蓝色月季转基因育种

Iris

×

hollandica DFR

gene

in addition to the viola

F3′5′H

gene in a rose cultivar. The resultant

roses exclusively accumulated delphinidin in the petals, and the

flowers had blue hues not achieved by hybridization breeding.

Moreover, the ability for exclusive accumulation of delphinidin was

inherited by the next generations.

Key words

Anthocyanin

花青素

Delphinidin

翠雀花素

Flavonoid

黄酮化合物

Flower color

Metabolic engineering

途径工程

Rosa hybrida

现代月季

Previous SectionNext Section

Introduction

Flower color plays important roles in plant sexual hybridization by

attracting pollinators, such as insects and birds. Flower color is also

attractive to people and is an important characteristic of flowers from

a floricultural viewpoint. Hybridization breeding has partially achieved

the diversification of flower color of floricultural crops.

Plant species have adopted versatile and ingenious ways to exhibit

flower color (Grotewold

2006

, Tanaka and Brugliera

2006

). Such

ingenuity has been studied and understood in terms of chemistry,

biochemistry and molecular biology (Forkmann and Heller

1999

, Goto

1987

, Tanaka and Brugliera

2006

). Among various pigments in petals,

anthocyanins, a class of flavonoids, are major constituents of flower

color from orange/red to violet/blue. Their chemical structures

primarily determine their color, i.e. the number of hydroxy groups on

the B-ring and/or aromatic acyl moieties modifying anthocyanins

increase, causing a bathochromic shift to blue (Honda and Saito

2002

). The majority of violet/blue flowers contain delphinidin-based

anthocyanins modified with one or more aromatic acyl moieties

(Honda and Saito

2002

). Anthocyanins change their color depending

on the pH of the vacuole in which anthocyanins localize; their color is

bluer in weakly acidic or neutral pH, and redder in acidic pH.

Co-pigments, usually flavones and flavonols, cause a bathochromic

shift of anthocyanins when they stack with anthocyanins (Goto and

Kondo

1991

). The formation of a complex with metal ions can give a

blue color (Kondo et al.

1992

, Yoshida et al.

2003

, Shiono et al.

2005

,

Shoji et al.

2007

).

Among the factors influencing flower color, flavonoid/anthocyanin

biosynthesis has been the most extensively studied. The pathway

leading to anthocyanidin 3-glucoside is generally conserved among

higher plant species (

Fig. 1

) (Forkmann and Heller

1999

, Grotewold

2006

, Tanaka and Brugliera

2006

). Each plant species usually

accumulates limited kinds of anthocyanins and exhibits limited flower

color by the expression of a specific set of biosynthetic genes, the

substrate specificity of key enzymes and/or the temporal and spatial

regulation of the biosynthetic genes. Therefore, it is rare for a single

species to have the entire spectrum of flower color, although

floricultural breeders have always sought novel flower colors (Tanaka

et al.

2005

, Tanaka and Brugliera

2006

). For example, roses,

carnations and chrysanthemum, which are the most important

floricultural crops, do not accumulate delphinidin-based anthocyanins.

Thus, they lack violet/blue varieties. This is attributed to their

deficienc

y of flavonoid 3′,5′

-

hydroxylase (F3′5′H), a key enzyme in

the synthesis of delphinidin (

Fig. 1

) (Holton and Tanaka

1994

)).

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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

F3′5′H, has hampered the generation of rose flowers having blue and violet hues. The expression

of a hetelorogous F3′5′H 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;

F3′H,

flavonoid

3′

-

hydroxylase;

F3′5′H,

flavonoid

3′,5′

-hydroxylase;

FLS,

flavonol synthase; FNS, flavone synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin

synthase; GT, anthocyanidin glucosyltransferase; AT, anthocyanin acyltransferase.

On

the

other

hand,

petunia

and

cymbidium

lack

brick

red/orange

varieties

due

to

the

lack

of

pelargonidin-based

anthocyanins

because

their

（二氢山奈酚）

dihydroflavonol

4-reductases

(DFRs) do not utilize dihydrokaempferol as a substrate (Forkmann and Heller 1999, Johnson et al.

1999).

Some

species

that

usually

accumulate

delphinidin-based

anthocyanins

and

do

not

accumulate pelargonidin-based anthocyanins in their petals, such as iris and gentian, are likely to

have

DFRs

with

a

similar

substrate

specificity

to

that

of

petunia

DFR.

Rose

DFR

can

utilize < /p>

dihydromyricetin

（

二 氢杨梅素）

as a substrate on the basis of feeding of dihydromyricetin to rose

petals (Holton and Tanaka 1994).

