r- 2006 phytoche flower colour and cytochromes p450

8/6/2019 R- 2006 Phytoche Flower Colour and Cytochromes p450

http://slidepdf.com/reader/full/r-2006-phytoche-flower-colour-and-cytochromes-p450 1/9

Abstract Flavonoids are major constituents of

flower colour. Plants accumulate specific flavo-noids and thus every species often exhibits a

limited flower colour range. Three cytochromesP450 play critical roles in the flavonoid biosyn-

thetic pathway. Flavonoid 3¢-hydroxylase (F3¢H,CYP75B) and flavonoid 3¢,5¢-hydroxylase

(F3¢5¢H, CYP75A) catalyze the hydroxylation of

the B-ring of flavonoids and are necessary tobiosynthesize cyanidin-(red to magenta) and del-

phinidin-(violet to blue) based anthocyanins,respectively. Pelargonidin-based anthocyanins(orange to red) are synthesized in their absence.

Some species such as roses, carnations and chry-santhemums do not have violet/blue flower colour

due to deficiency of F3¢5¢H. Successful expression

of heterologous F3¢ 5¢H genes in roses and car-nations results in delphinidin production, causing

a novel blue/violet flower colour. Down-regula-tion of F3¢H and F3¢ 5¢H genes has yielded orange

petunia and pink torenia colour that accumulate

pelargonidin-based anthocyanins. Flavone syn-thase II (CYP93B) catalyzes the synthesis of

flavones that contribute to the bluing of flowercolour, and modulation of FNSII gene expression

in petunia and tobacco changes their flower col-

our. Extensive engineering of the anthocyanin

pathway is therefore now possible, and can beexpected to enhance the range of flower colours.

Keywords Anthocyanin Æ Cytochrome P450 Æ

Flavonoid Æ Flavone Æ Flower colour Æ

Cyanidin Æ Delphinidin Æ Dihydroflavonol

4-reductase Æ Flavonoid 3¢-hydroxylase Æ

Flavonoid 3¢,5¢-hydroxylase Æ Flavone synthase II Æ

Flavonol hydroxylaseÆ

MonooxygenaseÆPelargonidin

Abbreviations

DFR dihydroflavonol 4-reductase

DHK dihydrokaempferol

DHQ dihydroquercetinDHM dihydromyricetin

F3H flavanone 3-hydroxylaseF3¢H flavonoid 3¢-hydroxylase

F3¢5¢H flavonoid 3¢,5¢-hydroxylase

FLS flavonol synthaseFNS flavone synthase

Flavonoids and anthocyanins

Flower colour is due to flavonoids, carotenoids,betalains and some other pigments (Tanaka and

Y. Tanaka (&)Institute for Advanced Core Technology,Suntory Ltd., 1-1-1, Wakayamadai, Shimamoto-cho,Mishima-gun, Osaka 618-8503, Japane-mail: [email protected]

Phytochem Rev (2006) 5:283–291

DOI 10.1007/s11101-006-9003-7

123

Flower colour and cytochromes P450

Yoshikazu Tanaka

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



Brugliera 2006). Cytochromes P450 play impor-tant roles in flavonoid biosynthesis that has been

well characterized (Fig. 1). Anthocyanins, a col-

oured class of flavonoids, produce orange, red,violet and blue. Anthocyanins are synthesized

from their aglycons, anthocyanidins (pelargoni-

din, cyanidin and delphinidin) after modificationwith glycosyl (Fig. 1), acyl and methyl moietiesin a species-specific manner. Such modifications

results in structural diversity of anthocyanins

that generally explain flower colour diversity.Anthocyanins are usually localized in the vacu-

oles of epidermal cells. Anthocyanins tend to beblue when the number of the hydroxyl groups on

the B-ring increases, the number of attachedaromatic acyl groups increases, the vacuolar pH

is elevated, and/or copigments (typically flavones

and flavonols) are present. Metal ions sometimescontribute to bluing (Harborne and Williams

2000).

The flavonoid/anthocyanin biosynthetic path-way involved in flower colour is well established,and the mainstream of the pathway is well con-

served among higher plants. The pathway hasbeen reviewed many times (Forkmann and Heller

1999; Tanaka et al. 2005a; Tanaka and Brugliera

2006), and the focus in this review is on issuesrelated to cytochrome P450.

