Phytochem Rev (2006) 5:283291 DOI 10.

1007/s11101-006-9003-7

Flower colour and cytochromes P450

Yoshikazu Tanaka

Received: 21 December 2005 / Accepted: 25 April 2006 / Published online: 31 October 2006 Springer Science+Business Media B.V. 2006

Abstract Flavonoids are major constituents of ower colour. Plants accumulate specic avonoids and thus every species often exhibits a limited ower colour range. Three cytochromes P450 play critical roles in the avonoid biosynthetic pathway. Flavonoid 3-hydroxylase (F3H, CYP75B) and avonoid 3,5-hydroxylase (F35H, CYP75A) catalyze the hydroxylation of the B-ring of avonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue ower colour due to deciency of F35H. Successful expression of heterologous F35H genes in roses and carnations results in delphinidin production, causing a novel blue/violet ower colour. Down-regulation of F3H and F35H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of avones that contribute to the bluing of ower colour, and modulation of FNSII gene expression

in petunia and tobacco changes their ower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of ower colours. Keywords Anthocyanin Cytochrome P450 Flavonoid Flavone Flower colour Cyanidin Delphinidin Dihydroavonol 4-reductase Flavonoid 3-hydroxylase Flavonoid 3,5-hydroxylase Flavone synthase II Flavonol hydroxylase Monooxygenase Pelargonidin Abbreviations DFR dihydroavonol 4-reductase DHK dihydrokaempferol DHQ dihydroquercetin DHM dihydromyricetin F3H avanone 3-hydroxylase F3H avonoid 3-hydroxylase F35H avonoid 3,5-hydroxylase FLS avonol synthase FNS avone synthase

Y. Tanaka (&) Institute for Advanced Core Technology, Suntory Ltd., 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan e-mail: Yoshikazu_Tanaka@suntory.co.jp

Flavonoids and anthocyanins Flower colour is due to avonoids, carotenoids, betalains and some other pigments (Tanaka and

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Brugliera 2006). Cytochromes P450 play important roles in avonoid biosynthesis that has been well characterized (Fig. 1). Anthocyanins, a coloured class of avonoids, produce orange, red, violet and blue. Anthocyanins are synthesized from their aglycons, anthocyanidins (pelargonidin, cyanidin and delphinidin) after modication with glycosyl (Fig. 1), acyl and methyl moieties in a species-specic manner. Such modications results in structural diversity of anthocyanins that generally explain ower colour diversity. Anthocyanins are usually localized in the vacuoles of epidermal cells. Anthocyanins tend to be blue when the number of the hydroxyl groups on

the B-ring increases, the number of attached aromatic acyl groups increases, the vacuolar pH is elevated, and/or copigments (typically avones and avonols) are present. Metal ions sometimes contribute to bluing (Harborne and Williams 2000). The avonoid/anthocyanin biosynthetic pathway involved in ower colour is well established, and the mainstream of the pathway is well conserved among higher plants. The pathway has been reviewed many times (Forkmann and Heller 1999; Tanaka et al. 2005a; Tanaka and Brugliera 2006), and the focus in this review is on issues related to cytochrome P450.

Fig. 1 The avonoid biosynthetic pathway leading to the production of the rst coloured anthocyanins, anthocyanidin (cyanidin, pelargonidin and delphinidin) 3-glucosides. Anthocyanidin 3-glucosides are further modied with glycosyl, acyl or methyl groups in a species-specic manner (Tanaka et al. 2005a, Tanaka and Brugliera 2006). Methylation of cyanidin glucoside yields peonidin (3-O-methyl cyanidin) glucoside and that of delphinidin glucoside yields petunidin (3-O-methyl delphinidin) and malvidin (3,5-O-

dimethyl delphinidin) glucosides. Cytochrome P450 enzymes are emphasized with squares. Abbreviations include CHS, chalcone synthase; CHI, chalcone isomerase; F3H, avanone 3-hydroxylase; F3H, avonoid 3-hydroxylase; F35H, avonoid 3,5-hydroxylase; DFR, dihydroavonol 4-reductase; ANS, anthocyanidin synthase; FNS, avone synthase; FLS, avonol synthase; 3GT, UDP-glucose: anthocyanidin 3-O-glucosyltransferase

