基于花青素苷合成和呈色机理的观赏植物花色改良分子育种

doi:10.3864/j.issn.0578-1752.2016.03.011

摘要/Abstract

摘要： 花色是观赏植物最重要的品质性状之一，是植物自然进化过程中最具适应意义的表型性状，也是表观遗传学研究的重要内容。花青素苷是使花朵呈色的重要色素之一，被子植物中约有80%的科的花朵颜色由花青素苷决定；迄今从自然界分离和鉴定出的花青素苷多达600种，主要由6种花青素苷元衍生而来。花青素苷合成途径是迄今为止研究得最为清楚的植物次生代谢途径之一，它的合成首先取决于类黄酮代谢途径的生成，花青素苷种类的多样性则源于其不同分支途径的形成，在花青素苷元基本骨架上不同位置取代基的差异形成了多种多样的花青素苷。在花青素苷生物合成过程中，分支点酶的竞争机制和关键酶的底物特异性使花青素苷的种类及相应的花色表型具有种属特异性。花青素苷合成后需要转运到液泡中被包裹成色素体，植物细胞中的液泡积累和贮存色素体的能力是影响花青素苷呈色的重要因素，因此，花青素苷在花瓣中的最终呈色还受液泡pH、助色素含量以及金属离子的络合作用等多种细胞内因素的影响。目前，已经在多种植物中获得了与花青素苷合成及呈色相关的结构基因和调节基因，并解析了其功能，成功获得了一些转基因花卉，但是这些基因调控表达的机制，包括转录水平和转录后水平的调控、DNA序列本身的差异和DNA甲基化修饰的调控机制等仍不清楚，转基因植株花色改良的程度也很有限，对于如何将这些机制应用于花色改良的转基因育种也是一个前沿的课题。花青素苷对园艺作物器官呈色机制的解析有助于对花朵呈色机制的理解，观赏植物中花色形成机理的研究对于园艺作物器官呈色机制的解析同样具有重要的参考价值。因此，本文以观赏植物为例，从花青素苷合成分支途径形成的机理、花青素苷生物合成途径的遗传调控机理以及影响花青素苷呈色的主要因素及其遗传调控机理3个方面，对影响植物花朵呈色的机制进行了综述，并对近年来基于花青素苷代谢和呈色机理的花色改良分子设计育种，尤其是国际上广泛关注的蓝色花育种进行了梳理和总结，以期为定向培育具有新奇花色的观赏植物新品种提供参考。

关键词: 观赏植物, 花青素苷, 生物合成, 呈色机理, 遗传调控, 分子育种

Abstract: Flower color, one of the most important quality traits for ornamental plants, is of great adaptive significance during the natural evolution process of plants. Moreover, flower color is also an important content for epigenetic researches. Anthocyanins, the most important pigments for flower coloration, designate the flower colors of approximately 80% plant families in angiosperm. Up to date, more than 400 anthocyanins have been isolated and identified from the natural world, which are mainly derived from six anthocyanidins. The biosynthetic pathway of anthocyanins has been well studied, which starts from the flavonoid metabolic pathway. Different branch pathways result in the diversity of anthocyanins, mainly due to the differences of substituent groups that are located on the basic skeleton of various anthocyanidins. During the biosynthetic process of anthocyanins, the competition forces of enzymes which are located on the branch nodes and the substrate specificity of some key enzymes result in the genus and species specificity of anthocyanins and the corresponding flower color phenotypes. Anthocyanins are transferred to vacuole and are packaged as chromatophore after being biosynthesized. The accumulation and conserve abilities on the chromatophore of vacuole affect the coloration of anthocyanins to a large extent. Therefore, many intracellular factors, such as the pH value of vacuole, the content of co-pigments and the complexation of metal ion, jointly affect the final coloration of anthocyanins in the petals. At present, some structural and regulatory genes that are related to the anthocyanins biosynthesis and coloration have been isolated, whose functions also have been well revealed. Based on these genes, some transgenic flowers have been successfully bred out. However, the mechanisms of gene regulating expression, including the regulation mechanisms on the transcriptional and post-transcriptional levels, and the differences of DNA sequences and the DNA methylation, still remain elusive. Moreover, the present modifications on the flower colors are still very limited. Therefore, how to apply these mechanisms on the transgenic breeding of flower color modification is a frontier topic. Revealing the organ coloration mechanisms based on anthocyanins in horticultural crops is conducive to the understanding of coloration mechanisms of flowers; studies on the mechanisms of flower color in ornamental plants is of important reference value for the reveal of the organ coloration mechanisms in horticultural crops. Therefore, in this paper, we reviewed the mechanisms of flower coloration in ornamental plants from three aspects, including the mechanisms of the branch pathways generation, the genetic regulation mechanisms of anthocyanins biosynthetic pathway, and the main factors affecting the anthocyanins coloration and the corresponding genetic regulation mechanisms. Finally, we summarized the successes of molecular design breeding on flower color modification based on these mechanisms, especially the international-concerned molecular breeding for blue flowers, aiming at providing references for the directive breeding of ornamental plants with novel flower colors.

