Plant Cell Reports

, Volume 32, Issue 12, pp 1925–1937 | Cite as

Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers

  • Takashi Nakatsuka
  • Eri Yamada
  • Misa Saito
  • Kohei Fujita
  • Masahiro Nishihara
Original Paper


Key message

Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color.


RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes—GtMYB1R and GtMYB1R9—predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in flowers of other floricultural plant species.


Antagonistic transcription factor Anthocyanin biosynthesis Floral pigmentation Japanese gentian MYB1R 





Anthocyanidin synthase


Basic helix-loop-helix


Cinnamate 4-hydroxylase


Cauliflower mosaic virus


Chalcone isomerase


Chalcone synthase


ERF-associated amphiphilic repression


Flavonoid 3-hydroxylase


Flavonoid 3′-hydroxylase


Flavonol synthase


Dihydroflavonol 4-reductase


Phenylalanine ammonia lyase


Quantitative reverse-transcription polymerase chain reaction


RNA interference



We thank Ms. Akiko Kubota, Iwate Biotechnology Research Center, for technical support. This work was financially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 24380024) and by the Iwate prefectural government.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aharoni A, De Vos CH, Wein M, Sun Z, Greco R, Kroon A, Mol JN, O’Connell AP (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28:319–332PubMedCrossRefGoogle Scholar
  2. Albert NW, Lewis DH, Zhang H, Schwinn KE, Jameson PE, Davies KM (2011) Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J 65:771–784PubMedCrossRefGoogle Scholar
  3. Baudry A, Heim MA, Dubreucq B, Caboche M, Weisshaar B, Lepiniec L (2004) TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J 39:366–380PubMedCrossRefGoogle Scholar
  4. Baudry A, Caboche M, Lepiniec L (2006) TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. Plant J 46:768–779PubMedCrossRefGoogle Scholar
  5. Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394PubMedGoogle Scholar
  6. Chen B, Wang X, Hu Y, Wang Y, Lin Z (2004) Ectopic expression of a c1-1 allele from maize inhibits pigment formation in the flower of transgenic tobacco. Mol Biotechnol 26:187–192PubMedCrossRefGoogle Scholar
  7. Cocciolone SM, Nettleton D, Snook ME, Peterson T (2005) Transformation of maize with the p1 transcription factor directs production of silk maysin, a corn earworm resistance factor, in concordance with a hierarchy of floral organ pigmentation. Plant Biotechnol J 3:225–235PubMedCrossRefGoogle Scholar
  8. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676PubMedCrossRefGoogle Scholar
  9. Davies K (2009) Modifying anthocyanin production in flowers. In: Gould K, Davies K, Winefield C (eds) Anthocyanins, biosynthesis, functions, and applications. Springer, New York, pp 49–83Google Scholar
  10. Dubos C, Le Gourrierec J, Baudry A, Huep G, Lanet E, Debeaujon I, Routaboul JM, Alboresi A, Weisshaar B, Lepiniec L (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J 55:940–953PubMedCrossRefGoogle Scholar
  11. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trend Plant Sci 15:573–581CrossRefGoogle Scholar
  12. Feller A, Machemer K, Braun EL, Grotewold E (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J 66:94–116PubMedCrossRefGoogle Scholar
  13. Grotewold E, Drummond BJ, Bowen B, Peterson T (1994) The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76:543–553PubMedCrossRefGoogle Scholar
  14. Grotewold E, Sainz MB, Tagliani L, Hernandez JM, Bowen B, Chandler VL (2000) Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proc Natl Acad Sci USA 97:13579–13584PubMedCrossRefGoogle Scholar
  15. Han Y, Vimolmangkang S, Soria-Guerra RE, Korban SS (2012) Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. J Exp Bot 63:2437–2447PubMedCrossRefGoogle Scholar
  16. Hanumappa H, Choi G, Ryu S, Choi G (2007) Modulation of flower colour by rationally designed dominant-negative chalcone synthase. J Exp Bot 58:2471–2478PubMedCrossRefGoogle Scholar
  17. Hartmann U, Valentine WJ, Christie JM, Hays J, Jenkins GI, Weisshaar B (1998) Identification of UV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homologous protoplast transient expression system. Plant Mol Biol 36:741–754PubMedCrossRefGoogle Scholar
  18. Hemm MR, Herrmann KM, Chapple C (2001) AtMYB4: a transcription factor general in the battle against UV. Trend Plant Sci 6:135–136CrossRefGoogle Scholar
  19. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that induce the EAR motif, a repression domain, in Arabidopsis. Plant J 34:733–739PubMedCrossRefGoogle Scholar
  20. Horsch RB, Fry JE, Hoffmann NL, Eicholtz D, Rogers SG, Fraley RT (1985) A simple method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  21. Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19:6150–6161PubMedCrossRefGoogle Scholar
  22. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trend Plant Sci 10:236–242CrossRefGoogle Scholar
  23. Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2012) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50CrossRefGoogle Scholar
  24. Martin C, Paz-Ares J (1997) MYB transcription factors in plants. Trend Genet 13:67–73CrossRefGoogle Scholar
  25. Matsui K, Umemura Y, Ohme-Takagi M (2008) AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J 55:954–967PubMedCrossRefGoogle Scholar
  26. Mehrtens F, Kranz H, Bednarek P, Weisshaar B (2005) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiol 138:1083–1096PubMedCrossRefGoogle Scholar