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Cytochromes P450 in avonoid biosynthetic pathway Hydroxylation of the B-ring The two cytochromes P450 avonoid 3-hyroxylase (F3H) and avonoid 3,5-hydroxylase (F35H) catalyze the 3 and 5-hydroxylation of dihydroavonols and eventually determine the hydroxylation pattern of the anthocyanin B-ring (Fig. 1). They also catalyze the hydroxylation of avanones, avones and avonols. The substrate specicity of dihydroavonol 4-reductase (DFR) often determines the structure of anthocyanidins that a plant species accumulates, i.e. petunia efciently accumulates delphinidin and not pelargonidin. This is because the petunia DFR preferably recognizes dihydromyricetin (DHM) and not dihydrokaempferol (DHK, Forkmann and Heller 1999). F3H activity was rst detected in the microsomal fraction of Happlopappus cell cultures, and then in Matthiola incana, Antirrhinum majus, Dianthus caryophyllus, Petunia hybrida and many other plant species (Forkmann and Heller 1999). F35H activity was rst demonstrated with the microsomal fraction from owers of Verbena hybrida, and then with those from the owers of Callistephus chinensis, Lathyrus odoratus and Petunia hybrida (Forkmann and Heller 1999). Holton et al. (1993) isolated the genes of petunia F35H (CYP75A1 and A3) for the rst time. Petunia has two F35H genes, regulated by two loci, Hf1 and Hf2. Hf1 plays a dominant role. F35H genes belonging to CYP75A have been isolated from eggplant, gentian, lisianthus, campanula, torenia and many other plants (CYP75A1-A24, http://www.drnelson.utmem.edu/ CytochromeP450.html). The F3H gene was also rst isolated from petunia (CYP75B2) and its homologues have since been isolated from Arabidopsis, torenia, gerbera, gentian, rose, carnation, chrysanthemum, C. chinensis and other species (CYP75B1-4, 6-27, http://www.drnelson.utmem.edu/CytochromeP450. html). CYP75A and CYP75B genes have been suggested to have diverted before the speciation of higher plants (Ueyama et al. 2002; Tanaka and

Brugliera 2006). Nevertheless, many plant species, such as rose, carnation and chrysanthemum, lack F35H activity and do not produce delphinidin, which indicates that these plants may have lost F35H genes during their evolution. Interestingly, a C. chinesis F3H homologue (CYP75B5) exhibited F35H activity when it was expressed in yeast (http://www.drnelson.utmem.edu/CytochromeP450. html). Flavone biosynthesis Flavones are common copigments that cause a bathochromic shift (bluing) of anthocyanins by forming complexes with the anthocyanins (Martens and Mithofer 2005). Flavone synthase catalyzes the synthesis of avones from avanones by introducing the double bond between C-2 and C-3 of avanones (Fig. 1, Forkmann and Heller 1999). Ayabe and Akashi (2006) describe the details of FNS in another review of this publication. Cytochrome P450 type FNSII genes have been isolated from A. majus, torenia (Akashi et al. 1999), gerbera (Martens and Forkmann 1999), perilla, C. chinesis, gentian and verbena (CYP93B2-6, 9, http:// www.drnelson.utmem.edu/CytochromeP450.html). Flavonol hydroxylase Rare 6 and/or 8 hydroxy avonols confer a pale yellow colour in owers. There are two kinds of avonol 6-hydroxylase (F6H); the cytochrome P450-type F6H has been reported from Tagetes sp. (Halbwirth et al. 2004) but has not been isolated. The gene of avonoid 6-hydroxylase (CYP71D9) was isolated from soybean, and its substrate specicity was examined. Its activity toward avonol was not strong (Latunde-Dada et al. 2001) and this gene is therefore unlikely to encode F6H. Flavonol 8-hydroxylase from Chrysanthemum segetum has been suggested to be a P450 enzyme (Forkmann and Heller 1999). Flower colour engineering by modulating the expression of cytochrome P450 genes Natural species often have a limited variation of anthocyanins. Although extensive breeding has signicantly contributed to expanding ower

