Abstract
Chalcone synthase (CHS) is the key enzyme in an early stage of the flavonoid biosynthetic pathway. In the present study, a full-length cDNA clone for CHS was isolated from flower tepals of the liliaceous ornamental Tricyrtis sp., in which tepals have many reddish-purple spots resulting from accumulation of cyanidin derivatives. The deduced amino acid sequence of the isolated cDNA clone, designated TrCHS1 (accession number AB478624 in the GenBank/EMBL/DDBJ databases), shows 79.4–91.4% identity with those of previously reported CHS genes. An RNA interference (RNAi) construct targeting TrCHS1 was introduced by Agrobacterium-mediated transformation in order to alter the flower color of Tricyrtis sp. Seven transgenic plants that produced flowers could be classified into three types according to flower color phenotype: one transgenic plant had tepals with as many reddish-purple spots as non-transgenic plants (Type I); one had tepals with reduced numbers of reddish-purple spots (Type II); and five had completely white tepals without any spots (Type III). High-performance liquid chromatography analysis showed that tepals of Type III transgenic plants did not accumulate detectable amounts of anthocyanidins. In addition, TrCHS1 mRNA levels in tepals of Type II and Type III transgenic plants decreased substantially compared with non-transgenic plants, as determined by quantitative real-time reverse transcription–polymerase chain reaction analysis. Our results indicate the validity of RNAi suppression of the flavonoid biosynthetic pathway genes for flower color alteration in Tricyrtis sp. To the best of our knowledge, this is the first report on flower color alteration by genetic transformation in monocotyledonous ornamentals.
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References
Adachi Y, Mori S, Nakano M (2005) Agrobacterium-mediated production of transgenic plants in Tricyrtis hirta (Liliaceae). Acta Hort 673:415–419
Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990
Courtney-Gutterson N, Napoli C, Lemieux C, Morgan A, Firoozabady E, Robinson KE (1994) Modification of flower color in florist’s chrysanthemum: production of a white-flowering variety through molecular genetics. Biotechnology 12:268–271
de Mesa MC, Santiago-Doménech N, Pliego-Alfaro F, Quesada MA, Mercado JA (2004) The CaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants. Plant Cell Rep 23:32–38
Deroles SC, Bradley JM, Schwinn KE, Markham KR, Bloor S, Manson DG, Davies KM (1998) An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers. Mol Breed 4:59–66
Durbin ML, Learn GH Jr, Huttley GA, Clegg MT (1995) Evolution of the chalcone synthase gene family in the genus Ipomoea. Proc Natl Acad Sci USA 92:3338–3342
Elomaa P, Honkanen J, Puska R, Seppäne P, Helariutta Y, Mehto M, Kotilainen M, Nevalainen L, Teeri TH (1993) Agrobacterium-mediated transfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower pigmentation. Biotechnology 11:508–511
Forkmann G (1991) Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering. Plant Breed 106:1–26
Gutterson N (1995) Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression. HortSci 30:964–966
Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083
Hoshi Y, Kondo M, Mori S, Adachi Y, Nakano M, Kobayashi H (2004) Production of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep 22:359–364
Johzuka-Hisatomi Y, Hoshino A, Mori T, Habu Y, Iida S (1999) Characterization of the chalcone synthase genes expressed in flowers of the common and Japanese morning glories. Genet Syst 74:141–147
Karimi M, Inze D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195
Katsumoto Y, Fukushi-Mizutani M, Fukui Y, Brugliera F, Holton TA, Karan M, Nakamura N, Yonekura-sakakibara K, Togami J, Pigeaire A, Tao G, Nehra N, Lu C, Dyson B, Tsuda S, Ashikari T, Kusumi T, Mason J, Tanaka Y (2007) Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol 48:1589–1600
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120
Koes RE, Spelt CE, van den Elzen PJM, Mol JNM (1989) Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene 81:245–257
Li J, Ou-Lee T-M, Raba R, Amundson RG, Last RL (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5:171–179
Mattanovich D, Rüker F, Machado AC, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res 17:6747
Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:667–678
Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495
Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138:1903–1913
Mori S, Asano S, Kobayashi H, Nakano M (2002) Analysis of anthocyanidins and anthocyanins in flowers of Muscari spp. Bull Fac Agric Niigata Univ 55:13–18
Mori S, Oka E, Umehara H, Suzuki S, Kobayashi H, Hoshi Y, Kondo M, Koike Y, Nakano M (2007) Somaclonal variation and stability of GUS gene expression in transgenic agapanthus (Agapanthus praecox ssp. orientalis) plants at the flowering stage. In Vitro Cell Dev Biol Plant 43:79–87
Mori S, Oka E, Umehara H, Kobayashi H, Hoshi Y, Kondo M, Koike Y, Nakano M (2008) Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation. Biol Plant 52:513–516
Nakamura N, Fukuchi-Mizutani M, Miyazaki K, Suzuki K, Tanaka Y (2006) RNAi suppression of the anthocyanidin synthase gene in Torenia hybrida yields white flowers with higher frequency and better stability than antisense and sense suppression. Plant Biotechnol 23:13–17
Nakano M, Mizunashi K, Tanaka S, Godo T, Nakata M, Saito H (2004) Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus Tricyrtis. In Vitro Cell Dev Biol Plant 40:274–278
Nakano M, Mori S, Suzuki S, Hoshi Y, Kobayashi H (2006) Production of transgenic plants via Agrobacterium-mediated transformation in Liliaceous ornamentals. In: da Silva JAT (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues, vol II. Global Science Books, Middlesex, pp 172–183
Nakatsuka T, Izumi Y, Yamagishi M (2003) Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily. Plant Sci 165:759–767
Nakatsuka T, Mishiba K, Abe Y, Kakizaki Y, Yamamura S, Nishihara M (2008) Flower color modification of gentian plants by RNAi-mediated gene silencing. Plant Biotechnol 25:61–68
Nishihara M, Nakatsuka T, Hosokawa K, Yokoi T, Abe Y, Mishiba K, Yamamura S (2006) Dominant inheritance of white-flowered and herbicide-resistant traits in transgenic gentian plants. Plant Biotechnol 23:25–31
Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. CABIOS 12:357–358
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160
Suzuki K, Xue H, Tanaka Y, Fukui Y, Fukuchi-Mizutani M, Murakami Y, Katsumoto Y, Tsuda S, Kusumi T (2000) Flower color modification of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes. Mol Breed 6:239–246
Tatsuzawa F, Saito N, Miyoshi K, Shinoda K, Shigihara A, Honda T (2004) Diacylated 8-C-glucosylcyanidin 3-glucoside from the flowers of Tricyrtis formosana. Chem Pharm Bull 52:631–633
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 22:4673–4680
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 hybrida commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386
Waterhouse PM, Graham MW, Wang M-B (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964
Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5:218–223
Acknowledgments
We gratefully acknowledge Prof. K. Shimamoto and Dr. D. Miki, Nara Institute of Science and Technology, Japan, for the gift of the vector pANDA35HK. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 23580037) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Kamiishi, Y., Otani, M., Takagi, H. et al. Flower color alteration in the liliaceous ornamental Tricyrtis sp. by RNA interference-mediated suppression of the chalcone synthase gene. Mol Breeding 30, 671–680 (2012). https://doi.org/10.1007/s11032-011-9653-z
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DOI: https://doi.org/10.1007/s11032-011-9653-z