Identification of functional flavonol synthase genes from fragrant wild cyclamen (Cyclamen purpurascens)

  • Yusuke Akita
  • Satoshi Kitamura
  • Riho Mikami
  • Hiroshi Ishizaka
Original Article

Abstract

Cyclamen purpurascens is considered suitable for horticultural breeding of cyclamens because it has an attractive fragrance that is not found in other wild species. To improve the commercial value of cyclamen flowers, this fragrance has been introduced into ornamental cultivars. However, variation in flower color is somewhat limited in these cultivars, and therefore understanding the genetic networks of flower coloration in C. purpurascens is required. We previously isolated DNA fragments of anthocyanin biosynthetic genes from C. purpurascens, broadening our understanding of the biosynthetic pathway of flavonols, which are co-pigments in flower coloration. In this study, we isolated complete open reading frames of flavonol synthase genes from C. purpurascens (CpurFLS1 and CpurFLS2) and analyzed the in planta functions of the genes by molecular complementation assay using the fls mutant of Arabidopsis thaliana. Expression patterns in several organs of C. purpurascens were also determined. The results strongly suggest that the CpurFLS genes participate in flavonol synthesis. We discuss the involvement of these two FLSs in flower coloration in C. purpurascens.

Keywords

Cyclamen Flavonol Flavonol synthase Flower 

Notes

Acknowledgements

We are grateful to Yukimi Yamaguchi, Chie Kurihara, and Tomomi Iwasaki for providing help and advice. We also thank Dr. Masayoshi Nakayama (NARO Institute of Vegetable and Floriculture Science) for helpful comments on flavonols in flowers.

