, Volume 234, Issue 5, pp 945–958 | Cite as

Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia

  • Sho Ohno
  • Munetaka Hosokawa
  • Misa Kojima
  • Yoshikuni Kitamura
  • Atsushi Hoshino
  • Fumi Tatsuzawa
  • Motoaki Doi
  • Susumu Yazawa
Original Article


Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar ‘Yuino’ and the two seedling cultivars ‘OriW1’ and ‘OriW2’ borne from a red-white bicolor cultivar, ‘Orihime’, indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in ‘Yuino’ petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.


CHS Dahlia variabilis PTGS Pure white flower siRNA 



Anthocyanidin 3-glucosyltransferase


Anthocyanidin synthase


Basic helix-loop-helix


Chalcone isomerase


Chalcone synthase


Dihydroflavonol 4-reductase


Flavanone 3-hydroxylase


Flavonol synthase


Flavone synthase


High-performance liquid chromatography


Open-reading frame


Post-transcriptional gene silencing


Small interfering RNA


Single nucleotide polymorphisms


Thin layer chromatography


Transcriptional gene silencing


WD40 repeats

Supplementary material

425_2011_1456_MOESM1_ESM.docx (1.1 mb)
Supplementary material (DOCX 1,079 kb)


  1. Bate-Smith EC, Swain T, Nördstrom CG (1955) Chemistry and inheritance of flower colour in the Dahlia. Nature 176:1016–1018CrossRefGoogle Scholar
  2. De Paoli E, Dorantes-Acosta A, Jixian Z, Accerbi M, Jeong DH, Sunhee P, Meyers BC, Jorgensen RA, Green PJ (2009) Distinct extremely abundant siRNAs associated with cosuppression in petunia. RNA 15:1965–1970PubMedCrossRefGoogle Scholar
  3. Ferrer JL, Jez JM, Bowman ME, Dixon RA, Noel JP (1999) Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat Struct Biol 6:775–784PubMedCrossRefGoogle Scholar
  4. Fukusaki EI, Kawasaki K, Kajiyama S, An CI, Suzuki K, Tanaka Y, Kobayashi A (2004) Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference. J Biotechnol 111:229–240PubMedCrossRefGoogle Scholar
  5. Gatt M, Ding H, Hammett K, Murray B (1998) Polyploidy and evolution in wild and cultivated Dahlia species. Ann Bot 81:647–656CrossRefGoogle Scholar
  6. Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Gen 10:94–108CrossRefGoogle Scholar
  7. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952PubMedCrossRefGoogle Scholar
  8. Harborne JB (1984) Phytochemical methods, 2nd edn. Chapman and Hall, LondonCrossRefGoogle Scholar
  9. Hemleben V, Dressel A, Epping B, Lukačin R, Martens S, Austin MB (2004) Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae). Plant Mol Biol 55:455–465PubMedCrossRefGoogle Scholar
  10. Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot. doi: 10.1093/jxb/erq442
  11. Hoshino A, Park KI, Iida S (2009) Identification of r mutations conferring white flowers in the Japanese morning glory (Ipomoea nil). J Plant Res 122:215–222PubMedCrossRefGoogle Scholar
  12. Jiang CZ, Chen JC, Reid M (2011) Virus-induced gene silencing in ornamental plants. Methods Mol Biol 744:81–96PubMedCrossRefGoogle Scholar
  13. Koseki M, Goto K, Masuta C, Kanazawa A (2005) The star-type color pattern in Petunia hybrida ‘Red Star’ flowers is induced by sequence-specific degradation of chalcone synthase RNA. Plant Cell Physiol 46:1879–1883PubMedCrossRefGoogle Scholar
  14. Kurauchi T, Matsumoto T, Taneda A, Sano T, Senda M (2009) Endogenous short interfering RNAs of chalcone synthase genes associated with inhibition of seed coat pigmentation in soybean. Breed Sci 59:419–426CrossRefGoogle Scholar
  15. Lawrence WJC (1929) The genetics and cytology of Dahlia species. J Genet 21:125–159CrossRefGoogle Scholar
  16. Lawrence RJ, Pikaard CS (2003) Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. Plant J 36:114–121PubMedCrossRefGoogle Scholar
  17. Ma LQ, Pang XB, Shen HY, Pu GB, Wang HH, Lei CY, Wang H, Li GF, Liu BY, Ye HC (2009) A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum. Planta 229:457–469PubMedCrossRefGoogle Scholar
  18. Martin C, Carpenter R, Sommer H, Saedler H, Coen ES (1985) Molecular analysis of instability in flower pigmentation of Antirrhinum majus, following isolation of the pallida locus by transposon tagging. EMBO J 4:1625–1630PubMedGoogle Scholar
  19. Mato M, Onozaki T, Ozeki Y, Higeta D, Itoh Y, Yoshimoto Y, Ikeda H, Yoshida H, Shibata M (2000) Flavonoid biosynthesis in white-flowered Sim carnations (Dianthus caryophyllus). Sci Hortic 84:333–347CrossRefGoogle Scholar
  20. McClaren M (2009) Dahlia: history and species. In: McClaren B (ed) Encyclopedia of dahlias. Timber Press, Portland, pp 161–166Google Scholar
  21. Metzlaff M, O’Dell M, Cluster PD, Flavell RB (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in Petunia. Cell 88:845–854PubMedCrossRefGoogle Scholar
