Biosci. Biotechnol. Biochem.

, 74 (9), 17601769, 2010

Award Review

Flower Color Modication by Engineering

of the Flavonoid Biosynthetic Pathway: Practical Perspectives
Yoshikazu T ANAKA,1; y Filippa B RUGLIERA,2 Gianna K ALC,2 Mick SENIOR,2 Barry D YSON,2
Noriko N AKAMURA,1 Yukihisa K ATSUMOTO,1 and Steve C HANDLER2
1
2

Institute for Plant Science, Suntory Holdings Ltd., 1-1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan
Florigene Pty Ltd., 1 Park Drive, Bundoora, Victoria 3083, Australia

Online Publication, September 7, 2010

[doi:10.1271/bbb.100358]

The status quo of avonoid biosynthesis as it relates

to ower color is reviewed together with a success in
modifying ower color by genetic engineering. Flavonoids and their colored class compounds, anthocyanins,
are major contributors to ower color. Many plant
species synthesize limited kinds of avonoids, and thus
exhibit a limited range of ower color. Since genes
regulating avonoid biosynthesis are available, it is
possible to alter ower color by overexpressing heterologous genes and/or down regulating endogenous
genes. Transgenic carnations and a transgenic rose that
accumulate delphinidin as a result of expressing a
avonoid 30 ,50 -hydroxylase gene and have novel blue
hued owers have been commercialized. Transgenic
Nierembergia accumulating pelargonidin, with novel
pink owers, has also been developed. Although it is
possible to generate white, yellow, and pink-owered
torenia plants from blue cultivars by genetic engineering, eld trial observations indicate diculty in obtaining stable phenotypes.
Key words:

anthocyanin; avonoid; ower; genetic

engineering; transgenic plants

Most currently cultivated oricultural crops have been

developed by a combination of extensive hybridization
and mutation breeding, but hybridization breeding
suers from genetic constraints within a species.
Genetic engineering liberates plant breeders from such
constraints. Genetically modied soybean, corn, canola,
and cotton varieties have gained considerable market
share since the rst commercial production in 1996, and
were cultivated on 134 million ha across the world in
2009 (http://www.isaaa.org).
Successful genetic engineering of plants required
several technological breakthroughs, including isolation
of useful genes, the establishment of an ecient
transformation system for a target species, and regulation of transgene expression. We have been working
on the genetic engineering of oricultural crops focusing
to produce novel ower colors. We have isolated many

genes encoding enzymes that act on compounds that

contribute to ower color, have established transformation systems for several oricultural crops, and have
modied the biosynthetic pathway of ower pigments.
Since this eld has been reviewed several times,15) we
focus here on recent progress in ower color and
avonoid biosynthesis relevant to ower color modication, describing major achievements in this eld,
including previously unpublished results.
Flower and fruit color are important in the attraction
of pollinators and animals for seed dispersal, and thus
are critical factors in plant survival. Flower and fruit
color is derived mainly from avonoids, carotenoids,
and betalains. Flavonoids and their colored class,
anthocyanins, contribute to a wide range of colors; pale
yellow, orange, red, magenta, violet, blue. Carotenoids
are ubiquitously distributed in plants as essential
components of photosynthesis and confer a yellow or
red color on owers when they are present. Betalains
also result in yellow and red color, but only the families
of Caryophyllales (except for Caryophyllaceae and
Molluginaceae) produce betalains. To date, no plants
producing both anthocyanins and betalains have been
discovered. This review focuses on the avonoids that
have been studied and engineered most extensively
among these three classes of pigments.
Flavonoids are a class of phenylpropanoids that have
a C3-C6-C3 structure. They are classied into more than
10 groups, mainly depending on the structure of the
C-ring (Fig. 1). The avonoids relevant to ower color
are usually localized in the vacuole, mostly in glycosylated and acylated forms. Anthocyanidins are the
aglycone form and are chromophore of anthocyanins,
and are also direct precursors of anthocyanins in their
biosynthesis (Fig. 1). In spite of the structural diversity
of anthocyanins, there are basically three major anthocyanidins: pelargonidin, cyanidin, and delphinidin. An
increase in the hydroxyl group number confers a color
shift to blue, and thus anthocyanins derived from
delphinidin, cyanidin, and pelargonidin tend to yield
violet/blue, red/magenta, and orange/intense red ower