Key words: ornamental plant, anthocyanin, biosynthesis, coloration mechanism, genetic regulation, molecular breeding

戴思兰，洪艳. 基于花青素苷合成和呈色机理的观赏植物花色改良分子育种[J]. 中国农业科学, 2016, 49(3): 529-542.

DAI Si-lan, HONG Yan. Molecular Breeding for Flower Colors Modification on Ornamental Plants Based on the Mechanism of Anthocyanins Biosynthesis and Coloration[J]. Scientia Agricultura Sinica, 2016, 49(3): 529-542.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.03.011

https://www.chinaagrisci.com/CN/Y2016/V49/I3/529

参考文献

[1] Tanaka Y, Ohmiya A. Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Current Opinion in Biotechnology, 2008, 19(2): 190-197.

[2] Tanaka Y, Brugliera F. Flower colour and cytochromes P450. Philosophical Transactions of the Royal Society B-Biological Sciences, 2013, 5(2/3): 283-291.

[3] Yoshida K, Mori M, Kondo T. Blue flower color development by anthocyanins: from chemical structure to cell physiology. Natural Product Reports, 2009, 26(7): 884-915.

[4] 胡可, 韩科厅, 戴思兰. 环境因子调控植物花青素苷合成及呈色的机理. 植物学报, 2010, 45(3): 307-317.

Hu K, Han K T, Dai S L. Regulation of plant anthocyanin synthesis and pigmentation by environmental factors. Chinese Bulletin of Botany, 2010, 45(3): 307-317. (in Chinese)

[5] Hong Y, Tang X J, Huang H, Zhang Y, Dai S L. Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum. BMC Genomics, 2015, 16: 202.

[6] Van der Krol A R, Lenting P E, Veenstra J, van der Meer I M, Koes R E, Gerats A G, Mol J N, Stuitje A R. An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature, 1988, 333: 866-869.

[7] Nishihara M, Nakatsuka T. Genetic engineering of flavonoid pigments to modify flower color in floricultural plants. Biotechnology Letters, 2011, 33(3): 433-441.

[8] Chandler S F, Sanchez C. Genetic modification; the development of transgenic ornamental plant varieties. Plant Biotechnology Journal, 2012, 10: 891-903.

[9] Kong J M, Chia S, Goh N K, Chia T F, Brouillard R. Analysis and biological activities of anthocyanins. Phytochemistry, 2003, 64(5): 923-933.

[10] Castañeda-Ovando A, de Lourdes Pacheco-Hernández M, Páez-Hernández M E, Rodríguez J A, Galán-Vidal C A. Chemical studies of anthocyanins: a review. Food Chemistry, 2009, 113(4): 859-871.

[11] Pelletier M K, Murrell J R, Shirley B W. Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis (further evidence for differential regulation of “early” and “late” genes). Plant Physiology, 1997, 113(4): 1437-1445.

