Molecular Breeding

, Volume 17, Issue 2, pp 91–99 | Cite as

Heterologous Expression of Two Gentian Cytochrome P450 Genes can Modulate the Intensity of Flower Pigmentation in Transgenic Tobacco Plants

  • Takashi Nakatsuka
  • Masahiro Nishihara
  • Keiichiro Mishiba
  • Saburo Yamamura
Article
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Accepted:

Abstract

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.

Key words

Cytochrome P450 Flavone synthase II Flavonoid 3′-hydroxylase Flavonoid biosynthesis Gentiana triflora Nicotiana tabacum 

Abbreviations

DHK

dihydrokaempferol

DHM

dihydromyricetin

DHQ

dihydroquercetin

F3′H

flavonoid 3′-hydroxylase

FSII

flavone synthase II

HPLC

high performance liquid chromatography

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References

  1. Aida, R., Kishimoto, S., Tanaka, Y., Shibata, M. 2000Modification of flower color in torenia (Torenia fournieri Lind.) by genetic transformationPlant Sci.1533342CrossRefGoogle Scholar
  2. Akashi, T., Aoki, T., Ayabe, S. 1998Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase IIFEBS Lett.431287290PubMedGoogle Scholar
  3. Akashi, T., Fukuchi-Mizutani, M., Aoki, T., Ueyama, Y., Yonekura-Sakakibara, K., Tanaka, Y., Kusumi, T., Ayabe, S. 1999Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavonesPlant Cell Physiol.4011821186PubMedGoogle Scholar
  4. Brugliera, F., Barri-Rewell, G., Holton, T.A., Mason, J.G. 1999Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybridaPlant J.19441451CrossRefPubMedGoogle Scholar
  5. Chapple, C. 1998Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenasesAnnu. Rev. Plant Physiol. Plant Mol. Biol.49311343CrossRefPubMedGoogle Scholar
  6. Fujiwara, H., Tanaka, Y., Yonekura-Sakakibara, K., Fukuchi-Mizutani, M., Nakao, M., Fukui, Y., Yamaguchi, M., Ashikari, T., Kusumi, T. 1998cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana trifloraPlant J.16421431CrossRefPubMedGoogle Scholar
  7. Fukuchi-Mizutani, M., Okuhara, H., Fukui, Y., Nakao, M., Katsumoto, Y., Yonekura-Sakakibara, K., Kusumi, T., Hase, T., Tanaka, Y. 2003Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3′-O-glucosyltransferasea key enzyme for blue anthocyanin biosynthesis, from gentianPlant Physiol.13216521663CrossRefPubMedGoogle Scholar
  8. Goto, T., Kondo, T., Tamura, H., Imagawa, H., Iino, A., Takeda, K. 1982Structure of gentiodelphin, an acylated anthocyanin isolated from Gentiana makinorithat is stable in dilute aqueous solutionTetrahedron Lett.2336953698Google Scholar
  9. Holton, T.A., Brugliera, F., Lester, D.R., Tanaka, Y., Hyland, C.D., Menting, J.G., Lu, C.Y., Farcy, E., Stevenson, T.W., Cornish, E.C. 1993Cloning and expression of cytochrome P450 genes controlling flower colourNature366276279CrossRefPubMedGoogle Scholar
  10. Holton, T.A., Cornish, E.C. 1995Genetics and biochemistry of anthocyanin biosynthesisPlant Cell710711083CrossRefPubMedGoogle Scholar
  11. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eicholtz, D., Rogers, S.G., Fraley, R.T. 1985A simple and general method for transferring genes into plantsScience22712291231Google Scholar
  12. Hosokawa, K., Fukushi, E., Kawabata, J., Fujii, C., Ito, T., Yamamura, S. 1997Seven acylated anthocyanins in blue flowers of GentianaPhytochemistry45167171CrossRefGoogle Scholar
  13. Kaltenbach, M., Schroder, G., Schmelzer, E., Lutz, V., Schroder, J. 1999Flavonoid hydroxylase from Catharanthus roseus: cDNAheterologous expression, enzyme properties and cell-type specific expression in plantsPlant J.19183193CrossRefPubMedGoogle Scholar
  14. Kitada, C., Gong, Z., Tanaka, Y., Yamazaki, M., Saito, K. 2001Differential expression of two cytochrome P450s involved in the biosynthesis of flavones and anthocyanins in chemo-varietal forms of Perilla frutescensPlant Cell Physiol.4213381344CrossRefPubMedGoogle Scholar
