Engineering of the Rose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers Accumulating Delphinidin

Plant Cell Physiol. 48(11): 1589–1600 (2007) doi:10.1093/pcp/pcm131, available FREE online at www.pcp.oxfordjournals.org ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Engineering of the Rose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers Accumulating Delphinidin Yukihisa Katsumoto 1, Masako Fukuchi-Mizutani 1, Yuko Fukui 1, Filippa Brugliera 2, Timothy A. Holton 2, Mirko Karan 2, Noriko Nakamura 1, Keiko Yonekura-Sakakibara 1, 3, Junichi Togami 1, Alix Pigeaire 2, Guo-Qing Tao 2, Narender S. Nehra 2, Chin-Yi Lu 2, Barry K. Dyson 2, Shinzo Tsuda 1, Toshihiko Ashikari 1, 2, Takaaki Kusumi 1, John G. Mason 2 and Yoshikazu Tanaka 1, * 1 Suntory Limited Research Center, Institute for Plant Science, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503 Japan 2 Florigene Pty. Ltd, 1 Park Drive, Bundoora, VIC 3083, Australia Flower color is mainly determined by anthocyanins. Rosa and birds. Flower color is also attractive to people and is hybrida lacks violet to blue flower varieties due to the absence an important characteristic of flowers from a floricultural of delphinidin-based anthocyanins, usually the major consti- viewpoint. Hybridization breeding has partially achieved tuents of violet and blue flowers, because roses do not possess the diversification of flower color of floricultural crops. flavonoid 30,50-hydoxylase (F3050H), a key enzyme for Plant species have adopted versatile and ingenious ways delphinidin biosynthesis. Other factors such as the presence to exhibit flower color (Grotewold 2006, Tanaka and of co-pigments and the vacuolar pH also affect flower color. Brugliera 2006). Such ingenuity has been studied and under- We analyzed the flavonoid composition of hundreds of rose stood in terms of chemistry, biochemistry and molecular cultivars and measured the pH of their petal juice in order to biology (Forkmann and Heller 1999, Goto 1987, Tanaka Editor-in-Chief’s choice select hosts of genetic transformation that would be suitable and Brugliera 2006). Among various pigments in petals, for the exclusive accumulation of delphinidin and the resulting anthocyanins, a class of flavonoids, are major constituents 0 0 color change toward blue. Expression of the viola F3 5 H gene of flower color from orange/red to violet/blue. Their in some of the selected cultivars resulted in the accumulation of chemical structures primarily determine their color, i.e. the a high percentage of delphinidin (up to 95%) and a novel bluish number of hydroxy groups on the B-ring and/or aromatic flower color. For more exclusive and dominant accumulation acyl moieties modifying anthocyanins increase, causing a of delphinidin irrespective of the hosts, we down-regulated the bathochromic shift to blue (Honda and Saito 2002). The endogenous dihydroflavonol 4-reductase (DFR) gene and majority of violet/blue flowers contain delphinidin-based overexpressed the Iris3hollandica DFR gene in addition to anthocyanins modified with one or more aromatic acyl 0 0 the viola F3 5 H gene in a rose cultivar. The resultant roses moieties (Honda and Saito 2002). Anthocyanins change exclusively accumulated delphinidin in the petals, and the their color depending on the pH of the vacuole in which flowers had blue hues not achieved by hybridization breeding. anthocyanins localize; their color is bluer in weakly acidic or Moreover, the ability for exclusive accumulation of delphini- neutral pH, and redder in acidic pH. Co-pigments, usually din was inherited by the next generations. flavones and flavonols, cause a bathochromic shift of anthocyanins when they stack with anthocyanins (Goto Keywords: Anthocyanin — Delphinidin — Flavonoid — and Kondo 1991). The formation of a complex with metal Flower color — Metabolic engineering — Rosa hybrida. ions can give a blue color (Kondo et al. 1992, Yoshida et al. Abbreviations: AT, hydroxycinnamoyl CoA:anthocyanin 2003, Shiono et al. 2005, Shoji et al. 2007). hydroxycinnamoyl transferase; CaMV, cauliflower mosaic virus; Among the factors influencing flower color, flavonoid/ 0 0 0 0 DFR, dihydroflavonol 4-reductase; F3 5 H, flavonoid 3 ,5 - anthocyanin biosynthesis has been the most extensively hydroxylase; NMR, nuclear magnetic resonance; NPTII, neomycin studied. The pathway leading to anthocyanidin 3-glucoside phosphotransferase; RHSCC, Royal Horticultural Society Color Chart; RNAi, RNA interference; siRNA, small interfering is generally conserved among higher plant species (Fig. 1) RNA; TFA, trifluoroacetic acid; TOF-MS, time-of-flight-mass (Forkmann and Heller 1999, Grotewold 2006, Tanaka and spectrometry. Brugliera 2006). Each plant species usually accumulates limited kinds of anthocyanins and exhibits limited flower Introduction color by the expression of a specific set of biosynthetic genes, the substrate specificity of key enzymes and/or the Flower color plays important roles in plant sexual temporal and spatial regulation of the biosynthetic genes. hybridization by attracting pollinators, such as insects Therefore, it is rare for a single species to have the entire 3Present address: RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, 230-0045 Japan. *Corresponding author. E-mail, [email protected]; Fax, þ81-75-962-3791. 