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

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Biologia Plantarum

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 expressions 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.

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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

References

  • Adachi, Y., Mori, S., Nakano, M.: Agrobacterium-mediated production of transgenic plants in Tricyrtis hirta (Liliaceae). - Acta Hort. 673: 415–419, 2005.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap. - Evolution 39: 783–791, 1985.

    Article  PubMed  Google Scholar 

  • Forkmann, G., Ruhnau, B. Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrid. - Z. Naturforsch C 42: 1146–1148, 1987.

    Article  CAS  Google Scholar 

  • Fornalé, S., Shi, X., Chai, C., Encina, A., Irar, S., Capellades, M., Fuguet, E., Torres, J.L., Rovira, P., Puigdomènech, 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Holton, T.A., Cornish, E.C.: Genetics andbBiochemistry of anthocyanin biosynthesis. - Plant Cell 7: 1071–1083, 1995.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Jaakola, L., Määttä, K., Pirttilä, A.M., Törrönen, R., Kärenlampi, 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Kumar, S., Stecher, G., Tamura, K.: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. - Mol. Biol. EVol. 33: 1870–1874, 2016.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Noda, N., Aida, R., Kishimoto, S., Ishiguro, K., Fukuchi-Mizutani, M., Tanaka, Y., Ohmiya, A.: Genetic engineering of novel bluer-colored chrysanthemums produced by accumulation of delphinidin-based anthocyanins. - Plant Cell Physiol. 54: 1684–1695, 2013.

    Article  PubMed  CAS  Google Scholar 

  • Nørbæk, R., Kondo, T.: Anthocyanins from flowers of Lilium (Liliaceae). - Phytochemistry 50: 1181–1184, 1999.

    Article  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Petroni, K., Tonelli, C.: Recent advances on the regulation of anthocyanin synthesis in reproductive organs. - Plant Sci. 181: 219–229, 2011.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rabino, I., Mancinelli, A.L.: Light, temperature, and anthocyanin production. - Plant Physiol. 81: 922–924, 1986.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Saitou, N., Nei, M.: The neighbor-joining method: A new method for reconstructing phylogenetic trees. - Mol. Biol. EVol. 4: 406–425, 1987.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Van Nocker, S., Ludwig, P.: The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function. - BMC Genomics 4: 50, 2003.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Winkel-Shirley, B.: Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. - Plant Physiol. 126: 485–493, 2001.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Graduate School of Science and Technology, Niigata University, 2-8050, Ikarashi, Nishi-ku, Niigata, 950-2181, Japan

    M. Otani, Y. Kanemaki, Y. Funayama & M. Nakano

  2. Faculty of Agriculture, Niigata University, 2-8050, Ikarashi, Nishi-ku, Niigata, 950-2181, Japan

    F. Oba & M. Shibuya

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  1. M. Otani
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  2. Y. Kanemaki
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  3. F. Oba
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  4. M. Shibuya
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  5. Y. Funayama
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  6. M. Nakano
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Correspondence to M. Nakano.

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Otani, M., Kanemaki, Y., Oba, F. et al. Comprehensive isolation and expression analysis of the flavonoid biosynthesis-related genes in Tricyrtis spp.. Biol Plant 62, 684–692 (2018). https://doi.org/10.1007/s10535-018-0802-7

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