Advances

in

molecular

biology

and

plant

transformation

technology

have

made

possible

the

engineering of an anthocyanin biosynthetic pathway and, thus, flower color by the overexpression

of heterologous flavonoid biosynthetic genes and/or the down- regulation of endogenous genes in

transgenic

plants,

including

petunia,

torenia

and

carnation,

as

reviewed

by

Tanaka

(2006).

In

carnation, the overexp

ression of the F3′5′H gene alone was insufficient to convert the metabolic

flux fully toward delphinidin biosynthesis. White carnation cultivars that specifically

lacked the

DFR gene were transformed with the petunia or viola F3′5′H gene in combination wit

h the petunia

DFR

gene.

As

a

result,

violet

carnations

accumulating

delphinidin- based

anthocyanins

were

almost

exclusively

generated

(Holton

1996,

Fukui

et

al.

2003).

These

results

indicate

that

it

is

possible

to

generate

a

novel

flower

color

by

changing

the

structure

of

anthocyanin,

more

specifically

the

number

of

hydroxyl

groups

on

the

B-ring,

with

genetic

modification

of

the

pathway. They also indicate that the selection of cultivars that have proper genetic background and

flavonoid compositions and/or the artificial down-regulation of a competing endogenous pathway

is necessary to obtain a desirable phenotype (Tanaka 2006, Tanaka and Brugliera 2006).

Roses are the most important flowers in today's global flower market and have been the center of

attraction for consumers and breeders for hundreds of years. Modern roses (Rosa hybrida) have

resulted from extensive hybridization of wild rose species and have various flower colors, except

those in the violet to blue range (Vries et al. 1974, Holton and Tanaka 1994). Rose breeders have

long

endeavored

to

create

blue

roses,

but

their

efforts

have

so

far

only

led

to

pink

and

pale

mauve-colored

flowers.

This

is

not

surprising

because

rose

petals

contain

pelargonidin/cyanidin

3-glucoside

or

3,5-diglucoside

(Biolley

and

Jay

1993,

Mikanagi

et

al.

2000)

and

carotenoids

(Vries et al. 1974). Some roses produce only small amounts of acylated anthocyanins, and roses do

not produce flavones

（

黄酮）

(Mikanagi et al. 2000), which are stronger co-pigments than flavonol

（黄酮醇）

(Yabuya et al. 1997). The vacuolar pH of rose petal epidermal cells is low (from pH

3.69

to

5.78)

(Biolley

and

Jay

1993).

Therefore,

roses

fundamentally

lack

the

components

required to yield violet/blue flowers.

More recently, some rose cultivars have been shown to contain a small amount of blue pigments

(rosacyanin)

that

are

derivatives

of

cyanidin

(Fukui

et

al.

2006). Blue coloration

in

the

cultivar

‘Rhapsody

in

Blue’

is

caused

by

the

accumulation

of

cyanin

(cyanidin

3,5

-O-di-glucoside)

in

anthocyanic

vacuolar

inclusions

(A

VIs)

(Gonnet

2003).

However,

the

molecular

mechanisms

of

rosacyanin and A

VIs is not yet understood, and their utilization to engineer blue/violet roses is not

currently possible.

Although there may be multiple ways to create blue roses, we have chosen delphinidin production

in rose petals because delphinidin biosynthesis is regulated by a single enzyme,

F3′5′H, and its

overexpression is unlikely to have a detrimental effect on plants. Delphinidin production presents

a major breakthrough in the achievement of blue roses (Holton and Tanaka 1994). The expression

of

viola

F3′5′H

genes

produced

delphinidin

(Brugliera

et

al.

2004).

However,

the

presence

of

pelargonidin

and

cyanidin-based

anthocyanins

derived

from

the

endogenous

pathway

prevented

sufficient

coloration

toward

blue.

Unlike

carnations,

we

were

not

able

to

obtain

white

rose

cultivars that specifically lacked DFR activity.

Here, we analyzed the flavonoid composition of many rose cultivars in order to select cultivars

which

will

exhibit

a

blue

hue

when

delphinidin

is

accumulated.

The

overexpression

of

a

viola

F3′5′H gene resulted in the efficient accumulation of delphinidin and color changes with a novel

blue hue in a few of the selected cultivars. Furthermore, the down-regulation of the rose DFR gene

and overexpression of the iris DFR gene, as well as

the overexpression of the viola F3′5′H gene,

resulted

in

more

efficient

and

exclusive

delphinidin

production

and

a

bluer

flower

color.

It

is

noteworthy that exclusive delphinidin production is heritable by the progeny, which should change

rose breeding as far as flower color is concerned.