Fig. 1 The flavonoid biosynthetic pathway leading to theproduction of the first coloured anthocyanins, anthocyani-din (cyanidin, pelargonidin and delphinidin) 3-glucosides.Anthocyanidin 3-glucosides are further modified withglycosyl, acyl or methyl groups in a species-specific manner(Tanaka et al. 2005a, Tanaka and Brugliera 2006). Meth-ylation of cyanidin glucoside yields peonidin (3¢-O-methylcyanidin) glucoside and that of delphinidin glucoside yieldspetunidin (3¢-O-methyl delphinidin) and malvidin (3¢,5¢-O-

dimethyl delphinidin) glucosides. Cytochrome P450 en-zymes are emphasized with squares. Abbreviations includeCHS, chalcone synthase; CHI, chalcone isomerase; F3H,flavanone 3-hydroxylase; F3¢H, flavonoid 3¢-hydroxylase;F3¢5¢H, flavonoid 3¢,5¢-hydroxylase; DFR, dihydroflavonol4-reductase; ANS, anthocyanidin synthase; FNS, flavonesynthase; FLS, flavonol synthase; 3GT, UDP-glucose:anthocyanidin 3-O-glucosyltransferase

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Cytochromes P450 in flavonoid biosynthetic

pathway

Hydroxylation of the B-ring

The two cytochromes P450 flavonoid 3¢-hyroxy-

lase (F3¢H) and flavonoid 3¢,5¢-hydroxylase(F3¢5¢H) catalyze the 3¢ and 5¢-hydroxylation of dihydroflavonols and eventually determine the

hydroxylation pattern of the anthocyanin B-ring

(Fig. 1). They also catalyze the hydroxylation of flavanones, flavones and flavonols. The substrate

specificity of dihydroflavonol 4-reductase (DFR)often determines the structure of anthocyanidins

that a plant species accumulates, i.e. petunia

efficiently accumulates delphinidin and notpelargonidin. This is because the petunia DFR

preferably recognizes dihydromyricetin (DHM)and not dihydrokaempferol (DHK, Forkmann

and Heller 1999).F3¢H activity was first detected in the micro-

somal 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). F3¢5¢H activity was first demonstrated withthe microsomal fraction from flowers of

Verbena hybrida, and then with those from the

flowers of Callistephus chinensis, Lathyrusodoratus and Petunia hybrida (Forkmann and

Heller 1999).Holton et al. (1993) isolated the genes of petu-

nia F3¢ 5¢H (CYP75A1 and A3) for the first time.Petunia has two F3¢ 5¢H genes, regulated by two

loci, Hf1 and Hf2. Hf1 plays a dominant role.

F3¢ 5¢H genes belonging to CYP75A have beenisolated from eggplant, gentian, lisianthus,

campanula, torenia and many other plants(CYP75A1-A24, http://www.drnelson.utmem.edu/

CytochromeP450.html).The F3¢H gene was also first isolated frompetunia (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 beensuggested 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

F3¢5¢H activity and do not produce delphinidin,

which indicates that these plants may have lost

F3¢ 5¢H genes during their evolution. Interestingly,

a C. chinesis F3¢H homologue (CYP75B5) exhib-

ited F3¢5¢H 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 byforming complexes with the anthocyanins (Martens

and Mithofer 2005). Flavone synthase catalyzes thesynthesis of flavones from flavanones by introduc-

ing the double bond between C-2 and C-3 of flav-anones (Fig. 1, Forkmann and Heller 1999). Ayabeand Akashi (2006) describe the details of FNS in

another review of this publication. CytochromeP450 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 flavonols confer a paleyellow colour in flowers. There are two kinds of flavonol 6-hydroxylase (F6H); the cytochrome

P450-type F6H has been reported from Tagetes

sp. (Halbwirth et al. 2004) but has not been iso-

lated. The gene of flavonoid 6-hydroxylase(CYP71D9) was isolated from soybean, and its

substrate specificity was examined. Its activity

toward flavonol was not strong (Latunde-Dadaet al. 2001) and this gene is therefore unlikely to

encode F6H. Flavonol 8-hydroxylase from Chry-

santhemum segetum has been suggested to be aP450 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

significantly contributed to expanding flower

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colour variation by the incorporation of variouswild species into breeding programs, progress is

limited by the gene pool for any given species.