Funding

This study was funded by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (Number JP15K18641) awarded to Y. Akita.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Akita Y, Ishizaka H, Nakayama M, Shimada S, Kitamura S, Hase Y, Narumi I, Tanaka Y (2010) Comparative analysis of floral pigmentation between wild-type and white-flowered varieties of Cyclamen graecum. J Hortic Sci Biotechnol 84:437–443CrossRefGoogle Scholar
  2. Akita Y, Kitamura S, Hase Y, Narumi I, Ishizaka H, Kondo E, Kameari N, Nakayama M, Tanikawa N, Morita Y, Tanaka A (2011) Isolation and characterization of the fragrant cyclamen O-methyltranseferase involved in flower coloration. Planta 234:1127–1136CrossRefPubMedGoogle Scholar
  3. Anderberg AA, Trift I, Källersjö M (2000) Phylogeny of Cyclamen L. (Primulaceae): evidence from morphology and sequence data from the internal transcribed spacers of nuclear ribosomal DNA. Plant Syst Evol 220:147–160CrossRefGoogle Scholar
  4. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis. Nature 408:796–815CrossRefGoogle Scholar
  5. Boase MR, Lewis DH, Davies KM, Marshall GB, Patel D, Schwinn KE, Deroles SC (2010) Isolation and antisense suppression of flavonoid 3′, 5′-hydroxylase modifies flower pigments and colour in cyclamen. BMC Plant Biol 10:107CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  7. Chua CS, Biermann D, Goo KS, Sim TS (2008) Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols. Phytochemistry 69:66–75CrossRefPubMedGoogle Scholar
  8. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  9. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP:cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97:3718–3723CrossRefPubMedPubMedCentralGoogle Scholar
  10. Davies KM, Bradley JM, Schwinn KE, Markham KR, Podivinsky E (1993) Flavonoid biosynthesis in flower petals of five lines of lisianthus (Eustoma grandiflorum Grise.). Plant Sci 95:67–77CrossRefGoogle Scholar
  11. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791CrossRefPubMedGoogle Scholar
  12. Forkmann G (1991) Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering. Plant Breed 106:1–26CrossRefGoogle Scholar
  13. Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fujita A, Goto-Yamamoto N, Aramaki I, Hashizume K (2006) Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins. Biosci Biotechnol Biochem 70:632–638CrossRefPubMedGoogle Scholar
  15. Grey-Wilson C (2002) Cyclamen: a guide for gardeners, horticulturists and botanists. B.T. Bastsford, EnglandGoogle Scholar
  16. Hase Y, Akita Y, Kitamura S, Narumi I, Tanaka A (2012) Development of an efficient mutagenesis technique using ion beams: Toward more controlled mutation breeding. Plant Biotechnol 29:193–200CrossRefGoogle Scholar
  17. Holton TA, Brugilera F, Tanaka Y (1993) Cloning and expression of flavonol synthase from Petunia hybrid. Plant J 4:1003–1010CrossRefPubMedGoogle Scholar
  18. Ishizaka H (2008) Interspecific hybridization by embryo rescue in the genus Cyclamen. Plant Biotechnol 25:511–519CrossRefGoogle Scholar
  19. Ishizaka H, Yamada H, Sasaki K (2002) Volatile compounds in the flowers of Cyclamen persicum, C. purpurascens and their hybrids. Sci Hortic (Amst) 94:125–135CrossRefGoogle Scholar
  20. Ishizaka H, Kondo E, Kameari N (2012) Production of novel flower color mutants from the fragrant cyclamen (Cyclamen persicum × C. purpurascens) by ion-beam irradiation. Plant Biotechnol 29:201–208CrossRefGoogle Scholar
  21. Ito T, Motohashi R, Kuromori T, Mizukado S, Sakurai T, Kanahara H, Seki M, Shinozaki K (2002) A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome. Plant Physiol 129:1695–1699CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kitamura S, Akita Y, Ishizaka H, Narumi I, Tanaka A (2012) Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen. J Plant Physiol 169(6):636–642CrossRefPubMedGoogle Scholar
  23. Kuromori T, Hirayama T, Kiyosue Y, Takabe H, Mizukado S, Sakurai T, Akiyama K, Kamiya A, Ito T, Shinozaki K (2004) A collection of 11800 single-copy Ds transposon insertion lines in Arabidopsis. Plant J 37:897–905CrossRefPubMedGoogle Scholar
  24. Li C, Bai Y, Li S, Chen H, Han X, Zhao H, Shao J, Park S, Wu Q (2012) Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in Tartary buckwheat. J Agric Food Chem 60:5161–5168CrossRefPubMedGoogle Scholar
  25. Miyajima I, Doi I, Kage T (1990) Floral pigment and flower colour expression in the petals of cyclamen. Sci Bull Fac Agric Kyushu Univ 45:83–89 (in Japanese) Google Scholar
