of stilbenes: seguing towards the clinic. Curr Clin Pharmacol 1:81–101

    Article  PubMed  CAS  Google Scholar 

  • Schröder J, Schröder G (1990) Stilbene and chalcone synthases: related enzymes with key function in plant-specific pathways. Z Naturforsch 45c:1–8

    Google Scholar 

  • Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C (1994) Cloning and molecular analysis of structural genes for flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 24:743–755

    Article  PubMed  CAS  Google Scholar 

  • Yu CKY, Shih CH, Chu IK, Lo C (2008) Accumulation of trans-piceid in sorghum seedling infected with Colletotrichum sublineolum. Phytochemistry 69:700–706

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Li SZ, Li J, Pan X, Cahoon RE, Jaworski JG, Wang X, Jez JM, Cheng F, Yu O (2006) Using unnatural protein fusion to engineer resveratrol biosynthesis in yeast and mammalian cells. J Am Chem Soc 128:13030–13031

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Arkansas Biosciences Institute, National Science Foundation-EPSCoR (grant # EPS-0701890; Center for Plant-Powered Production-P3), Arkansas ASSET Initiative and the Arkansas Science and Technology Authority.

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Authors and Affiliations

  1. Arkansas Biosciences Institute, Arkansas State University, State University, P.O. Box 639, AR, 72467, USA

    Jose Condori, Giuliana Medrano, Ganapathy Sivakumar, Vipin Nair, Carole Cramer & Fabricio Medina-Bolivar

  2. Department of Biological Sciences, Arkansas State University, State University, AR, 72467, USA

    Fabricio Medina-Bolivar

Authors
  1. Jose Condori
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  2. Giuliana Medrano
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  3. Ganapathy Sivakumar
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  4. Vipin Nair
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  5. Carole Cramer
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  6. Fabricio Medina-Bolivar
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Corresponding author

Correspondence to Fabricio Medina-Bolivar.

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Communicated by G. Phillips.

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Condori, J., Medrano, G., Sivakumar, G. et al. Functional characterization of a stilbene synthase gene using a transient expression system in planta . Plant Cell Rep 28, 589–599 (2009). https://doi.org/10.1007/s00299-008-0664-0

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  • DOI: https://doi.org/10.1007/s00299-008-0664-0

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