This review was written in response to the corresponding authors receipt of The Japan Prize of Agricultural Science in 2009.
To whom correspondence should be addressed. Tel: +81-75-962-8807; Fax: +81-75-962-3791; E-mail:Yoshikazu Tanaka@suntory.co.jp
Abbreviations: THC, 20 ,40 ,60 ,4-tetrahydroxychalcone; ODG, 2-oxoglutarate dependent dioxygenase; DHK, dihydrokaempferol; FNS, avone
synthase; P450, cytochrome P450; F2H, avanone 2-hydroxylase; F30 H, avonoid 30 -hydroxylase; F30 50 H, avonoid 30 ,50 -hydroxylase; FLS, avonol
synthase; DFR, dihydroavonol 4-reductase; DHM, dihydromyricetin; ANS, anthocyanidin synthase; F3GT, UDP-glucose: avonoid
3-glucosyltransferae; AT, acyltransferase; GST, glutathione S-transferase; MRP, multidrug resistance-associated protein; ABC, ATP binding
cassette; MATE, multidrug and toxic extrusion; bHLH, basic helix loop helix; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase
y

Flower Color Modication

1761

Fig. 1. Schematic of the General Flavonoid Biosynthetic Pathway Relevant to Flower Color.
Anthocyanidin is further modied with glycosyl, acyl, or methyl groups catalyzed by glycosyltransferase (GT), acyltransferase (AT), and
methyltransferase (MT). Classication of avonoids is shown in parentheses. Abbreviations: CHS, chalcone synthase; F3H, avanone
3-hydroxylase; F30 H, avonoid 30 -hydroxylase; F30 50 H, avonoid 30 ,50 -hydroxylase; DFR, dihydroavonol 4-reductase; ANS, anthocyanidin
synthase; THC40 GT, tetrahydroxychalcone 40 -glucosyltransferase; AS, aurusidin synthase; FLS, avonol synthasae; FNS, avone synthase;
LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase.

color, respectively.6) Classically bred roses, carnations,

chrysanthemums, and lilies lack delphinidin-based
anthocyanins, and this is the primary reason there are
no blue/violet varieties in these species.
Plants adopt many sophisticated mechanisms to
exhibit and stabilize the ower color they need,
especially blue colors. The mechanism of blue ower
colur has attracted the attention of chemists and
biologists for many years, and is discussed in a recent
excellent review.6) The glycosylation, acylation, and
methylation of anthocyanidins are the primary sources
of structural diversity. The glycosylation and methylation of anthocyanins causes the color to become
slightly redder. Aromatic acylation of anthocyanins
shifts their absorption maxima toward a longer wavelength (bluer). Intermolecular stacking of anthocyanins
modied with plural aromatic acyl groups, polyacylated
anthocyanins,7) such as in gentian, buttery pea, and
cineraria, results in a stable blue color. Stacking with
co-pigments, typically avones and avonols, results in
a bathochromic shift to a deeper and bluer color.
Complexation with metal ions also results in a blue

color. Anthocyanin color also depends on pH to a large

extent. Acidic and neutral vacuolar pH gives redder and
bluer color respectively. Recent progress is described
below.