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colour variation by the incorporation of various wild species into breeding programs, progress is limited by the gene pool for any given species. The top-selling cut-ower species (rose, chrysanthemum and carnation) do not produce delphinidin in their petals and lack violet/blue ower colour varieties due to the deciency of F35H. Petunia and cymbidium do not accumulate pelargonodin, as their DFR does not catalyze the reduction of dihydrokaempferol (DHK). Genetic engineering, or molecular breeding, is therefore is an additional technology to alter ower colour (Tanaka et al. 2005a). Basic tactics to engineer ower colour using cytochrome P450 genes Bluer ower colour can be obtained by an increase in the number of hydroxyl groups on the B-ring by over-expression of F35H and/or F3H genes and an increase in the concentration of avones by over-expression of the FNSII gene. A decrease of hydroxyl groups and/or avones should create a redder ower colour. Over-expression of the F35H gene is not sufcient to achieve efcient accumulation of delphinidin because endogenous enzymes, such as DFR, F3H and FLS, often compete against the introduced F35H. In order to avoid the competition, it is desirable to obtain natural mutants of these competing genes or downregulate them. Since ower colour is not exclusively determined by P450 enzymes, engineering an anthocyanin modication pathway as well as pH and metal uptake, which are not discussed in this review, should also be considered (Harborne and Williams 2000; Tanaka et al. 2005a; Tanaka and Brugliera 2006). Engineering of F3H and F35H gene expression in transgenic plants Solanaceae species-petunia, tobacco and Nierembergia Petunia has been the most commonly used plant to study the expression of F35H and F3H genes because its genetics, avonoid composition and genetic transformation protocol have been well

established (Tanaka and Brugliera 2006). Holton et al. (1993) reported that the expression of the petunia F35H genes in a pale-pink petunia (decient in F3H and F35H) elevated the amount of delphinidin-based anthocyanins and yielded reddish purple owers. The similar colour changes can be achieved by expression of a F35H gene from other species (Fig. 2A). Pink to magenta colour changes in petunia were achieved using petunia and lisianthus F35H genes (Shimada et al. 1999). Expression of a Vinca major F35H gene in red-owered petunia yielded deep-red owers with deep-purple sectors (Mori et al. 2004). Interestingly, the expression of a Campanula medium F35H gene in tobacco yielded a higher percentage of delphinidin (up to 99%) than the petunia and lisianthus F35H genes. This is probably due to a more efcient enzymatic character of the campanula F35H (Okinaka et al. 2003). Similarly a buttery pea F35H gene worked better than a verbena F35H gene in transgenic verbena plants (Togami et al. 2006). These results indicate that the choice of the gene source is important for an efcient change of the pathway. When the F35H genes in a red-purple petunia that mainly accumulated delphinidin-based anthocyanins were down-regulated, pink owers accumulating cyanidin-based anthocyanins and avonols were obtained (Tsuda et al. 2004). Flower colour changes from deep blue to pale blue/pink were also reported (Shimada et al. 2001). However, the down-regulation of the F35H gene in Nierembergia, a close relative of petunia, only resulted in a white ower containing increased avonols, but no cyanidin, due to its low F3H and high FLS expression (Ueyama et al. 2006). These results indicate that the down-regulation of the competing pathway is necessary to obtain desirable phenotypes. The expression of the petunia F3H gene in a pale-pink petunia (decient in F3H and F35H) altered the ower colour to dark-pink, due to accumulation of cyanidin-based anthocyanins. Accumulation of quercetin was also observed (Brugliera et al. 1999). An increase of cyanidin and quercetin has been achieved in tobacco by the expression of the gentian F3H gene (Nakatsuka et al. 2006).

