Comprehensive isolation and expression analysis of the flavonoid biosynthesis-related genes in Tricyrtis spp.

  • M. Otani
  • Y. Kanemaki
  • F. Oba
  • M. Shibuya
  • Y. Funayama
  • M. Nakano
Article
  • 16 Downloads

Abstract

Tricyrtis spp., which belong to the family Liliaceae, produce unique flowers, whose tepals have many reddish-purple spots. Although elucidation of a molecular mechanism of tepal spot formation and molecular breeding for flower colour alteration are desired for Tricyrtis spp., only one flavonoid biosynthesis-related gene, TrCHS encoding chalcone synthase (CHS), has been isolated so far. In the present study, comprehensive isolation and expression analysis of the other flavonoid biosynthesis-related genes were carried out in Tricyrtis sp. Six genes (TrCHI, TrF3H, TrF3′H, TrFLS, TrDFR, and TrANS) encoding biosynthetic enzymes chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H); flavonoid 3′-hydroxylase (F3′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanin synthase (ANS) as well as three genes (TrMYB1, TrbHLH2 and TrWDR) encoding transcription factors myeloblastosis 1 (MYB1), basic helix-loop-helix (bHLH), and WD40 repeats (WDRs) were newly isolated. Phylogenetic analysis showed that each isolated gene was classified into the monocotyledonous clade. Deduced amino acid sequences of DFRs showed that TrDFR has no substrate specificity. “Early” genes in the flavonoid biosynthetic pathway (TrCHS, TrCHI, and TrF3H) were constantly expressed in tepals during flower development, whereas expression of “late” genes (TrF3′H, TrFLS, TrDFR, and TrANS) varied with the flower developmental stage. Expression patterns of the late genes were mostly correlated with those of transcriptional factor genes, indicating that the late genes may be under the control of a transcription factor complex consisted of TrMYB1, TrbHLH2, and TrWDR. Accumulation of anthocyanins in tepals occurred slightly after transcriptional upregulation of the late genes. Results obtained in the present study may be valuable for further studies on flower colour and flower colour pattern in Tricyrtis spp.

Additional key words

anthocyanins flavonoids flower colours flower development transcription factors 

Abbreviations

AN1

anthocyanin1

AN2

anthocyanin2

ANS

anthocyanin synthase

bHLH

basic helix-loop-helix

CHI

chalcone isomerase

CHS

chalcone synthase

DFR

dihydroflavonol 4-reductase

FLS

flavonol synthase

F3H

flavanone-3-hydroxylase

F3′H

flavonoid 3′-hydroxylase

F3′5′H

flavonoid 3′,5′-hydroxylase

HPLC

high performance liquid chromatography

MBW

MYB-bHLH-WDR

MYB

myeloblastosis

RACE

rapid amplification of cDNA ends

RT-PCR

reverse transcription-polymerase chain reaction

TF

transcription factor

TTG1

transparent testa glabra 1

WDRs

WD40 repeats

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10535_2018_802_MOESM1_ESM.pdf (302 kb)
Supplementary material, approximately 302 KB.