[12] Quattrocchio F, Wing J F, Leppen H, Mol J, Koes R E. Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell, 1993, 5: 1497-1512.

[13] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 2001, 126(2): 485-493.

[14] Nakatsuka T, Sasaki N, Nishihara M. Transcriptional regulators of flavonoid biosynthesis and their application to flower color modification in Japanese gentians. Plant Biotechnology, 2014, 31: 389-399.

[15] Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E. Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant Journal, 1991, 1: 37-49.

[16] Kubasek W L, Shirley B W, McKillop A, Goodman H M, Briggs W, Ausubel F M. Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. Plant Cell, 1992, 4: 1229-1236.

[17] Pelletier M K, Shirley B W. Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase. Plant Physiology, 1996, 111: 339-345.

[18] Spribille R, Forkmann G. Genetic control of chalcone synthase activity in flowers of Antirrhinum majus. Phytochemistry, 1982, 21: 2231-2234.

[19] Hemleben V, Dressel A, Epping B, Lukacin R, Martens S, Austin M B. Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae). Plant Molecular Biology, 2004, 55: 455-465.

[20] Hoshino A, Park K I, Iida S. Identification of r mutations conferring white flowers in the Japanese morning glory (Ipomoea nil). Journal of Plant Research, 2009, 122: 215-222.

[21] Koes R E, Spelt C E, van den Elzen P J M, Mol J N M. Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene, 1989, 81: 245-257.

[22] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co- suppression of homologous genes in trans. Plant Cell, 1990, 2: 279-289.

[23] Deng X, Bashandy H, Ainasoja M, Kontturi J, Pietiainen M, Laitinen R A E, Albert V A, Valkonen J P T, Elomaa P, Teeri T H. Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida. New Phytologist, 2014, 201(4): 1469-1483.

[24] Tanaka Y, Brugliera F, Kalc G, Senior M, Dyson B, Nakamura N, Katsumoto Y, Chandler S. Flower color modification by engineering of the flavonoid biosynthetic pathway: practical perspectives. Bioscience, Biotechnology, and Biochemistry, 2010, 74(9): 1760-1769.

[25] 孟丽, 戴思兰. F3′5′H基因与蓝色花的形成. 分子植物育种, 2004, 2(3): 413-420.

Meng L, Dai S L. F3′5′H genes regulation and blue flowers formation. Molecular Plant Breeding, 2004, 2(3): 413-420. (in Chinese)

[26] 徐清燏, 戴思兰. 蓝色花卉分子育种. 分子植物育种, 2004, 2(1): 93-99.

Xu Q Y, Dai S L. Blue flowers’ molecular breeding. Molecular Plant Breeding, 2004, 2(1): 93-99. (in Chinese)

[27] Nakatsuka T, Nishihara M, Mishiba K, Hirano H, Yamamura S. Two different transposable elements inserted in flavonoid 3′, 5′-hydroxylase gene contribute to pink flower coloration in Gentiana scabra. Molecular Genetics & Genomics, 2006, 275(3): 231-241.

[28] Sato M, Kawabe T, Hosokawa M, Tatsuzawa F, Doi M. Tissue culture-induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3′, 5′ hydroxylase (F3′5′H) promoter. Plant Cell Reports, 2011, 30: 929-939.

[29] Brugliera F, Barri-Rewell G, Holton T A, Mason J G. Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant Journal, 1999, 19: 441-451.

[30] Sun Y, Huang H, Meng L, Hu K, Dai S L. Isolation and functional analysis of a homolog of flavonoid 3′, 5′-hydroxylase gene from Pericallis×hybrida. Physiologia Plantarum, 2013, 149(2): 151-159.

[31] Seitz C, Eder C, Deiml B, Kellner S, Martens S, Forkmann G. Cloning, functional identification and sequence analysis of flavonoid 3′- hydroxylase and flavonoid 3′, 5′-hydroxylase cDNAs reveals independent evolution of flavonoid 3′,5′-hydroxylase in the Asteraceae family. Plant Molecular Biology, 2006, 61(3): 365-381.