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DFRs of many plants can catalyze the reduction of DHK to leucopelargonidin. The expression of the gene of such a DFR in petunia lines that lack F35H and F3H activity yielded orange petunias producing pelargonidin. However, the expression of the gene in a petunia that is dominant in F3H and/or F35H genes did not change the ower colour (reviewed by Tanaka and Brugliera 2006). This observation again indicates that the down-regulation of the competing enzyme (F3H and F35H) is necessary to re-direct the pathway toward pelargonidin. The down-regulation of the endogenous F3H gene and over-expression of a rose DFR gene in red petunia (producing cyanidin) yielded an orange petunia containing pelargonidin (Fig. 2B, Tsuda et al. 2004). The down-regulation of a target gene has been shown to be efciently achieved by RNAi technology (Nakamura et al. 2006). Torenia Torenia is a popular garden plant. Torenia hybrida Summerwave is an interspecies hybrid that is sterile. Genetic engineering is a useful way to increase ower colour variation in this product. Summerwave Blue petals mainly contain delphinidin-based anthocyanins (mainly a malvidin (3,5-O-dimethyl delphinidin) derivative) and some cyanidin-based anthocyanins (a peonidin (3-O-methyl cyanidin) derivative) (Suzuki et al. 2000). The down-regulation of the F35H gene in the torenia changed the colour from blue to pale pink, as a result of a decrease of the malvidin derivatives and an increase in the peonidin derivative (Suzuki et al. 2000). The over-expression of the torenia F3H gene resulted in darkerpink colour ower with an increase of the peonidin derivative (Ueyama et al. 2002). The down-regulation of torenia F35H and F3H and over-expression of a rose or pelargonium DFR gene in Summerwave Blue yielded various pink ower colour plants that predominantly contained pelargonidin (manuscript in preparation, Fig. 2C). Pelargonium DFR seemed to yield pelargonidin more efciently than rose DFR.

Carnation Over-expression of an F35H gene in carnation varieties generates delphinidin, which shifts the ower colour towards blue. However, the introduced F35H gene can only partly direct the metabolic pathway toward delphinidin synthesis because of the presence of endogenous DFR and/ or F3H activity. In order to avoid such competition, carnation varieties that specically lacked the DFR gene were obtained by screening commercial varieties. Expression of the petunia or pansy F35H gene and the petunia DFR gene in the varieties yielded blue/violet carnations that exclusively produced delphinidin (Holton 1996, Fig. 2D). The resultant ower colour was a unique blue hue, unachieved by traditional breeders. The amount of delphinidin and ower colour varied to a large extent depending on the hosts, gene constructs and transgenic events. These carnations have been vegetatively propagated and sold in the USA, Japan, Australia and some European countries for 610 years. They are the only examples of the commercialization of a plant cytochrome P450. Petunia contains a cytochrome b5 that specically transfers electrons to Hf1 F35H (de Vetten et al. 1999). The coexpression of the cytochrome b5 and Hf1 F35H genes efciently yielded delphinidin even in carnations expressing endogenous F3H and DFR (the photo was shown in Tanaka et al. 2005a). Rose Roses are, by far, man-kinds most beloved owers. Roses red and roses white Plucked I for my loves delight. She would none of all my posies.. Bade me gather her blue roses.... The words of the Victorian era poet Rudyard Kipling were penned at a time when oriography, the language of owers, was at its peak, and when rose was as symbolic as any ower. The coded signicance of a gift of owers was

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Fig. 2 (A) The expression of a buttery pea F35H gene in a pale-pink petunia (left below, decient in F3H and F35H) generated purple colour as a result of increase of delphinidinbased anthocyanins (malvidin and petunidin derivatives are mainly accumulated in this case since the petunia expresses anthocyanin methyltransferases). The phenotypes vary to a large degree depending in transgenic lines. (B) An orange petunia that produces pelargonidin-based anthocyanins was made from a red petunia accumulating cyanidin-based anthocyanins by down-regulation of the F3H gene and overexpression of a rose DFR gene. (C) Down-regulation of F3H and F35H genes and over-expression of a pelargonium DFR