  22. Morita Y, Saitoh M, Hoshino A, Nitasaka E, Iida S (2006) Isolation of cDNAs for R2R3-MYB, bHLH and WDR transcriptional regulators and identification of c and ca mutations conferring white flowers in the Japanese morning glory. Plant Cell Physiol 47:457–470PubMedCrossRefGoogle Scholar
  23. Nakatsuka T, Nishihara M, Mishiba K, Yamamura S (2005) Two different mutations are involved in the formation of white-flowered gentian plants. Plant Sci 169:949–958CrossRefGoogle Scholar
  24. Nakatsuka T, Mishiba KI, Kubota A, Abe Y, Yamamura S, Nakamura N, Tanaka Y, Nishihara M (2010) Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian. J Plant Physiol 167:231–237PubMedCrossRefGoogle Scholar
  25. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289PubMedCrossRefGoogle Scholar
  26. Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L (2000) The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 12:1863–1878PubMedCrossRefGoogle Scholar
  27. Nordström CG, Swain T (1956) The flavonoid glycosides of Dahlia variabilis. II. Glycosides of yellow varieties Pius IX and Coton. Arch Biochem Biophys 60:329–344PubMedCrossRefGoogle Scholar
  28. Nordström CG, Swain T (1958) The flavonoid glycosides of Dahlia variabilis. III. Glycosides from white varieties. Arch Biochem Biophys 73:220–223PubMedCrossRefGoogle Scholar
  29. Ohno S, Hosokawa M, Hoshino A, Kitamura Y, Morita M, Park KI, Nakashima A, Deguchi A, Tatsuzawa F, Doi M, Iida S, Yazawa S (2011) bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis). J Exp Bot (in press)Google Scholar
  30. Onozaki T, Mato M, Shibata M, Ikeda H (1999) Differences in flower color and pigment composition among white carnation (Dianthus caryophyllus L.) cultivars. Sci Hortic 82:103–111CrossRefGoogle Scholar
  31. Park KI, Choi JD, Hoshino A, Morita Y, Iida S (2004) An intragenic tandem duplication in a transcriptional regulatory gene for anthocyanin biosynthesis confers pale-colored flowers and seeds with fine spots in Ipomoea tricolor. Plant J 38:840–849PubMedCrossRefGoogle Scholar
  32. Park KI, Ishikawa N, Morita Y, Choi JD, Hoshino A, Iida S (2007) A bHLH regulatory gene in the common morning glory, Ipomoea purpurea, controls anthocyanin biosynthesis in flowers, proanthocyanidin and phytomelanin pigmentation in seeds, and seed trichome formation. Plant J 49:641–654PubMedCrossRefGoogle Scholar
  33. Quattrocchio F, Wing JF, Leppen HTC, Mol JNM, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5:1497–1512PubMedCrossRefGoogle Scholar
  34. Saito N, Ishizuka K, Osawa Y (1970) Paper-chromatographic identification of flavonoids from a scarlet-flowering dahlia and crystallization of pelargonidin and butein. Bot Mag Tokyo 83:229–232Google Scholar
  35. Senda M, Jumonji A, Yumoto S, Ishikawa R, Harada T, Niizeki M, Akada S (2002) Analysis of the duplicated CHS1 gene related to the suppression of the seed coat pigmentation in yellow soybeans. Theor Appl Genet 104:1086–1091PubMedCrossRefGoogle Scholar
  36. Spelt C, Quattrocchio F, Mol JNM, Koes R (2000) anthocyanin1 of Petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619–1631PubMedCrossRefGoogle Scholar
  37. Spelt C, Quattrocchio F, Mol J, Koes R (2002) ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms. Plant Cell 14:2121–2135PubMedCrossRefGoogle Scholar
  38. Spribille R, Forkmann G (1982) Genetic control of chalcone synthase activity in flowers of Antirrhinum majus. Phytochemistry 21:2231–2234CrossRefGoogle Scholar
  39. Stam M (1997) Post-transcriptional silencing of flower pigmentation genes in Petunia hybrida by (trans)gene repeats. PhD thesis, Vrije UniversiteitGoogle Scholar
  40. Suzuki H, Nakayama T, Yonekura-Sakakibara K, Fukui Y, Nakamura N, Yamaguchi MA, Tanaka Y, Kusumi T, Nishino T (2002) cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6′-O-malonyltransferase from dahlia flowers. Plant Physiol 130:2142–2151PubMedCrossRefGoogle Scholar
  41. Tuteja JH, Zabala G, Varala K, Hudson M, Vodkin LO (2009) Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats. Plant Cell 21:3063–3077PubMedCrossRefGoogle Scholar
  42. Van Der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291–299PubMedCrossRefGoogle Scholar
  43. Zheng D, Schröder G, Schröder J, Hrazdina G (2001) Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus. Plant Mol Biol 46:1–15PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Sho Ohno
    • 1
  • Munetaka Hosokawa
    • 1
  • Misa Kojima
    • 1
  • Yoshikuni Kitamura
    • 2
  • Atsushi Hoshino
    • 3
  • Fumi Tatsuzawa
    • 4
  • Motoaki Doi
    • 1
  • Susumu Yazawa
    • 1
  1. 1.Laboratory of Vegetable and Ornamental Horticulture, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Faculty of AgricultureShinshu UniversityNaganoJapan
  3. 3.Division of Molecular GeneticsNational Institute for Basic BiologyOkazakiJapan
  4. 4.Department of Agriculture and Life ScienceIwate UniversityIwateJapan

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