The top-selling cut-flower species (rose, chrysan-themum and carnation) do not produce delphin-

idin in their petals and lack violet/blue flower

colour varieties due to the deficiency of F3¢5¢H.Petunia and cymbidium do not accumulate pe-largonodin, as their DFR does not catalyze the

reduction of dihydrokaempferol (DHK). Genetic

engineering, or molecular breeding, is therefore isan additional technology to alter flower colour

(Tanaka et al. 2005a).

Basic tactics to engineer flower colour using

cytochrome P450 genes

Bluer flower colour can be obtained by anincrease in the number of hydroxyl groups on

the B-ring by over-expression of F3¢ 5¢H and/or

F3¢H genes and an increase in the concentrationof flavones by over-expression of the FNSII

gene. A decrease of hydroxyl groups and/or

flavones should create a redder flower colour.Over-expression of the F3¢ 5¢H gene is not

sufficient to achieve efficient accumulation of

delphinidin because endogenous enzymes, suchas DFR, F3¢H and FLS, often compete against

the introduced F3¢5¢H. In order to avoid thecompetition, it is desirable to obtain natural

mutants of these competing genes or down-regulate them. Since flower colour is not exclu-

sively determined by P450 enzymes, engineeringan anthocyanin modification pathway as well as

pH and metal uptake, which are not discussed in

this review, should also be considered (Harborneand Williams 2000; Tanaka et al. 2005a; Tanaka

and Brugliera 2006).

Engineering of F3¢H and F3

¢ 5¢H geneexpression in transgenic plants

Solanaceae species-petunia, tobacco

and Nierembergia

Petunia has been the most commonly used plant

to study the expression of F3¢ 5¢H and F3¢H genesbecause its genetics, flavonoid composition and

genetic transformation protocol have been well

established (Tanaka and Brugliera 2006). Holtonet al. (1993) reported that the expression of the

petunia F3¢ 5¢H genes in a pale-pink petunia

(deficient in F3¢H and F3¢5¢H) elevated theamount of delphinidin-based anthocyanins and

yielded reddish purple flowers. The similar colour

changes can be achieved by expression of aF3¢ 5¢H gene from other species (Fig. 2A). Pink tomagenta colour changes in petunia were achieved

using petunia and lisianthus F3¢ 5¢H genes (Shi-

mada et al. 1999). Expression of a Vinca major

F3¢ 5¢H gene in red-flowered petunia yielded

deep-red flowers with deep-purple sectors (Moriet al. 2004). Interestingly, the expression of a

Campanula medium F3¢ 5¢H gene in tobacco

yielded a higher percentage of delphinidin (up to99%) than the petunia and lisianthus F3¢ 5¢H

genes. This is probably due to a more efficientenzymatic character of the campanula F3¢5¢H(Okinaka et al. 2003). Similarly a butterfly pea

F3¢ 5¢H gene worked better than a verbena F3¢ 5¢H

gene in transgenic verbena plants (Togami et al.2006). These results indicate that the choice of the

gene source is important for an efficient change of the pathway.

When the F3¢ 5¢H genes in a red-purple petuniathat mainly accumulated delphinidin-based an-

thocyanins were down-regulated, pink flowers

accumulating cyanidin-based anthocyanins andflavonols were obtained (Tsuda et al. 2004).

Flower colour changes from deep blue to paleblue/pink were also reported (Shimada et al.

2001). However, the down-regulation of the

F3¢ 5¢H gene in Nierembergia, a close relative of petunia, only resulted in a white flower containing

increased flavonols, but no cyanidin, due to its

low F3¢H and high FLS expression (Ueyama et al.2006). These results indicate that the down-reg-

ulation of the competing pathway is necessary to

obtain desirable phenotypes.The expression of the petunia F3¢H gene in a

pale-pink petunia (deficient in F3¢H and F3¢5¢H)altered the flower colour to dark-pink, due to

accumulation of cyanidin-based anthocyanins.Accumulation of quercetin was also observed

(Brugliera et al. 1999). An increase of cyanidinand quercetin has been achieved in tobacco by

the expression of the gentian F3¢H gene (Nakat-

suka et al. 2006).

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DFRs of many plants can catalyze thereduction of DHK to leucopelargonidin. The

expression of the gene of such a DFR in petu-

nia lines that lack F3¢5¢H and F3¢H activityyielded orange petunias producing pelargonidin.