[32] Huang H, Hu K, Han K T, Xiang Q Y, Dai S L. Flower colour modification of chrysanthemum by suppression of F3′H and overexpression of the exogenous Senecio cruentus F3′5′H gene. Plos One, 2013, 8: e74395.

[33] 胡可, 孟丽, 韩科厅, 戴思兰. 瓜叶菊花青素苷合成关键基因的分离及表达的初步分析. 园艺学报, 2009, 36(7): 1013-1022.

Hu K, Meng L, Han K T, Sun Y, Dai S L. Isolation and expression analysis of key genes involved in anthocyanin biosynthesis of cineraria. Acta Horticulturae Sinica, 2009, 36(7): 1013-1022. (in Chinese)

[34] Nielsen K, Deroles S C, Markham K R, Bradley M J, Podivinsky E, Manson D. Antisense flavonol synthase alters copigmentation and flower color in lisianthus. Molecular Breeding, 2002, 9: 217-229, 2002.

[35] Davies K M, Schwinn K E, Deroles S C, Manson D G, Lewis D H, Bloor S J, Bradley J M. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica, 2003, 131(3): 259-268.

[36] Tsuda S, Fukui Y, Nakamura N, Katsumoto Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Ohira K, Ueyama Y, Ohkawa H, Holton T A, Kusumi T, Tanaka Y. Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnology, 2004, 21(5): 377-386.

[37] Forkmann G, Ruhnau B. Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrida. Zeitschrift Fuer Naturforschung C, 1987, 42(9/10): 1146-1148.

[38] Johnson E T, Yi H, Shin B, Oh B J, Cheong H, Choi G. Cymbidium hybrid a dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins. Plant Journal, 1999, 19(1): 81-85.

[39] Zhang Y, Cheng S, De Jong D, Griffiths H, Halitschke R, De Jong W. The potato R locus codes for dihydroflavonol 4-reductase. Theoretical & Applied Genetics, 2009, 119(5): 931-937.

[40] Leonard E, Yan Y, Chemler J, Matern U, Martens S, Koffas M A G. Characterization of dihydroflavonol 4-reductases for recombinant plant pigment biosynthesis applications. Biocatalysis and Biotransformation, 2008, 26(3): 243-251.

[41] Trabelsi N, d’Estaintot B L, Sigaud G, Gallois B, Chaudière J. Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from Vitis vinifera and its unusually strong substrate inhibition. Journal of Biophysical Chemistry, 2011, 2(3): 332-344.

[42] He F, Mu L, Yan G, Liang N, Pan Q, Wang J, Reeves M J, Duan C. Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules, 2010, 15(12): 9057-9091.

[43] Yamazaki M, Gong Z Z, Fukuchi-Mizutani M, FukuiiY, Tanaka Y, Kusumi T, Saito K. Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin. Journal of Biological Chemistry, 1999, 274(11): 7405-7411.

[44] Tanaka Y, Yonekura K, Fukuchi-Mizutani M, Fukui Y, Fujiwara H, Ashikari T, Kusumi T. Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora. Plant Cell Physiology, 1996, 37: 711-716.

[45] Fukuchi-Mizutani M, Okuhara H, Fukui Y, Nakao M, Katsumoto Y, Yonekura-Sakakibara K, Kusumi T, Hase T, Tanaka Y. Biochemical and molecular characterization of a novel UDP-glucose: anthocyanin 3′-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian. Plant Physiology, 2003, 132: 1652-1663.

[46] Nakatsuka T, Sato K, Takahashi H, Yamamura S, Nishihara M. Cloning and characterization of the UDP-glucose: anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian. Journal of Experimental Botany, 2008, 59(6): 1241-1252.

[47] Nakatsuka T, Nishihara M. UDP-glucose: 3-deoxyanthocyanidin 5-O-glucosyltransferase from Sinningia cardinalis. Planta, 2010, 232(2): 383-392.