Previous SectionNext Section

Results

Cultivar screening for engineering a flavonoid pathway

Hundreds of rose cultivars that claimed to be ‘blue’ and/or have names associated with ‘blue’ have

been commercialized. Their flower color ranges are relatively blue among roses and in fact range

from pink/red purple to gray/mauve. Some of these cultivars were subjected to flavonoid analysis

and

pH

measurements,

and

the

results

are

shown

in

Table

1.

Their

petals

contained

mainly

cyanidin

as

an

anthocyanidin,

but

not

delphinidin.

They

contained

a

large

amount

of

flavonols.

Their bluish color may be due to a high flavonol/anthocyanidin ratio, and flavonols may function

as co-pigments. Their higher pH than that of red roses may also contribute to their flower color

(Table

1).

These

results

are

consistent

with

a

previous

report

(Biolley

and

Jay

1993).

Table

1

includes

some

red

cultivars

for

comparison,

which

contain

cyanidin

and/or

pelargonidin.

They

tend

to

have

fewer

flavonols

and

a

lower

pH.

We

recently

discovered

that

some

of

these

roses

contain small amounts of novel cyanidin derivatives (rosacyanins) exhibiting a blue color (Fukui

et al. 2006), but the degree of their contribution to flower color is not clear.

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Table 1

Flavonoid composition of commercial bluish rose varieties

The criteria for the selection of possible host cultivars to achieve flower color modification toward

blue

by

delphinidin

production

were:

(i)

they

accumulated

flavonols

that

were

expected

to

be

co-

pigments; (ii) they had a higher vacuolar pH; (iii) ideally, they did not have F3′H activity; and

(iv) they accumulated pelargonidin rather than cyanidin.

In order to choose cultivars to meet these criteria, several hundreds of rose flower cultivars were

screened

initially

by

visual

observation;

pink

to

mauve

roses

were

mainly

selected.

Dark

color

cultivars and red cultivars were not selected, since they did not contain much flavonol and their

pH tends to be low. Yellow and white cultivars were not selected either, as they are not able to

accumulate

anthocyanins

in

the

petals.

The

bluish

rose

cultivars

shown

in

Table

1

were

not

subjected

to

further

study

because

of

their

low

transformation

frequencies

or

their

low

anthocyanin

contents

to

achieve

clear

color

change.

The

petals

of

169

selected

cultivars

were

harvested

and

subjected

to

flavonoid

analysis

and

pH

measurements.

Some

of

the

results

are

shown in Table 2 (see controls).

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Table 2

Flavonoid composition of Keisei rose varieties transformed with pSPB130

Only pelargonidin and cyanidin were detected as anthocyanidins, and kaempferol and quercetin as

flavonols, which is consistent with previous reports (Biolley and Jay 1993, Mikanagi et al. 2000).

The absence of delphinidin and myricetin confirmed the deficiency of F3′5′H activity in the petals

of these cultivars. The absence of flavones, which are stronger co-pigments, was also confirmed.

The

cultivars

WKS77,

WKS82,

WKS100,

WKS116,

WKS124

and

WKS140

were

selected

for

genetic transformation.

Cloning of flavonoid biosynthetic genes and construction of binary vectors

The

aromatic

acylation

of

anthocyanin

shifts

its

color

toward

blue

by

3

–

4

nm

(Fujiwara

et

al.

1998).

For

co-

expression

with

the

F3′5′H

gene

in

rose,

the

cDNA

of

hydroxycinnamoyl

CoA:anthocyanin 5-hydroxycinnamoyl transferase (5AT) was obtained from the Torenia hybrida

petal cDNA library. Torenia 5AT exhibited 37 and 35% amino acid sequence identity to gentian

5AT

(Fujiwara

et

al.

1998)

and

perilla

3AT

(Yonekura-Sakakibara

et

al.

2000),

respectively.

Its

function

was

confirmed

by

expressing

the

cDNA

in

Escherichia

coli

and

yeast

as

previously

described

(Fujiwara

et

al.

1998,

Yonekura-Sakakibara

et

al.

2000).

The

enzyme

catalyzed

the

transfer of the coumaroyl or caffeoyl moiety to 5-glucose of anthocyanidin 3,5-diglucosides (data

not shown).

The substrate specificity of the DFR often determines which anthocyanidins a plant accumulates

(Forkmann and Heller 1999). The DFR of some species, such as iris and gentian, which mainly

accumulate delphinidin and lack pelargonidin, is expected to have a similar substrate to petunia

DFR,

which

efficiently

reduces

dihydromyricetin.

Iris

DFR

cDNA

was

isolated

from

the

Iris×

hollandica

petal

cDNA

library.

The

iris

DFR

exhibited

reasonable

amino

acid

sequence

identity

to

the

DFRs

of

other

plants

(61,

58

and

56%

to

cymbidium,

rose

and

petunia

DFR,

respectively)

and

had

a

specific

feature,

namely

that

it

did

not

utilize

dihydrokaempferol

as

a

substrate

(Johnson

et

al.