gene in a torenia (left) resulted in pink ower colour ower that (middle, right). (D) Transgenic violet/blue carnation owers accumulating delphinidin-based anthocyanins by expressing F35H and petunia DFR genes. Two more varieties (Moonaqua and Moonvista) are in market (http://www.origene.com.au). (E) Transgenic rose owers accumulating delphinidin. Overexpression of a pansy F35H in a pink variety yielded a violet/ blue ower (left). Over-expression of pansy F35H and iris DFR genes and down-regulation of the endogenous DFR gene in a pink variety (middle) yielded more efcient delphinidin production and violet/blue ower colour (right)

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understood, and then, just as now, there were no blue or violet roses. The blue rose symbolized mystery, fantasy and the attainment of the impossible. The Victorian symbolism, maintained today in various exotic guises (including a very popular fantasy role-playing game), was itself derived from centuries old mythology, including that of the infamous Slavic magician Baba Yaga, who drank tea made from blue roses to stop herself aging. The obvious parallels between the technical challenges that must be overcome to develop a blue rose by genetic modication and the emotions that the blue rose embodies are most compelling. The unattainable blue rose has been a dream of rose breeders, who have made exhaustive breeding efforts to breed them with the eight species of wild rose and hybrids that all cultivated roses are derived from. Unfortunately they have failed, because we now know that the lack of violet/blue colour in rose is primarily attributed to lack of delphinidin based anthocyanins, due to deciency of F35H (Holton and Tanaka 1994). In a signicant milestone towards fullling the dream of a blue rose we have now obtained transgenic roses that display a novel blue/violet ower colour impossible to obtain by breeding. This was achieved through expression of a pansy F35H gene, to yield delphinidin-based anthocyanins (mainly delphinidin 3,5-diglucoside) in the petals (Brugliera et al. 2004). The colour and delphinidin content greatly depended on the host varieties, but in some transgenic lines delphinidin accounted for more than 90% of the total anthocyanidins with a novel violet/blue ower colour (unpublished results, Fig. 2E). Unlike carnation, rose varieties that lacked functional DFR genes were not identied and in order to change ower colour it was necessary to extensively engineer the anthocyanin biosynthesis pathway. A pink background variety was selected for transformation and the endogenous DFR gene was down-regulated and the pansy F35H and an iris DFR genes were over-expressed. Many of the transgenic roses contained delphinidin almost exclusively and displayed a blue/violet ower colour (Fig. 2E). Most importantly, the ability to synthesize delphinidin is heritable (Tanaka et al. 2005b). This

will change rose breeding history as far as ower colour is concerned because rose breeders can now utilize delphinidin in combination with cyanidin, pelargonidin and carotenoids. We can expect that rose owers will be more colourful in the future! Engineering the FNSII gene expression Over-expression of the torenia FNSII gene in a petunia successfully produced avones that native petunias do not produce. The ower colour changed from deep violet to pale violet. The amount of anthocyanins decreased, presumably because the supply of the substrates decreased due to a conversion of avanones to avones by the introduced FNSII gene (Tsuda et al. 2004). Flavone production was also reported in tobacco with the use of the gentian FNSII gene (Nakatsuka et al. 2006). The down-regulation of the FNSII gene in torenia generated a ower containing fewer avones. Curiously, the amount of anthocyanins decreased, and the ower colour become paler. The avones may stabilize anthocyanis in torenia owers (Ueyama et al. 2002). The copigment (bluing) effect of avone has been conrmed in transgenic carnations. Although both the transgenic spray carnations (Moondust and Moonshadow, Fig. 2D) and the standard carnations (Moonlite and Moonshade, Fig. 2D) produce the same delphinidin-based anthocyanins, the spray carnations are bluer. This is because the spray carnations accumulate a avone derivative that exhibits a strong copigment effect (Fukui et al. 2003). The ability of avone biosynthesis comes from the host carnation in this case. Future challenges and perspectives Although the key cytochrome P450 genes regulating ower colour are available, it is not always easy to obtain desirable phenotypes. Cytochrome P450 genes are not the only ones that determine ower colour, and extensive engineering of the pathway is often necessary. Efcient transformation protocols for commercially important species must be established. Gene expression in a target species should be optimized. Still, molecular breeding may overcome the limitations of the