However, the expression of the gene in a

petunia that is dominant in F3¢H and/or F3¢ 5¢H genes did not change the flower colour(reviewed by Tanaka and Brugliera 2006). This

observation again indicates that the down-reg-

ulation of the competing enzyme (F3¢H andF3¢5¢H) is necessary to re-direct the pathway

toward pelargonidin. The down-regulation of the endogenous F3¢H gene and over-expression

of a rose DFR gene in red petunia (producing

cyanidin) yielded an orange petunia containingpelargonidin (Fig. 2B, Tsuda et al. 2004). The

down-regulation of a target gene has beenshown to be efficiently achieved by RNAitechnology (Nakamura et al. 2006).

Torenia

Torenia is a popular garden plant. Torenia hyb-

rida ‘‘Summerwave’’ is an interspecies hybrid thatis sterile. Genetic engineering is a useful way to

increase flower colour variation in this product.‘‘Summerwave Blue’’ petals mainly contain del-

phinidin-based anthocyanins (mainly a malvidin(3¢,5¢-O-dimethyl delphinidin) derivative) andsome cyanidin-based anthocyanins (a peonidin

(3¢-O-methyl cyanidin) derivative) (Suzuki et al.

2000). The down-regulation of the F3¢ 5¢H gene inthe torenia changed the colour from blue to pale

pink, as a result of a decrease of the malvidin

derivatives and an increase in the peonidinderivative (Suzuki et al. 2000). The over-expres-

sion of the torenia F3¢H gene resulted in darker-pink colour flower with an increase of the

peonidin derivative (Ueyama et al. 2002).The down-regulation of torenia F3¢ 5¢H and

F3¢H and over-expression of a rose or pelargonium

DFR gene in ‘‘Summerwave Blue’’ yielded variouspink flower colour plants that predominantly

contained pelargonidin (manuscript in prepara-tion, Fig. 2C). Pelargonium DFR seemed to yield

pelargonidin more efficiently than rose DFR.

Carnation

Over-expression of an F3¢ 5¢H gene in carnation

varieties generates delphinidin, which shifts the

flower colour towards blue. However, the intro-duced F3¢ 5¢H gene can only partly direct the

metabolic pathway toward delphinidin synthesisbecause of the presence of endogenous DFR and/

or F3¢H activity. In order to avoid such competi-

tion, carnation varieties that specifically lackedthe DFR gene were obtained by screening com-

mercial varieties. Expression of the petunia or

pansy F3¢ 5¢H gene and the petunia DFR gene inthe varieties yielded blue/violet carnations that

exclusively produced delphinidin (Holton 1996,

Fig. 2D). The resultant flower colour was aunique blue hue, unachieved by traditional

breeders. The amount of delphinidin and flowercolour varied to a large extent depending on the

hosts, gene constructs and transgenic events.These carnations have been vegetatively propa-

gated and sold in the USA, Japan, Australia andsome European countries for 6–10 years. They

are the only examples of the commercialization of

a plant cytochrome P450.Petunia contains a cytochrome b5 that specifi-

cally transfers electrons to Hf1 F3¢5¢H (de Vetten

et al. 1999). The coexpression of the cytochrome

b5 and Hf1 F3¢ 5¢H genes efficiently yieldeddelphinidin even in carnations expressing endog-

enous F3¢H and DFR (the photo was shown inTanaka et al. 2005a).

Rose

Roses are, by far, man-kinds most beloved

flowers.

Roses red and roses white

Plucked I for my love’s delight .

She would none of all my posies..Bade me gather her blue roses....

The words of the Victorian era poet RudyardKipling were penned at a time when floriogra-

phy, the language of flowers, was at its peak,and when rose was as symbolic as any flower.

The coded significance of a gift of flowers was

Phytochem Rev (2006) 5:283–291 287

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Fig. 2 (A) The expression of a butterfly pea F3¢ 5¢H gene in apale-pink petunia (left below, deficient in F3¢H and F3¢5¢H)

generated purple colour as a result of increase of delphinidin-

based 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 antho-

cyanins by down-regulation of the F3¢H gene and over-

expression of a rose DFR gene. (C) Down-regulation of F3¢H

and F3¢ 5¢H genes and over-expression of a pelargonium DFR

gene in a torenia (left) resulted in pink flower colour flower that(middle, right). (D) Transgenic violet/blue carnation flowers

accumulating delphinidin-based anthocyanins by expressing

F3¢ 5¢H and petunia DFR genes. Two more varieties (Moonaqua

and Moonvista) are in market (http://www.florigene.com.au).