  27. Mitsuda N, Hiratsu K, Todaka D, Nakashima K, Yamaguchi-Shinozaki K, Ohme-Takagi M (2006) Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice. Plant Biotechnol J 4:325–332PubMedCrossRefGoogle Scholar
  28. Nakamura N, Fukuchi-Mizutani M, Miyazaki K, Suzuki K, Tanaka Y (2006) RNAi suppression of the anthocyanin synthase gene in Torenia hybrid yields white flowers with higher frequency and better stability than antisense ad sense suppression. Plant Biotechnol 23:13–17CrossRefGoogle Scholar
  29. Nakatsuka T, Nishihara M, Mishiba K, Yamamura S (2005) Temporal expression of flavonoid biosynthesis-related genes regulates flower pigmentation in gentian plants. Plant Sci 168:1309–1318CrossRefGoogle Scholar
  30. Nakatsuka T, Abe Y, Kakizaki Y, Yamamura S, Nishihara M (2007) Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes. Plant Cell Rep 26:1951–1959PubMedCrossRefGoogle Scholar
  31. Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant Cell Physiol 49:1818–1829PubMedCrossRefGoogle Scholar
  32. Nakatsuka T, Saito M, Yamada E, Nishihara M (2011) Production of picotee-type flowers in Japanese gentian by CRES-T. Plant Biotechnol 28:173–180CrossRefGoogle Scholar
  33. Nakatsuka T, Saito M, Yamada E, Fujita K, Kakizaki Y, Nishihara M (2012) Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers. J Exp Bot 63:6505–6517PubMedCrossRefGoogle Scholar
  34. Nishihara M, Nakatsuka T (2011) Genetic engineering of flavonoid pigments to modify flower color in floricultural plants. Biotechnol Lett 33:433–441PubMedCrossRefGoogle Scholar
  35. Nishihara M, Nakatsuka T, Yamamura S (2005) Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Lett 579:6074–6078PubMedCrossRefGoogle Scholar
  36. Pattanaik S, Kong Q, Zaitlin D, Werkman JR, Xie CH, Patra B, Yuan L (2010) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076PubMedCrossRefGoogle Scholar
  37. Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11:1433–1444PubMedGoogle Scholar
  38. Quattrocchio F, Baudry A, Lepiniec L, Grotewold E (2006) The regulation of flavonoid biosynthesis. In: Grotewold E (ed) The science of flavonoids. Springer, New York, pp 97–122CrossRefGoogle Scholar
  39. Ramsay NA, Glover (2005) MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trend Plant Sci 10:63–70CrossRefGoogle Scholar
  40. Salvatierra A, Pimentel P, Moya-Leon MA, Herrera R (2013) Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene. Phytochemistry 90:25–36PubMedCrossRefGoogle Scholar
  41. Shikata M, Narumi T, Yamaguchi H, Sasaki K, Aida R, Oshima Y, Takiguchi Y, Ohme-Takagi M, Mitsuda N, Ohtsubo N (2011) Efficient production of novel floral traits in torenia by collective transformation with chimeric repressors of Arabidopsis transcription factors. Plant Biotechnol 28:189–199CrossRefGoogle Scholar
  42. Spelt C, Quattrocchio F, Mol JN, Koes R (2000) anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619–1632PubMedGoogle Scholar
  43. Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456PubMedCrossRefGoogle Scholar
  44. Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677PubMedCrossRefGoogle Scholar
  45. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  46. Tanaka Y, Brugliera F, Kalc G, Senior M, Dyson B, Nakamura N, Katsumoto Y, Chandler S (2010) Flower color modification by engineering of the flavonoid biosynthetic pathway: practical perspectives. Biosci Biotechnol Biochem 74:1760–1769PubMedCrossRefGoogle Scholar
  47. Tsuda S, Fukui Y, Nakamura N, Katsumoto Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Ohira K, Ueyama Y, Ohkawa H, Holton TA, Kusumi T, Tanaka Y (2004) Flower color modification of Petunia hybrid commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386CrossRefGoogle Scholar
  48. Wada T, Tachibana T, Shimura Y, Okada K (1997) Epidermal cell differentiation in Arabidopsis determined by a Myb homolog, CPC. Science 277:1113–1116PubMedCrossRefGoogle Scholar
  49. Winkel BSJ (2006) The biosynthesis of flavonoid. In: Grotewold E (ed) The science of flavonoids. Springer, New York, pp 71–96CrossRefGoogle Scholar
  50. Xu W, Grain D, Gourrierec JL, Harscoët E, Berger A, Jauvion V, Scagnelli A, Berger N, Bidzinski P, Kelemen Z, Salsac F, Baudry A, Routaboul JM, Lepiniec L, Dubos C (2013) Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis. New Phytol 198:59–70PubMedCrossRefGoogle Scholar
  51. Yuan YW, Sagawa JM, Young RC, Christensen BJ (2013) Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics 194:255–263PubMedCrossRefGoogle Scholar
  52. Zhang W, Ning G, Lv H, Liao L, Bao M (2009) Single MYB-type transcription factor AtCAPRICE: a new efficient tool to engineer the production of anthocyanin in tobacco. Biochem Biophy Res Comm 388:742–747CrossRefGoogle Scholar
  53. Zhu HF, Fitzsimmons K, Khandelwal A, Kranz RG (2009) CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Mol Plant 2:790–802PubMedCrossRefGoogle Scholar
  54. Zhu L, Shan H, Chen S, Jiang J, Gu C, Zhou G, Chen Y, Song A, Chen F (2013) The heterologous expression of the chrysanthemum R2R3-MYB transcription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana. PLoS ONE 8:e65680PubMedCrossRefGoogle Scholar
  55. Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40:22–34PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Takashi Nakatsuka
    • 1
  • Eri Yamada
    • 2
  • Misa Saito
    • 2
  • Kohei Fujita
    • 2
  • Masahiro Nishihara
    • 2
  1. 1.Department of Biological and Environmental Science, Graduate School of AgricultureShizuoka UniversityShizuokaJapan
  2. 2.Iwate Biotechnology Research CenterKitakamiJapan

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