1999).

Such

substrate

specificity

is

suitable

for

the

conversion

of

the

metabolic flux toward delphinidin biosynthesis when it is co-

expressed with the F3′5′H gene.

The binary vectors used in this study (pSPB130, pSFL207, pSFL236 and pSPB919) are shown in

Fig.

2

.

The

vector

pSPB130

is

designed

for

the

constitutive

overexpression

of

the

viola

F3′5′H

BP40

gene

and

the

torenia

5AT

gene

in

rose.

kdThe

binary

vector

pSFL207

is

used

for

the

constitutive overexpression of the viola F3′5′H gene alone, and pSFL

236 is for the co-expression

of

the

viola

F3′5′H

and

the

iris

DFR

genes.

The

vector

pSPB919

is

to

down

-regulate

the

endogenous rose DFR gene using RNA interference (RNAi) and to overexpress the iris DFR and

the viola F3′5′H genes.

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Fig. 2

Schematic representation of binary vectors constructed for color modification. Only some T-DNA

regions are shown. The directions of the cDNA sense strand are shown by arrows.

All of them

have the nptII gene as the selectable marker for plant transformation. E35S Pro., enhanced CaMV

35S promoter; mas Ter., terminator region from manopine synthase; nos Ter., nopaline synthase

gene terminator; D8 Ter., terminator region from a petunia phospholipid transfer protein gene (D8)

(Holton

1996

);

F3′5′H,

flavonoid

3′,5′

-hydroxylase;

DFR,

dihydroflavonol

4-reductase;

5A

T,

anthocyanin 5-acyltransferase.

Generation of transgenic roses and their phenotype

Embryogenic calli were induced from the selected cultivars and subjected to transformation with

Agrobacterium

tumefaciens

containing

pSPB130

to

assess

how

efficiently

these

cultivars

can

accumulate delphinidin and the effect of delphinidin on flower color. At the same time, the effect

of 5-acylation catalyzed by torenia 5AT on flower color could be evaluated. Transgenic roses were

obtained,

and

flowers

with

altered

color

were

subjected

to

flavonoid

analysis.

The

results

are

summarized in Table 2. The production of delphinidin and myricetin indicates that the introduced

F3′5′H

gene

functioned

in

transgenic

roses.

The

delphinidin

contents

(percentage

of

the

total

anthocyanidins and amount) and the changes in flower color varied with the transgenic hosts and

events.

The accumulation of delphinidin conferred flower color changes. Some of them, such as WKS82,

WKS100, WKS116 and WKS140 transformants (Fig. 3B, C, D, F), exhibited a novel blue hue that

has not been achieved in hybridization breeding. First, WKS82/130-4-1 and WKS82/130-9-1 were

selected from these transgenic roses on the basis of flower color and performance in a greenhouse,

and

were

subjected

to

a

field

trial.

Transgenic

WKS77

and

WKS124

also

changed their

flower

color

with

delphinidin

production.

The

amount

of

anthocyanins

increased,

and

the

flower

color

became darker. The color of the flowers remained magenta, probably because the pH of the petals

was lower than that of other cultivars.

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Fig. 3

Flower color changes by delphinidin production. The rose cultivars WKS77, WKS82, WKS100,

WKS116,

WKS124

and

WKS140

were

transformed

with

pSPB130,

and

their

flower

color

changed (left, host; right, a transformant). A flower of the line exhibiting the most significant color

change

is

shown.

(A)

WKS77,

(B)

WKS82,

(C)

WKS100,

(D)

WKS116,

(E)

WKS124,

(F)

WKS140.

These results indicated that delphinidin production confers a blue hue to the flowers. They also

revealed that both the delphinidin contents and the resultant color depend on the host cultivars. A

dominant production of delphinidin is not always easy to achieve by overexpression of the F3′5′H

gene even in the selected cultivars.

Only some anthocyanins (up to 44%) were revealed to be aromatically acylated by the introduced

torenia

5AT

gene.

The

effects

of

anthocyanin

acylation

on

flower

color

were

not

visually

observable

when

the

anthocyanins

were

partly

acylated

because

the

5-acylation

of

anthocyanin

shifts only 4 nm to a longer wavelength (Fujiwara et al. 1998).

Transgenic plants can be used as breeding material, since the transgene is expected to be heritable

by

progeny.

Dominant

production

of

delphinidin

irrespective

of

the

cultivars

is

a

desirable

characteristic to incorporate into rose breeding. However, since the delphinidin content depends

on

cultivars

to

a

large

extent,

the

incorporation

of

t

he

overexpression

of

the

viola

F3′5′H

gene