[48] Cheng J, Wei G, Zhou H, Gu C, Vimolmangkang S, Liao L, Han Y. Unraveling the mechanism underlying the glycosylation and methylation of anthocyanins in peach. Plant Physiology, 2014, 166(2): 1044-1058.

[49] Du H, Wu J, Ji K X, Bhuiya M W, Su S, Shu Q Y, Ren H X, Liu Z A, Wang L S. Methylation mediated by an anthocyanin, O- methyltransferase, is involved in purple flower coloration in Paeonia. Journal of Experimental Botany, 2015, doi: 10.1093/jxb/erv365.

[50] Ramsay N A, Glover B J. MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends in Plant Science, 2005, 10(2): 63-70.

[51] Xu W, Grain D, Le Gourrierec J, Harscoet E, Berger A, Jauvion V, Scagnelli A, Berger N, Bidzinski P, Kelemen Z K, Salsac F, Baudry A, Routaboul J, Lepiniec L, Dubos C. Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis. New Phytologist, 2013, 198: 59-70.

[52] Tohge T, Nishiyama Y, Hirai M Y, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H, Goodenowe D B, Kitayama M, Noji M, Yamazaki M, Saito K. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants overexpressing an MYB transcription factor. Plant Journal, 2005, 42: 218-235.

[53] Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant Journal, 2008, 53: 814-827.

[54] 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 Physiology, 2008, 49(12): 1818-1829.

[55] Nakatsuka T, Saito M, Yamada E, Fujita K, Kakizaki Y, Nishihara M. Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers. Journal of Experimental Botany, 2012, 63: 6505-6517.

[56] Kobayashi S, Ishimaru M, Hiraoka K, Honda C. Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis. Planta, 2002, 215(6): 924-933.

[57] Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris N N, Walker A R, Robinson S P, Bogs J. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiology, 2009, 151(3): 1513-1530.

[58] Quattrocchio F, Verweij W, Kroon A C, Mol J, Koes R. PH4 of petunia is an R2R3 MYB protein that activates vacuolar acidification through interactions with basic-helix-loop-helix transcription factors of the anthocyanin pathway. Plant Cell, 2006, 18(5): 1274-1291.

[59] Espley R V, Hellens R P, Putterill J, Stevenson D E, Kutty-Amma S, Allan A C. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal, 2007, 49(3): 414-427.

[60] Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant and Cell Physiology, 2007, 48(7): 958-970.

[61] Lai Y S, Li H X, Yamagishi M. A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity. Frontiers in Biology, 2013, 8(6): 577-598.

[62] Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie T K, Espley R V, Hellens R P, Allan A C. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biology, 2010, 10(2): 549-553.

[63] Aharoni A, De Vos C H, Wein M , Sun Z, Greco R, Kroon A, Moi J N, O’Connell A P. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal, 2001, 28(3): 319-332.

[64] Matsui K, Umemura Y, Ohme-Takagi M. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant Journal, 2008, 55: 954-967.

[65] Albert N W, Davies K M, Lewis D H, Zhang H, Montefiori M, Brendolise C, Boase M R, Ngo H, Jameson P E, Schwinn K E. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell, 2014, 26: 962-980.

[66] Li Y Y, Mao K, Zhao C, Zhao X Y, Zhang H L, Shu H R, Hao Y J. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiology, 2012, 160(2): 1011-1022.

[67] Shin D H, Choi M, Kim K, Bang G, Cho M, Choi S, Choi G, Park Y. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Letters, 2013, 587 (10): 1543-1547.

[68] Yang F, Cai J, Yang Y, Liu Z. Overexpression of microRNA828 reduces anthocyanin accumulation in Arabidopsis. Plant Cell, Tissue and Organ Culture, 2013, 115(2): 159-167.

[69] Gou J Y, Felippes F F, Liu C J, Weigel D, Wang J W. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell, 2011, 23(4): 1512-1522.

[70] Gasciolli V, Mallory A C, Bartel D P, Vaucheret H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Current Biology, 2005, 15(16): 1494-1500.