References

  1. Adachi, Y., Mori, S., Nakano, M.: Agrobacterium-mediated production of transgenic plants in Tricyrtis hirta (Liliaceae). — Acta Hort. 673: 415–419, 2005.CrossRefGoogle Scholar
  2. Chen, K., Liu, H., Lou, Q., Liu, Y.: Ectopic expression of the grape hyacinth (Muscari armeniacum) R2R3-MYB transcription factor gene, MaAN2, induces anthocyanin accumulation in tobacco. — Front. Plant Sci. 8: 965, 2017.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen, S.M., Li, C.H., Zhu, X.R., Deng, Y.M., Sun, W., Wang, L.S., Chen, F.D., Zhang, Z.: The identification of flavonoids and the expression of genes of anthocyanin biosynthesis in the chrysanthemum flowers. — Biol. Plant 56: 458–464, 2012.CrossRefGoogle Scholar
  4. Chiou, C.Y., Yeh, K.W.: Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey. — Plant mol. Biol. 66: 379–388, 2008.CrossRefPubMedGoogle Scholar
  5. Chiu, L.W., Zhou, X., Burke, S., Wu, X., Prior, R.L., Li, L.: The purple cauliflower arises from activation of a MYB transcription factor. — Plant Physiol. 154: 1470–1480, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cone, K. C., Cocciolone, S.M., Burr, F.A., Burr, B.: Maize anthocyanin regulatory gene pl is a duplicate of c1 that functions in the plant. — Plant Cell 5: 1795–1805, 1993.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap. — Evolution 39: 783–791, 1985.CrossRefPubMedGoogle Scholar
  8. Forkmann, G., Ruhnau, B. Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrid. — Z. Naturforsch C 42: 1146–1148, 1987.Google Scholar
  9. Fornalé, S., Shi, X., Chai, C., Encina, A., Irar, S., Capellades, M., Fuguet, E., Torres, J.L., Rovira, P., Puigdomenech, P., Rigau, J., Grotewold, E., Gray, J., Caparrós-Ruiz, D.: ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. — Plant J. 64: 633–644, 2010.CrossRefPubMedGoogle Scholar
  10. Gerats, A.G., De Vlaming, P., Doodeman, M., Al, B., Schram, A.W.: Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida. — Planta 155: 364–368, 1982.CrossRefPubMedGoogle Scholar
  11. Guo, N., Cheng, F., Wu, J., Liu, B., Zheng, S., Liang, J., Wang, X.: Anthocyanin biosynthetic genes in Brassica rapa. — BMC Genomics 15: 426, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hichri, I., Barrieu, F., Bogs, J., Kappel, C., Delrot, S., Lauvergeat, V.: Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. — J. exp. Bot. 62: 2465–2483, 2011.CrossRefPubMedGoogle Scholar
  13. Holton, T.A., Cornish, E.C.: Genetics andbBiochemistry of anthocyanin biosynthesis. — Plant Cell 7: 1071–1083, 1995.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Jaakola, L., Määttä, K., Pirttilä, A.M., Torronen, R., Karenlampi, S., Hohtola, A.: Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. — Plant Physiol. 130: 729–739, 2002.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiang, C., Gu, X., Peterson, T.: Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sativa L. ssp. indica. — Genome Biol. 5: R46, 2004.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Johnson, E.T., Ryu, S., Yi, H., Shin, B., Cheong, H., Choi, G.: Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase. — Plant J. 25: 325–333, 2001.CrossRefPubMedGoogle Scholar
  17. Johnson, E.T., Yi, H., Shin, B., Oh, B.J., Cheong, H., Choi, G.: Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins. — Plant J. 19: 81–85, 1999.CrossRefPubMedGoogle Scholar
  18. Kamiishi, Y., Otani, M., Takagi, H., Han, D.S., Mori, S., Tatsuzawa, F., Okuhara, H., Kobayashi, H., Nakano, M.: Flower color alteration in the liliaceous ornamental Tricyrtis sp. by RNA interference-mediated suppression of the chalcone synthase gene. — Mol. Breed. 30: 671–680, 2012.CrossRefGoogle Scholar
  19. Katsumoto, Y., Fukuchi-Mizutani, M., Fukui, Y., Brugliera, F., Holton, T.A., Karan, M., Nakamura, N., Yonekura-Sakakibara, K., Togami, J., Pigeaire, A., Tao, G.Q., Nehra, N.S., Lu, C.Y., Dyson, B.K., Tsuda, S., Ashikari, T., Kusumi, T., Mason, J.G., Tanaka, Y.: Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. — Plant Cell Physiol. 48: 1589–1600, 2007.CrossRefPubMedGoogle Scholar