  26. Muir SR, Collins GJ, Robinson S, Hughes S, Bovy A, Ric De Vos CH, van Tune AJ, Verhoeyen ME (2001) Overexpression of petunia chalcone isomerase in tomato results in furit containing increased levels of flovonols. Nat Biotechnol 19:470–474CrossRefPubMedGoogle Scholar
  27. Nakayama M, Tanikawa N, Morita Y, Ban Y (2012) Comprehensive analyses of anthocyanin and related compounds to understand flower color change in ion-beam mutants of cyclamen (Cyclamen spp.) and carnation (Dianthus caryophyllus). Plant Biothechnol 29:215–221CrossRefGoogle Scholar
  28. Nielsen K, Deroles SC, Markham KR, Bradley MJ, Podivinsky E, Manson D (2002) Antisense flavonol synthase alters copigmentation and flower color in lisianthus. Mol Breed 9:217–229CrossRefGoogle Scholar
  29. Owens DK, Alerding AB, Crosby KC, Bandara AB, Westwood JH, Winkel BSJ (2008) Functional analysis of a predicted favonol synthase gene family in Arabidopsis. Plant Physiol 147:1046–1061CrossRefPubMedPubMedCentralGoogle Scholar
  30. Park S, Kim DH, Lee JY, Ha SH, Lim SH (2017) Comparative analysis of two flavonol synthases from different-colored onions provides insight into flavonos biosynthesis. J Agric Food Chem 65:5287–5298CrossRefPubMedGoogle Scholar
  31. Peer WA, Brown DE, Tague BW, Muday GK, Taiz L, Murphy AS (2001) Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis. Plant Physiol 126:536–548CrossRefPubMedPubMedCentralGoogle Scholar
  32. Preuß A, Stracke R, Weisshaar B, Hillebrecht A, Matern U, Martens S (2009) Arabidopsis thaliana expresses a second functional flavonol synthase. FEBS Lett 583:1981–1986CrossRefPubMedGoogle Scholar
  33. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  34. Schwinn K, Venail J, Shang Y, Mackay S, Alm V, Butelli E, Oyama R, Bailey P, Davies K, Martin C (2006) A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18:831–851CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sheahan JJ, Rechnitz GA (1993) Differential visualization of transparent testa mutants in Arabidopsis thaliana. Anal Chem 65:961–963CrossRefGoogle Scholar
  36. Takamura T (2006) Cyclamen. Cyclamen persicum Mill. In: Anderson NO (ed) Flower breeding and genetics. Springer, Dordrecht, pp 459–478Google Scholar
  37. Takamura T, Omi S, Sugimura T, Tanaka M (1997) Flower color and anthocyanins in the petal of Cyclamen species. J Jpn Soc Hort Sci 66(Suppl 2):508–509 (in Japanese) Google Scholar
  38. Takamura T, Nakayama M, Ishizaka H (2005) Inheritance of flower color pigment in crosses between cyclamen cultivars and Cyclamen purpurascens. Acta Hortic 673:437–441CrossRefGoogle Scholar
  39. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  40. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedPubMedCentralGoogle Scholar
  41. Tian J, Han ZY, Zhang J, Hu Y, Song T, Yao Y (2015) The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapple. Sci Rep 5:12228CrossRefPubMedPubMedCentralGoogle Scholar
  42. Turnbull JJ, Nakajima J, Welford RW, Yamazaki M, Saito K, Schofield CJ (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3β-hydroxylase. J Biol Chem 279:1206–1216CrossRefPubMedGoogle Scholar
  43. Xu F, Li L, Zhang W, Cheng H, Sun N, Cheng S, Wang Y (2012) Isolation, characterization, and function analysis of flavonol synthase gene from Ginkgo biloba. Mol Biol Rep 39:2285–2296CrossRefPubMedGoogle Scholar
  44. Yazaki Y (1976) Co-pigmentation and the color change with age in petals of Fuchsia hybrida. Bot Mag Tokyo 89:45–57CrossRefGoogle Scholar
  45. Zhang C, Liu H, Jia C, Liu Y, Wang F, Wang J (2016) Cloning, characterization and functional analysis of a flavonol synthase from Vaccinium corymbosum. Trees 30:1595–1605CrossRefGoogle Scholar
  46. Zhou XY, Fan ZQ, Chen Y, Zhu YL, Li JY, Yin HF (2013) Functional analyses of a flavonol synthase-like gene from Camellia nitidissima reveal its roles in flavonoid metabolism during floral pigmentation. J Biosci 38:593–604CrossRefPubMedGoogle Scholar
  47. Zuckerkandl E, Pauling L (1965) Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ (eds) Evolving genes and proteins. Academic Press, New York, pp 97–166CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2017

Authors and Affiliations

  • Yusuke Akita
    • 1
  • Satoshi Kitamura
    • 2
  • Riho Mikami
    • 1
  • Hiroshi Ishizaka
    • 3
  1. 1.Department of Life Science and Green Chemistry, Faculty of EngineeringSaitama Institute of TechnologyFukayaJapan
  2. 2.Quantum Beam Science Research DirectorateNational Institutes for Quantum and Radiological Science and TechnologyTakasakiJapan
  3. 3.Saitama Agricultural Technology Research CenterKumagayaJapan

Personalised recommendations