[71] Ohno S, Hosokawa M, Kojima M, Kitamura Y, Hoshino A, Tatsuzawa F, Doi M, Yazawa S. Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia. Planta, 2011, 234: 945-958.

[72] Morita Y, Saito R, Ban Y, Tanikawa N, Kuchitsu K, Ando T, Yoshikawa M, Habu Y, Ozeki Y, Nakayama M. Tandemly arranged chalcone synthase A genes contribute to the spatially regulated expression of siRNA and the natural bicolor floral phenotype in Petunia hybrida. Plant Journal, 2012, 70: 739-749.

[73] Espley R, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J, Schouten H J, Gardiner S E, Hellens R P, Allan A C. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apple. Plant Cell, 2009, 21: 168-183.

[74] Kobayashi S, Goto-Yamamoto N, Hirochika H. Retrotransposon- induced mutations in grape skin color. Science, 2004, 304(5673): 982-982.

[75] Durbin M L, Denton A L, Clegg M T. Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea). Proceedings of the National Academy of Sciences of the USA, 2001, 98(9): 5084-5089.

[76] Butelli E, Licciardello C, Zhang Y, Liu J, Mackay S, Bailey P, Reforgiato-Recupero G, Martin C. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. The Plant Cell Online, 2012, 24(3): 1242-1255.

[77] Cocciolone S M, Cone K C. Pl-Bh, an anthocyanin regulatory gene of maize that leads to variegated pigmentation. Genetics, 1993, 135: 575-588.

[78] Sekhon R S, Chopra S. Progressive loss of DNA methylation releases epigenetic gene silencing from a tandemly repeated maize Myb gene. Genetics, 2009, 181: 81-91.

[79] Telias A, Lin-Wang K, Stevenson D E, Cooney J M, Hellens R P, Allan A C, Hoover E E, Bradeen J M. Apple skin patterning is associated with differential expression of MYB10. BMC Plant Biology, 2011, 11: 93-107.

[80] Xu Y, Feng S, Jiao Q, Liu C, Zhang W, Chen W, Chen X. Comparison of MdMYB1 sequences and expression of anthocyanin biosynthetic and regulatory genes between Malus domestica Borkh. cultivar ‘Ralls’ and its blushed sport. Euphytica, 2012, 185: 157-170.

[81] Liu X J, Chuang Y N, Chiou C Y, Chin D C, Shen F Q, Yeh K W. Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars. Planta, 2012, 236(2): 401-409.

[82] Wang Z, Meng D, Wang A, Li T, Jiang S, Cong P, Li T. The methylation of PcMYB10 promoter is associated with green skinned sport in ‘Max Red Bartlett’ pear. Plant Physiology, 2013, 162: 885-896.

[83] Qian M J, Sun Y, Allan A C, Teng Y W, Zhang D. The red sport of ‘Zaosu’ pear and its red-striped pigmentation pattern are associated with demethylation of the PyMYB10 promoter. Phytochemistry, 2014, 107: 16-23.

[84] Schenke D, Cai D, Scheel D. Suppression of UV-B stress responses by flg22 is regulated at the chromatin level via histone modification. Plant, Cell & Environment, 2014, 37(7): 1716-1721.

[85] Holton T A, Brugliera F, Tanaka Y. Cloning and expression of flavonol synthase from Petunia hybrid. Plant Journal, 1993, 4(6): 1003-1010.

[86] Mol J, Grotewold E, Koes R. How genes paint flowers and seeds. Trends in Plant Science, 1998, 3(6): 212-217.

[87] Fukada-Tanaka S, Inagaki Y, Yamaguchi T, Saito N, Iida S. Colour- enhancing protein in blue petals. Nature, 2000, 407(6804): 581-581.

[88] Yamaguchi T, Fukada-Tanaka S, Inagaki Y, Saito N, Yonekura- Sakakibara K, Tanaka Y, Kusumi T, Iida S. Genes encoding the vacuolar Na⁺/H⁺exchanger and flower coloration. Plant and Cell Physiology, 2001, 42(5): 451-461.