1589 1590 Metabolic engineering of blue roses coumaroyl-CoA + 3 x malonyl-CoA CHS apigenin (a flavone) CHI FNS F3′5′H FLAVONOLS naringenin quercetin myricetin F3′H F3′5′H F3H FLS FLS FLS ′ ′ OH 3′ F3 5 H OH OH OH OH HO O ′ HO O HO O 5 OH OH F3′HF3OH ′5′H OH OH O OH O OH O dihydrokaempferol dihydroquercetin dihydromyricetin DFR DFR DFR ANS ANS ANS OH OH OH OH OH + + + HO O HOO HO O OH OH OH OH 5 3 OH OH OH pelargonidin cyanidin delphinidin GT, AT GT, AT GT, AT pelargonidin-based cyanidin-based delphinidin-based anthocyanins anthocyanins anthocyanins Fig. 1 Generalized flavonoid biosynthetic pathway relevant to flower color. Native rose petals only accumulate pelargonodin and cyanidin-based anthocyanins, mainly pelargonidin and cyanidin 3,5-diglucoside. Lack of delphinidin-based anthocynanins, which is attributed to deficiency of F3050H, has hampered the generation of rose flowers having blue and violet hues. The expression of a hetelorogous F3050H gene in rose is expected to generate delphinidin and, thus, a novel flower color with a blue hue. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F30H, flavonoid 30-hydroxylase; F3050H, flavonoid 30,50-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; GT, anthocyanidin glucosyltransferase; AT, anthocyanin acyltransferase. spectrum of flower color, although floricultural breeders anthocyanins in their petals, such as iris and gentian, have always sought novel flower colors (Tanaka et al. 2005, are likely to have DFRs with a similar substrate specificity Tanaka and Brugliera 2006). For example, roses, carnations to that of petunia DFR. Rose DFR can utilize and chrysanthemum, which are the most important dihydromyricetin as a substrate on the basis of feeding of floricultural crops, do not accumulate delphinidin-based dihydromyricetin to rose petals (Holton and Tanaka 1994). anthocyanins. Thus, they lack violet/blue varieties. This is Advances in molecular biology and plant transforma- attributed to their deficiency of flavonoid 30,50-hydroxylase tion technology have made possible the engineering of an (F3050H), a key enzyme in the synthesis of delphinidin anthocyanin biosynthetic pathway and, thus, flower color (Fig. 1) (Holton and Tanaka 1994)). by the overexpression of heterologous flavonoid biosyn- On the other hand, petunia and cymbidium lack brick thetic genes and/or the down-regulation of endogenous red/orange varieties due to the lack of pelargonidin-based genes in transgenic plants, including petunia, torenia and anthocyanins because their dihydroflavonol 4-reductases carnation, as reviewed by Tanaka (2006). In carnation, the (DFRs) do not utilize dihydrokaempferol as a substrate overexpression of the F3050H gene alone was insufficient to (Forkmann and Heller 1999, Johnson et al. 1999). convert the metabolic flux fully toward delphinidin Some species that usually accumulate delphinidin-based biosynthesis. White carnation cultivars that specifically anthocyanins and do not accumulate pelargonidin-based lacked the DFR gene were transformed with the petunia or Metabolic engineering of blue roses 1591 viola F3050H gene in combination with the petunia DFR gene. Here, we analyzed the flavonoid composition of many As a result, violet carnations accumulating delphinidin- rose cultivars in order to select cultivars which will exhibit a based anthocyanins were almost exclusively generated blue hue when delphinidin is accumulated. The over- (Holton 1996, Fukui et al. 2003). These results indicate expression of a viola F3050H gene resulted in the efficient that it is possible to generate a novel flower color by changing accumulation of delphinidin and color changes with a novel the structure of anthocyanin, more specifically the number of blue hue in a few of the selected cultivars. Furthermore, the hydroxyl groups on the B-ring, with genetic modification of down-regulation of the rose DFR gene and overexpression the pathway. They also indicate that the selection of cultivars of the iris DFR gene, as well as the overexpression of the

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