  20. Koes, R., Verweij, W., Quattrocchio, F.: Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. — Trends Plant Sci. 10: 236–242, 2005.CrossRefPubMedGoogle Scholar
  21. Kranz, H.D., Denekamp, M., Greco, R., Jin, H., Leyva, A., Meissner, R.C., Petroni, K., Urzainqui, A., Bevan, M., Martin, C., Smeekens, S., Tonelli, C., Paz-Ares, J., Weisshaar, B.: Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. — Plant J. 16: 263–276, 1998.CrossRefPubMedGoogle Scholar
  22. Kumar, S., Stecher, G., Tamura, K.: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. — Mol. Biol. Evol. 33: 1870–1874, 2016.CrossRefPubMedGoogle Scholar
  23. Kunihiro, S., Tanabe, D., Niwa, Y., Kitamura, K., Abe, J., Yamada, T.: Isolation and molecular characterization of a Lotus japonicus R2R3-MYB subgroup 7 transcription factor gene. — Plant Biotechnol. 34: 45–49, 2017.CrossRefGoogle Scholar
  24. Lai, Y.S., Shimoyamada, Y., Nakayama, M., Yamagishi, M.: Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.). — Plant Sci. 193–194: 136–147, 2012.CrossRefPubMedGoogle Scholar
  25. Li, Q., Wang, J., Sun, H.Y., Shang, X.: Flower color patterning in pansy (Viola × wittrockiana Gams.) is caused by the differential expression of three genes from the anthocyanin pathway in acyanic and cyanic flower areas. — Plant Physiol. Biochem. 84: 134–141, 2014.CrossRefPubMedGoogle Scholar
  26. Liu, C., Long, J., Zhu, K., Liu, L., Yang, W., Zhang, H., Li, L., Xu, Q., Deng, X.: Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis. — Sci. Rep. 6: 25352, 2016.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Montefiori, M., Brendolise, C., Dare, A.P., Lin-Wang, K., Davies, K.M., Hellens, R.P., Allan, A.C.: In the Solanaceae, a hierarchy of bHLHs confer distinct target specificity to the anthocyanin regulatory complex. — J. exp. Bot. 66: 1427–1436, 2015.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mori, S., Asano, S., Kobayashi, H., Nakano, M.: Analyses of anthocyanidins and anthocyanins in flowers of Muscari spp. — Bull. Fac. Agr. Niigata Univ. 55: 13–18, 2002.Google Scholar
  29. Mori, S., Oka, E., Umehara, H., Kobayashi, H., Hoshi, Y., Kondo, M., Ogata, K., Nakano, M.: Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation. — Biol. Plant 52: 513–516, 2008.CrossRefGoogle Scholar
  30. Nakano, M., Nomizu, T., Mizunashi, K., Suzuki, M., Mori, S., Kuwayama, S., Hayashi, M., Umehara, H., Oka, E., Kobayashi, H., Asano, M., Sugawara, S., Takagi, H., Saito, H., Nakata, M., Godo, T., Hara, Y., Amano, J.: Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures. — Sci. Hort. 110: 366–371, 2006.CrossRefGoogle Scholar
  31. Nakatsuka, A., Mizuta, D., Kii, Y., Miyajima, I., Kobayashi, N.: Isolation and expression analysis of flavonoid biosynthesis genes in evergreen azalea. — Sci. Hort. 118: 314–320, 2008a.CrossRefGoogle Scholar
  32. Nakatsuka, T., Haruta, K.S., Pitaksutheepong, C., Abe, Y., Kakizaki, Y., Yamamoto, K., Shimada, N., Yamamura, S., Nishihara, M.: Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. — Plant Cell Physiol. 49: 1818–1829, 2008b.CrossRefPubMedGoogle Scholar
  33. Nakatsuka, T., Nishihara, M., Mishiba, K., Yamamura, S.: Temporal expression of flavonoid biosynthesis-related genes regulates flower pigmentation in gentian plants. — Plant Sci. 168: 1309–1318, 2005.CrossRefGoogle Scholar
  34. Noda, N., Aida, R., Kishimoto, S., Ishiguro, K., Fukuchi-Mizutani, M., Tanaka, Y., Ohmiya, A.: Genetic — Phytochemistry 50: 1181–1184, 1999.CrossRefGoogle Scholar
  35. Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P.A., Saedler, H.: The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. — EMBO J. 6: 3553–3558, 1987.PubMedPubMedCentralGoogle Scholar
  36. Perfus-Barbeoch, L., Jones, A.M., Assmann, S.M.: Plant heterotrimeric G protein function: insights from Arabidopsis and rice mutants. — Curr. Opin. Plant Biol. 7: 719–731, 2004.CrossRefPubMedGoogle Scholar
  37. Petroni, K., Tonelli, C.: Recent advances on the regulation of anthocyanin synthesis in reproductive organs. — Plant Sci. 181: 219–229, 2011.CrossRefPubMedGoogle Scholar