(E) Transgenic rose flowers accumulating delphinidin. Over-

expression of a pansy F3¢ 5¢H in a pink variety yielded a violet/

blue flower (left). Over-expression of pansy F3¢ 5¢H and iris

DFR genes and down-regulation of the endogenous DFR gene

in a pink variety (middle) yielded more efficient delphinidin

production and violet/blue flower colour (right)

288 Phytochem Rev (2006) 5:283–291

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understood, and then, just as now, there wereno blue or violet roses. The blue rose symbol-

ized mystery, fantasy and the attainment of the

impossible. The Victorian symbolism, main-tained 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 magicianBaba Yaga, who drank tea made from blue

roses to stop herself aging.

The obvious parallels between the technicalchallenges that must be overcome to develop a

blue rose by genetic modification and the emo-tions that the blue rose embodies are most com-

pelling. The ‘‘unattainable’’ blue rose has been a

dream of rose breeders, who have made exhaus-tive breeding efforts to breed them with the eight

species of wild rose and hybrids that all cultivatedroses are derived from. Unfortunately they havefailed, because we now know that the lack of

violet/blue colour in rose is primarily attributed tolack of delphinidin based anthocyanins, due to

deficiency of F3¢5¢H (Holton and Tanaka 1994).

In a significant milestone towards fulfilling thedream of a blue rose we have now obtained

transgenic roses that display a novel blue/violetflower colour impossible to obtain by breeding.

This was achieved through expression of a pansy

F3¢ 5¢H gene, to yield delphinidin-based anthocy-anins (mainly delphinidin 3,5-diglucoside) in the

petals (Brugliera et al. 2004). The colour anddelphinidin content greatly depended on the host

varieties, but in some transgenic lines delphinidin

accounted for more than 90% of the total anth-ocyanidins with a novel violet/blue flower colour

(unpublished results, Fig. 2E).

Unlike carnation, rose varieties that lackedfunctional DFR genes were not identified and in

order to change flower colour it was necessary to

extensively engineer the anthocyanin biosynthesispathway. A pink background variety was selectedfor transformation and the endogenous DFR gene

was down-regulated and the pansy F3¢ 5¢H and an

iris DFR genes were over-expressed. Many of thetransgenic roses contained delphinidin almost

exclusively and displayed a blue/violet flowercolour (Fig. 2E).

Most importantly, the ability to synthesize del-phinidin is heritable (Tanaka et al. 2005b). This

will change rose breeding history as far as flowercolour is concerned because rose breeders can now

utilize delphinidin in combination with cyanidin,pelargonidin and carotenoids. We can expect that

rose flowers will be more colourful in the future!

Engineering the FNSII gene expression

Over-expression of the torenia FNSII gene in apetunia successfully produced flavones that native

petunias do not produce. The flower colourchanged from deep violet to pale violet. The

amount of anthocyanins decreased, presumablybecause the supply of the substrates decreased

due to a conversion of flavanones to flavones bythe introduced FNSII gene (Tsuda et al. 2004).

Flavone production was also reported in tobacco

with the use of the gentian FNSII gene (Nakat-suka et al. 2006). The down-regulation of the

FNSII gene in torenia generated a flower con-

taining fewer flavones. Curiously, the amount of anthocyanins decreased, and the flower colour

become paler. The flavones may stabilize antho-

cyanis in torenia flowers (Ueyama et al. 2002).The copigment (bluing) effect of flavone has

been confirmed 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. Thisis because the spray carnations accumulate a fla-

vone derivative that exhibits a strong copigment

effect (Fukui et al. 2003). The ability of flavonebiosynthesis comes from the host carnation in this

case.

Future challenges and perspectives

Although the key cytochrome P450 genes regu-lating flower colour are available, it is not always

easy to obtain desirable phenotypes. CytochromeP450 genes are not the only ones that determine

flower colour, and extensive engineering of the

pathway is often necessary. Efficient transforma-tion protocols for commercially important species

must be established. Gene expression in a target

species should be optimized. Still, molecularbreeding may overcome the limitations of the

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species gene pool, and modulating cytochromeP450 gene expression will create more colourful

flowers.

Acknowledgements The author is grateful for the col-laboration received from Suntory Ltd. (Japan) and Flori-gene 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 acknowledgedfor improving the manuscript, especially the rose section,and providing a preprint, respectively. Due to space con-straints, only a limited number of publications have beencited.

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