  15. Koes, R., Verweij, W., Quattrocchio, F. 2005Flavonoids: a colorful model for the regulation and evolution of biochemical pathwaysTrends Plant Sci.10236242CrossRefPubMedGoogle Scholar
  16. Kobayashi, H., Oikawa, Y., Koiwa, H., Yamamura, S. 1998Flower-specific expression directed by the promoter of a chalcone synthase gene from Gentiana triflora in Petunia hybridaPlant Sci.131173180CrossRefGoogle Scholar
  17. Martens, S., Forkmann, G. 1999Cloning and expression of flavone synthase II from Gerbera hybridsPlant J.20611618CrossRefPubMedGoogle Scholar
  18. Mol, J., Grotewold, E., Koes, R. 1998How genes paint flowers and seedsTrends Plant Sci.3212216CrossRefGoogle Scholar
  19. Muir, S.R., Collins, G.J., Robinson, S., Hughes, S., Bovy, A., Ric De Vos, C.H., Tunen, A.J., Verhoeyen, M.E. 2001Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonolsNat. Biotechnol.19470474CrossRefPubMedGoogle Scholar
  20. Nakatsuka, T., Nishihara, M., Mishiba, K., Yamamura, S. 2005Temporal expression of flavonoid biosynthesis-related genes regulates flower pigmentation in gentian plantsPlant Sci.16813091318CrossRefGoogle Scholar
  21. Pompon, D., Louerat, B., Bronine, A., Urban, P. 1996Yeast expression of animal and plant P450s in optimized redox environmentsMethods Enzymol.2725164PubMedGoogle Scholar
  22. Ro, D.K., Arimura, G., Lau, S.Y., Piers, E., Bohlmann, J. 2005Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenaseProc. Natl. Acad. Sci. USA10280608065CrossRefPubMedGoogle Scholar
  23. Springob, K., Nakajima, J., Yamazaki, M., Saito, K. 2003Recent advances in the biosynthesis and accumulation of anthocyaninsNat. Prod. Rep.20288303CrossRefPubMedGoogle Scholar
  24. Suzuki, K., Xue, H.M., Tanaka, Y., Fukui, Y., Fukuchi-Mizutani, M., Murakami, Y., Katsumoto, Y., Tsuda, S., Kusumi, T. 2000Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genesMol. Breed.6239246CrossRefGoogle Scholar
  25. Takesawa, T., Ito, M., Kanzaki, H., Kameya, N., Nakamura, I. 2002Over-expression of (zeta)glutathione S-transferase in transgenic rice enhances germination and growth at low temperatureMol. Breed.993101CrossRefGoogle Scholar
  26. Tanaka, Y., Yonakura, K., Fukuchi-Mizutani, M., Fukui, Y., Fujiwara, H., Ashikari, T., Kusumi, T. 1996Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana trifloraPlant Cell Physiol.37711716PubMedGoogle Scholar
  27. Tsuda, S., Fukui, Y., Nakamura, N., Katsumoto, Y., Yonekura-Sabakibara, K., Fukuchi-Mizutani, M., Ohira, K., Ueyama, Y., Ohkawa, H., Holton, T.A., Kusumi, T., Tanaka, Y. 2004Flower color modification of Petunia hybrida commercial varieties by metabolic engineeringPlant Biotechnol.21377386Google Scholar
  28. Ueyama, Y., Suzuki, K., Fukuchi-Mizutani, M., Fukui, Y., Miyazaki, K., Ohkawa, H., Kusumi, T., Tanaka, Y. 2002Molecular and biochemical characterization of torenia flavonoid 3′-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of those genesPlant Sci.163253263CrossRefGoogle Scholar
  29. Urban, P., Werck-Reichhart, D., Teutsch, H.G., Durst, F., Regnier, S., Kazmaier, M., Pompon, D. 1994Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathwayEur. J. Biochem.222843850CrossRefPubMedGoogle Scholar
  30. van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N., Stuitje, A.R. 1990Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expressionPlant Cell2291299CrossRefPubMedGoogle Scholar
  31. Winkel-Shirley, B. 2001Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnologyPlant Physiol.126485493CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Takashi Nakatsuka
    • 1
  • Masahiro Nishihara
    • 1
  • Keiichiro Mishiba
    • 1
    • 2
  • Saburo Yamamura
    • 1
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversitySakaiJapan

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