  38. Quattrocchio, F., Wing, J., Van der Woude, K., Souer, E., De Vetten, N., Mol, J., Koes, R.: Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. — Plant Cell 11: 1433–1444, 1999.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Rabino, I., Mancinelli, A.L.: Light, temperature, and anthocyanin production. — Plant Physiol. 81: 922–924, 1986.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Saitou, N., Nei, M.: The neighbor-joining method: A new method for reconstructing phylogenetic trees. — Mol. Biol. Evol. 4: 406–425, 1987.PubMedGoogle Scholar
  41. Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., Oyama, R., Bailey, P., Davies, K., Martin, C.: A Small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. — Plant Cell 18: 831–851, 2006.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Schwinn, K.E., Ngo, H., Kenel, F., Brummell, D.A., Albert, N.W., McCallum, J.A., Pither-Joyce, M., Crowhurst, R.N., Eady, C., Davies, K.M.: The onion (Allium cepa L.) R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis. — Front. Plant Sci. 7: 1865, 2016.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Smith, T.F., Gaitatzes, C., Saxena, K., Neer, E.J.: The WD repeat: a common architecture for diverse functions. — Trends Biochem. Sci. 24: 181–185, 1999.CrossRefPubMedGoogle Scholar
  44. Spelt, C., Quattrocchio, F., Mol, J.N., Koes, R.: Anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. — Plant Cell 12: 1619–1632, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Stracke, R., Ishihara, H., Huep, G., Barsch, A., Mehrtens, F., Niehaus, K., Weisshaar, B.: Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. — Plant J. 50: 660–677, 2007.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Suzuki, K., Suzuki, T., Nakatsuka, T., Dohra, H., Yamagishi, M., Matsuyama, K., Matsuura, H.: RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies (Lilium spp.). — BMC Genomics 17: 611, 2016.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Tatsuzawa, F., Saito, N., Miyoshi, K., Shinoda, K., Shigihara, A., Honda, T.: Diacylated 8-C-glucosylcyanidin 3-glucoside from the flowers of Tricyrtis formosana. — Chem. Pharm. Bull. 52: 631–633, 2004.CrossRefPubMedGoogle Scholar
  48. Van Nocker, S., Ludwig, P.: The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function. — BMC Genomics 4: 50, 2003.CrossRefPubMedPubMedCentralGoogle Scholar
  49. Walker, A.R., Davison, P.A., Bolognesi-Winfield, A.C., James, C.M., Srinivasan, N., Blundell, T.L., Esch, J.J., Marks, M.D., Gray, J.C.: The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. — Plant Cell 11: 1337–1350, 1999.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Wang, L.M., Zhang, J., Dong, X.Y., Fu, Z.Z., Jiang, H., Zhang, H.C.: Identification and functional analysis of anthocyanin biosynthesis genes in Phalaenopsis hybrids. — Biol. Plant. 62: 45–54, 2018.CrossRefGoogle Scholar
  51. Winkel-Shirley, B.: Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. — Plant Physiol. 126: 485–493, 2001.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Yamagishi, M., Shimoyamada, Y., Nakatsuka, T., Masuda, K.: Two R2R3-MYB genes, homologs of Petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of asiatic hybrid lily. — Plant Cell Physiol. 51: 463–474, 2010.CrossRefPubMedGoogle Scholar
  53. Zhao, D., Tang, W., Hao, Z., Tao, J.: Identification of flavonoids and expression of flavonoid biosynthetic genes in two coloured tree peony flowers. — Biochem. Biophys. Res. Commun. 459: 450–456, 2015a.CrossRefPubMedGoogle Scholar
  54. Zhao, X., Yuan, Z., Feng, L., Fang, Y.: Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate. — J. Plant Res. 128: 687–696, 2015b.CrossRefPubMedGoogle Scholar

Copyright information

© The Institute of Experimental Botany 2018

Authors and Affiliations

  • M. Otani
    • 1
  • Y. Kanemaki
    • 1
  • F. Oba
    • 2
  • M. Shibuya
    • 2
  • Y. Funayama
    • 1
  • M. Nakano
    • 1
  1. 1.Graduate School of Science and TechnologyNiigata UniversityNiigataJapan
  2. 2.Faculty of AgricultureNiigata UniversityNiigataJapan

Personalised recommendations