I. Anthocyanins and Their Biosynthesis: An

Update of Recent Progress
1. Recent progress in understanding ower color:
metal ions
Although tulip petals accumulate delphinidin-based
anthocyanins, there are no completely blue varieties.
Some purple cultivars of tulip have blue coloration at the
bottom of the perianth, and the blue and the purple parts
of the ower contain the same anthocyanin (delphinidin
3-rutinoside) and avonols, and have a similar vacuolar
pH, but the concentration of iron is 25 times higher in
the blue perianth parts than in the purple parts. In-vitro
reconstruction with delphinidin 3-rutinoside, avonols,
and Fe3 reproduce the blue color.8) An iron transporter
located in the vacuolar membrane (TgVit1) has been
found to be responsible for the blue coloration. Transient

1762

Y. TANAKA et al.

expression of the gene in a purple tulip petal resulted in

a small number of cells turning blue.9) Expression of
TgVit1 and suppression of the ferritin (an iron storage
protein) gene, TgFer1, was necessary to reproduce the
blue color of the bottom of the perianth. TgVit1
specically expresses in the epidermal cells of the
perianth bottom, and TgFer1 is suppressed at the
bottom.10)
Hydrangea macrophylla changes in sepal color
depending on the availability of Al3 , whose importance to color has been claried by precise chemical
analysis. The extract of the colored protoplasts derived
from blue and red sepals were subjected to identify the
compounds they contained. Blue cells contain 13
equivalents of 5-acylquinic acids (co-pigments) and
1.2 equivalents of Al3 to anthocyanin (delphinidin
3-glucoside, 12.9 mM), while red cells contain only 3.6
equivalents of 5-acylquinic acids and a 0.03 equivalent
of Al3 . In vitro reconstruction using 10 mM delphinidin
3-glucoside with 22.5 equivalents of 5-acylquinic acids
and 1 equivalent of Al3 at pH 4.0 reproduced the color
of the blue cells, and mixing of 10 mM delphinidin
3-glucoside with 21.5 equivalents of 5-acylquinic acids
and a 0.03 equivalent of Al3 at pH 3.0 reproduced
the color of the red cells.11) Although Fe3 and Al3
resulted in clear bluing eects on anthocyanin color and
knowledge of metal iron transfer from the roots to the
aerial parts of plants is rapidly accumulating, especially
for iron,12) utilization of these ions to generate blue
owers in transgenic plants remains challenging since
an excess of heavy metal ions is generally detrimental
to plants.
2. Recent progress in the biosynthesis of avonoids
relevant to ower color
A major pathway relevant to ower color is shown in
Fig. 1.3,13) Chalcone synthase is the rst committed
enzyme in avonoid biosynthesis. 20 ,40 ,60 ,4-tetrahydroxychalone (THC) is stereospecically isomerized to (2S)naringenin (a avanone) by the catalysis of chalcone
isomerase. Flavanone 3
-hydroxylase, a 2-oxoglutarate
dependent dioxygenase (ODG), catalyzes 3-hydroxylation of avanones to the corresponding dihydroavonols, e.g., naringenin to dihydrokaempferol (DHK).
Flavanones are direct precursors of the corresponding
avones. Flavones act as co-pigments, and contribute to
a bathochromic shift (bluing and intensifying of colors)
of anthocyanins. Flavone synthesis is catalyzed by
avone synthase (FNS). There are two kinds of FNS:
an ODG type (FNSI) found in parsley14) and rice,15) and
a more ubiquitous cytochrome P450 (P450) type. In
legumes, avones are biosynthesized from avanones in
two steps, by the reaction of avanone 2-hydroxylase
(F2H) and then dehydration. In other plants (torenia,
snapdragon, gentian, perilla, gerbera, etc.) avones are
directly biosynthesized by the action of avone synthase
II (FNSII) on avanones.16) F2H and FNS belong to the
same subfamily (CYP93B) of the P450 superfamily.
Recently a P450 that is more closely related to FNSII
than F2H was identied in soybean, suggesting that
legume plants have both FNSII and F2H.17)
Flavone C-glucoside (e.g., isovitexin) has a strong
co-pigment eect, as in Japanese iris18) and a transgenic
carnation.19) Recent work on rice has revealed that