[89] Yoshida K, Miki N, Momonoi K, Kawachi M, Katou K, Okazaki Y, Uozumi N, Maeshima M, Kondo T. Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue. Proceedings of the Japan Academy Series B, 2009, 85(6): 187-197.

[90] Verweij W, Spelt C, Di Sansebastiano G P, Vermeer J, Reale L, Ferranti F, Koes R E, Quattrocchio F. A novel type of tonoplast localized H⁺-ATPase is required for vacuolar acidification and coloration of flowers and seeds. Nature Cell Biology, 2008, 10: 1456-1462.

[91] Faraco M, Spelt C, Bliek M, Verweij W, Hoshino A, Espen L, Prinsi B, Jaarsma R, Tarhan E, de Boer A H, Di Sansebastiano G P, Koes R, Quattrocchio F M. Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower color. Cell Reports, 2014, 6(1): 32-43.

[92] Shoji K, Miki N, Nakajima N, Momonoi K, Kato C, Yoshida K. Perianth bottom-specific blue color development in tulip cv. Murasakizuisho requires ferric ions. Plant & Cell Physiology, 2007, 48(2): 243-251.

[93] Momonoi K, Yoshida K, Mano S, Takahashi H, Nakamori C, Shoji K, Nitta A, Nishimura M. A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation. Plant Journal, 2009, 59(3): 437-447.

[94] Shoji K, Momonoi K, Tsuji T. Alternative expression of vacuolar iron transporter and ferritin genes leads to blue/purple coloration of flowers in Tulip cv. ‘Murasakizuisho’. Plant & Cell Physiology, 2010, 51(2): 215-224.

[95] Yoshida K, Negishi T. The identification of a vacuolar iron transporter involved in the blue coloration of cornflower petals. Phytochemistry, 2013, 94(5): 60-67.

[96] Ito D, Shinkai Y, Kato Y, Kondo T, Yoshida K. Chemical studies on different color development in blue-and red-colored sepal cells of Hydrangea macrophylla. Bioscience, Biotechnology, & Biochemistry, 2009, 73(5): 1054-1059.

[97] 韩科厅, 胡可, 戴思兰. 观赏植物花色的分子设计. 分子植物育种, 2008, 6(1): 16-24.

Han K T, Hu K, Dai S L. Flower color breeding by molecular design in ornamentals. Molecular Plant Breeding, 2008, 6(1): 16-24. (in Chinese)

[98] Dare A P, Tomes S, Jones M, McGhie T K, Stevenson D E, Johnson R A, Greenwood D R, Hellens R P. Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus× domestica). Plant Journal, 2013, 74: 398-410.

[99] Fukusaki E I, Kawasaki K, Kajiyama S, An C, Suzuki K, Tanaka Y, Kobayashi A. Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference. Journal of Biotechnology, 2004, 111(3): 229-240.

[100] Nakamura N, Fukuchi-Mizutani M, Miyazaki K, Suzuki K, Tanaka Y. 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 Biotechnology, 2006, 23(1): 13-17.

[101] Nakamura N, Masako F M, Fukui Y, Ishiguro K, Suzuki K, Suzuki H, Okazaki K, Shibata D, Tanaka T. Generation of pink flower varieties from blue Torenia hybrida by redirecting the flavonoid biosynthetic pathway from delphinidin to pelargonidin. Plant Biotechnology, 2010, 27(5): 375-383.

[102] Nakatsuka T, Mishiba K I, Kubota A, Abe Y, Yamamura S, Nakamura N, Tanaka Y, Nishihara M. Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian. Journal of Plant Physiology, 2010, 167(3): 231-237.

[103] Chen W H, Hsu C Y, Cheng H Y, Chang H, Chen H H, Ger M J. Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis. Plant Cell Reports, 2011, 30(6): 1007-1017.