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Phytochem Rev (2006) 5:283291 Holton TA, Brugliera F, Lester DR, Tanaka Y, Hyland CD, Menting JGT, Lu C-Y, Farcy E, Stevenson TW, Cornish EC (1993) Cloning and expression of cytochrome P450 genes controlling ower colour. Nature 366:276279 Latunde-Dada AO, Cabbello-Hurtado F, Czittrich N, Didierjean L, Schopfer C, Hertkorn N, Werk-Reichhart D, Ebel J (2001) Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase. J Biol Chem 276:16881695 Martens S, Forkmann G (1999) Cloning and expression of avone synthase II from Gerbera hybrida. Plant J 20:611618 Martens S, Mithofer A (2005) Flavones and avone synthases. Phytochem 66:23992407 Mori S, Kobayashi H, Hoshi Y, Kondo M, Nakano M (2004) Heterologous expression of the avonoid 3,5-hydroxylase gene of Vinca major alters ower color in transgenic Petunia hybrida. Plant Cell Rep 22:415421 Nakamura N, Fukuchi-Mizutani M, Suzuki K, Miyazaki K, Tanaka Y (2006) RNAi suppression of the anthocyanidin synthase gene in Torenia hybrida yields white owers with higher frequency and better stability than antisense and sense suppression. Plant Biotechnol 23:1318 Nakatsuka T, Nishihara M, Mishiba K, Yamamura S (2006) Heterologous expression of two gentian cytochrome P450 genes can modulate the intensity of ower pigmentation in transgenic tobacco plants. Mol Breeding 17:9199 Okinaka Y, Shimada Y, Nakano-Shimada R, Ohbayashi M, Kiyokawa S, Kikuchi Y (2003) Selective accumulation of delphinidin derivatives in tobacco using a putative avonoid 3,5-hydroxylase cDNA from Campanula medium. Biosci Biotechnol Biochem 67:161165 Shimada Y, Nakano-Shimada R, Ohbayashi M, Okinaka Y, Kiyokawa S, Kikuchi Y (1999) Expression of chimeric P450 genes encoding avonoid-3,5-hydroxylase in transgenic tobacco and petunia plants. FEBS Lett 461:241245 Shimada Y, Ohbayashi M, Nakano-Shimada R, Okinaka Y, Kiyokawa S, Kikuchi Y (2001) Genetic engineering of the anthocyanin biosynthetic pathway with avonoid 3,5-hydroxylase: specic switching of the pathway in petunia. Plant Cell Rep 20:456462 Suzuki K, Zue H, Tanaka Y, Fukui Y, Fukuchi-Mizutani M, Murakami Y, Katsumoto Y, Tsuda S, Kusumi T (2000) Flower color modications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes. Mol Breeding 6:239246 Tanaka Y, Brugliera F (2006) Flower colour. In: Ainsworth C (ed) Flowering and its manipulation. Blackwell, London, pp 201239 Tanaka Y, Katsumoto Y, Brugliera F, John M (2005a) Genetic engineering in oriculture. Plant Cell Tiss Org Cult 80:124 Tanaka Y, Fukui Y, Togami J, Mizutani M, Katsumoto Y (2005b) Process for producing rose with modied color, Patent publication number WO 2005/017147

species gene pool, and modulating cytochrome P450 gene expression will create more colourful owers.
Acknowledgements The author is grateful for the collaboration received from Suntory Ltd. (Japan) and Florigene Ltd. (Australia) and for research grants provided by Bio-oriented Technology Research Advancement Institute (Japan). Dr Chandler (Florigene Ltd.) and Dr Yamamura (Iwate Biotechnology Research Center) are acknowledged for improving the manuscript, especially the rose section, and providing a preprint, respectively. Due to space constraints, only a limited number of publications have been cited.

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