2-hydroxyavanone is glucosylated by the catalysis a

UDP-glucose dependent C-glucosyltransferase, and then
dehydrated to avone C-glucoside.20) It is yet to be
determined whether this pathway is the same in plant
species that accumulate a avone C-glucoside.
Flavonoid 30 -hydroxylase (F30 H) and avonoid 30 ,50 hydroxylase (F30 50 H) catalyze the hydroxylation of
avanones, dihydroavonols, avones, and avonols.
They determine the hydroxylation pattern of the B-ring
of anthocyanins, and are thus critical enzymes in ower
color engineering.16) They belong to the same family,
CYP75, of the P450 superfamily. In the chrysanthemum
family, the F30 50 H gene evolved from the F30 H gene
after the speciation of the chrysanthemum21) and a few
amino acid substitutions of F30 H resulted in F30 50 H
activity.22)
Dihydroavonols are also at a branching point of
avonoid biosynthesis, and are the direct precursors of
avonols. Flavonols are colorless or pale yellow to
human eyes by themselves, and contribute to bluing as
co-pigments. Flavonol biosynthesis is catalyzed by
avonol synthase (FLS), an ODG. Dihydroavonols
are reduced to leucoanthocyanidins by the action of
dihydroavonol 4-reductase (DFR). Competition between FLS and DFR aects ower color and the
amounts of avonols and anthocyanins are often
reciprocal in transgenic plants.23,24) Native plants can
avoid such competition, since FLS expression (avonol
biosynthesis) usually precedes DFR expression (anthocyanin biosynthesis) in petal development.
The DFRs of many plant species recognize DHK,
dihydroquercetin, and dihydromyricetin (DHM). Even
the DFR of rose, carnation, and chrysanthemum can
catalyze DHM, although they do not normally produce
DHM due to a lack to F30 50 H. On the other hand,
petunia25) and cymbidium26) DFR cannot utilize DHK as
a substrate. Petunia DFR catalyzes DHM eciently, and
is thus a suitable molecular tool to enhance the pathway
leading to delphinidin, as described below.
Leucoanthocyanidins are converted to the corresponding anthocyanidins by the catalysis of anthocyanidin
synthase (ANS, often called leucoanthocyanidin dioxygenase). FLS and ANS are multifunctional enzymes
(FLS has ANS activity and ANS has FLS activities) in
Arabidopsis, as recently reviewed.27) It is still an open
question whether such multifunctionality is applicable to
other plant species and how signicant it is for ower
color.
Anthocyanidins are unstable at neutral pH and must
be stabilized by glycosylation and acylation. The rst
glucosylation reaction is usually 3-glucosylation catalyzed by UDP-glucose:avonoid glucosyltransferase
(F3GT) resulting in the production of colored anthocyanins. Species specic glycosylation and acylation of
anthocyanins can then occur to result in diverse
anthocyanins. It was thought that anthocyanin biosynthesis proceeds in the cytosol, but recently some
acylation reactions have been found most likely to
proceed in the vacuole, catalyzed by acyl-glucose
dependent serine carboxypeptidase like enzymes, as
recently reviewed.1) Another anthocyanin acyltransferase (AT), one better characterized, is acyl CoA dependent BAHD type anthocyanin AT.28)