[104] Okinaka Y, Shimada Y R, Ohbayashi M, Kiyokawa S. Selective accumulation of delphinidin derivatives in tobacco using a putative flavonoid 3′,5′-hydroxylase cDNA from Campanula medium. Bioscience, Biotechnology & Biochemistry, 2003, 67(1): 161-165.

[105] Mori S, Kobayashi H, Hoshi Y, Kondo M, Nakano M. Heterologous expression of the flavonoid 3′, 5′-hydroxylase gene of Vinca major alters flower color in transgenic Petunia hybrida. Plant Cell Reports, 2004, 22(6): 415-421.

[106] Han Y P, Vimolmangkang S, Soria-Guerra R E, Korban S S. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. Journal of Experimental Botany, 2012, 63(7): 2437-2447.

[107] Abeynayake S W, Panter S, Chapman R, Webster T, Rochfort S, Mouradov A, Spangenberg G. Biosynthesis of proanthocyanidins in white clover flowers: cross talk within the flavonoid pathway. Plant Physiology, 2012, 158(2): 666-678.

[108] Zhou X W, Fan Z Q, Chen Y, Zhu Y L, Li J Y, Yin H F. Functional analyses of a flavonol synthase-like gene from Camellia nitidissima reveal its roles in flavonoid metabolism during floral pigmentation. Journal of Biosciences, 2013, 38: 593-604.

[109] Vetten N D, Horst J T, Schaik H P V, de Boer A, Mol J, Koes R. A cytochrome b5 is required for full activity of flavonoid 3′, 5′- hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proceedings of the National Academy of Sciences of the USA, 1999, 96(2): 778-783.

[110] Yukihisa K, Masako F M, Yuko F, 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 and Cell Physiology, 2007, 48(11): 1589-1600.

[111] Brugliera F, Tao G Q, Tems U, Kalc G, Mouradova E, Price K, Stevenson K, Nakamura N, Stacey I, Katsumoto Y, Tanaka Y, Mason J G. Violet/blue chrysanthemums-metabolic engineering of anthocyanin biosynthetic pathway results in novel petal colours. Plant and Cell Physiology, 2013, 54(10): 1696-1710.

[112] Seitz C, Ameres S, Forkmann G. Identification of the molecular basis for the functional difference between flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase. FEBS Letters, 2007, 581(18): 3429-3434.

[113] Ben Zvi M M, Negre-Zakharov F, Masci T, Ovadis M, Shklarman E, Ben-Meir H, Tzfira T, Dudareva N, Vainstein A. Interlinking showy traits: co-engineering of scent and colour biosynthesis in flowers. Plant Biotechnology Journal, 2008, 6: 403-415.

[114] Ben Zvi M M, Shklarman E, Masci T, Kalev H, Debener T, Shafir S, Ovadis M, Vainstein A. PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers. New Phytologist, 2012, 195: 335-345.

[115] Luo J, Butelli E, Hill L, Parr A, Niggeweg R, Bailey P, Weisshaar B, Martin C. AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol. Plant Journal, 2008, 56: 316-326.

[116] Nakatsuka T, Yamada E, Saito M, Fujita K, Nishihara M. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers. Plant Cell Reports, 2013, 32: 1925-1937.

[117] Nakatsuka T, Saito M, Yamada E, Nishihara M. Production of picotee- type flowers in Japanese gentian by CRES-T. Plant Biotechnology, 2011, 28(2): 173-180.

[118] Aguilar-Barragán A, Ochoa-Alejo N. Virus-induced silencing of MYB and WD40 transcription factor genes affects the accumulation of anthocyanins in chilli pepper fruit. Biologia Plantarum, 2014, 58(3): 567-574.

[119]王璐, 戴思兰, 金雪花, 黄河, 洪艳. 植物花青素苷转运机制的研究进展. 生物工程学报, 2014, 30(6): 848-863.

Wang L, Dai S L, Jin X H, Huang H, Hong Y. Advances in plant anthocyanin transport mechanism. Chinese Journal of Biotechnology, 2014, 30(6): 848-863. (in Chinese)