Flower Color Modication

3. Transport of anthocyanins to the vacuole and

regulation of vacuolar pH
An excellent review29) is available on this topic, and
we summarize this subject only briey here. Floral
avonoids accumulate in the vacuole, while biosynthesis
of them occurs mainly in the cytosol. The transport
mechanism to the vacuole is not as clear as the
biosynthesis. A better understanding of the sequestration
of anthocyanins and avonoid glycosides to the vacuole
may be important in the further alteration of ower
color, since blue owers tend to accumulate larger
anthocyanin molecules than red owers, and it may be
that the transport system in red-ower species is not
compatible with the larger anthocyanin molecules.
Two routes to vacuolar storage have been described.
One is membrane transporter-mediated, and the other is
vesicle-mediated. These are not necessarily exclusive,
and are dependent on plant species, organ type, and stage
of plant development. The membrane transportermediated route in maize has been found to consist of a
glutathione S-transferase (GST, BZ2) and a multidrug
resistance-associated protein (MRP)-type ATP binding
cassette (ABC) transporter (MRP3). Various results
indicate that GST is involved in anthocyanin transport
in other plants. The involvement of MRP has been
conrmed only for maize.30) Proton-gradient dependent
multidrug and toxic extrusion (MATE) transporters have
been found to facilitate epicatechin 30 -O-glucoside in
Medicago truncatula and Arabidopsis.31) Grapevine
(Vitis vinifera) MATE transporters (AM1 and AM3)
carry acylated anthocyanins (3-p-coumaroylglucosylated
anthocyanins) but not malvidin 3-glucoside, the predominant anthocyanin in grape skin.32) Malvidin 3-glucoside
might be carried into vacuoles by another mechanism.
Anthocyanin color depends on vacuolar pH. Lower pH
confers red color and neutral pH, blue. Intense red rose
petals tend to have lower vacuolar pH (about pH 4), and
hence the ability to manipulate vacuolar pH is important
in engineering ower color. Pelargonium and impatiens
petals contain delphinidin, but these species lack violet/
blue varieties, probably due to their lower vacuolar pH.
Regulation of vacuolar pH in petals has been studied in
morning glory and petunia. They regulate vacuolar pH in
the reverse direction. The vacuolar pH in wild-type
morning glory owers increases from pH 6.6 to 7.7
before ower opening through the expression of sodium
proton antiporter homolog33,34) recently shown to be a
potassium proton antiporter.35) Wild-type petunias acidify the petal vacuole by expressing a P3A -H -ATPase
localized on the vacuolar membrane,36) due to which
ower color changes from violet to magenta.
A recent example of vacuolar pH aecting ower
color was reported for Veronica petals. The petals
contain delphinidin 3-di-p-coumaroylsophoroside-5-malonylglucoside and 7-glucuronyl or 7-glucuronyl glucuronyl apigenin. Purple, violet, and blue cells have been
observed in the petals and in in vitro reconstitution
experiments, indicating that the vacuolar pH levels of
these cells are pH 5, 6, and 7, respectively.37)
4. Transcriptional control of anthocyanin biosynthesis
in owers and fruits
A complex consisting of two transcriptional factors,
basic helix loop helix (bHLH) and R2R3-MYB, and

1763

WD40 (MBW complex) activates the transcription of

avonoid biosynthetic genes.38,39) In the case of petunia,
they are ANTHOCYANIN1 (AN1), AN2, and AN11
respectively. bHLH and R2R3-MYB bind specic sequence motifs in regulatory regions of the transcription
of avonoid biosynthetic genes. The WD40 protein
facilitates protein-protein interaction. bHLH factors
and WD40 proteins are ubiquitously expressed, while
specic MYB proteins regulate specic biosynthetic
pathways. This process of regulation has been studied in
model species such as petunia, Arabidopsis, and snapdragon.38) The basic regulatory mechanisms of anthocyanin biosynthesis are well conserved and are regulated
dierently in distinct species.40) The biosynthesis of
anthocyanins, avones/avonols, and proanthocyanidins is regulated dierently depending on the species.
One plant species has more than 100 R2R3-MYB genes
and forms clusters consisting of orthologs from various
plants. R2R3-MYB, regulating anthocyanins and proanthocyanidins, forms distinct but related clusters.
GtMYB3 and GtbHLH1, which belong to the same
clusters as petunia AN2 and AN1 respectively, have been
isolated from gentian petals. Their expression proles
matched the accumulation of gentian anthocyanin. The
GtMYB3 and GtbHLH1 proteins have been found to
interact in a yeast two-hybrid system. Co-expression of
these genes up-regulated the transcription of gentian
anthocyanin biosynthetic genes. A white cultivar was
shown to have a transposon inserted in the GtMYB3
gene.41) Two R2R3-Myb genes, LhMyb6 and LhMyb12
(homolog of petunia AN2), were isolated from an Asiatic
hybrid lily. Their proteins interacted with a lily bHLH
(LhbHLH2), and transient coexpression of LhMyb6 and
LhbHLH2 or LhMyb12 and LhbHLH2 activated the
transcription of anthocyanin biosynthetic genes in lily
bulb scales. LhMyb12 positively regulates anthocyanin
biosynthesis in the tepals, laments, and styles, and
LhMyb6 in the anthocyanin spots in the tepals and lightinduced pigmentation in leaves.42)
Genes of R2R3-MYB10 belonging to the same cluster
as petunia AN2 have been isolated from many members
of the rosaceous family (apple, pear, plum, cherry, peach,
raspberry, rose, and strawberry). The expression of these
genes correlates with fruit and ower anthocyanin levels.
Constitutive expression of strawberry MYB10 in the
strawberry resulted in elevated anthocyanin levels in the
leaves, fruits, red roots, and stigmas.43) Rearrangement in
the upstream regulatory region of apple MYB10 activated
anthocyanin production, producing red foliage and fruit
esh. This rearrangement generated an autoregulatory
locus, and this autoregulation was sucient to increase
MYB10 transcript levels and the accumulate anthocyanins throughout the plant ectopically.44)
Gerbera hybrida R2R3-MYB10 is highly homologous
to petunia AN2. Transgenic gerbera expressing the gene
constitutively showed enhanced accumulation of anthocyanin pigments and an altered pigmentation pattern.
Microarray analysis of transgenic gerbera indicated that
it activated the transcription of a GST involved in
anthocyanin transport and a serine carboxypeptidaselike gene, in addition to avonoid biosynthetic genes.45)
Ectopic expression of Arabidopsis PAP1 (AtMYB75), an
R2R3-MYB gene, activates anthocyanin biosynthesis in
tobacco, but not in Medicago truncatula or alfalfa. On

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Y. TANAKA et al.

the other hand, the expression of Legume Anthocyanin

Production 1 (LAP1), an R2R3-MYB gene, from
M. truncatula, induced an accumulation of anthocyanins
and proanthocyanidins by elevating transcripts of two
avonoid and anthocyanin biosynthetic genes, a MATElike antiporter and ABC transporter.46)
Overexpression of Arabidopsis PAP2 (AtMYB90)
also activates anthocyanin biosynthesis in tobacco.
AtMYB90, lacking a C-terminal 78 amino acid sequence, converted the R2R3-MYB gene that activates
plant-wide anthocyanin biosynthesis to a dominantnegative allele that interfered with normal tobacco
pigment production.47) Overexpression of the bHLH or
R2R3-MYB gene resulted in ectopic expression of
anthocyanins and often darker owers, as reviewed.2)
Expression of a bHLH (snapdragon Delila) and a R2R3MYB (snapdragon Rosea 1) under a fruit-specic
promoter in tomato yielded purple fruits accumulating
anthocyanins at a concentration comparable to the
anthocyanin levels found in blackberries and blueberries.48) Such tomatoes might be benecial to health.
Grapevine R2R3 MYB genes have been analyzed
comprehensively.49) Grape skin color is determined by
the genotype of a Myb gene, R2R3 Myb (VvmybA1c),
which regulates anthocyanin biosynthesis by controlling the transcription of the F3GT gene. Insertion of a
retrotransposon into the promoter region of VvmybA1c
results in VvmybA1a and acyanic skin color.50) Analysis of the grapevine genome has revealed that it
contains more than 100 R2R3 MYB genes. They are
classied in terms of their structures and similarity to
their Arabidopsis orthologs.49) Anthocyanin related
MYBA clusters are located on chromosomes 2 and
14.49) Another R2R3-MYB protein, VvMYBPA1, regulates proanthocyanidin biosynthesis in berry skin and
seeds by controlling the transcription of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase
(ANR), both of which are critical enzymes in proanthocyanidin biosynthesis (Fig. 1). VvMYBPA1 does not
activate the F3GT gene.51) Proanthocyanidin biosynthesis in persimmon fruit is also regulated by an R2R3MYB, DkMyb4, that belongs to the same cluster as
VvMYBPA1. Ectopic expression of DkMyb4 in kiwifruit
induces proanthocyanidin biosynthesis but not anthocyanin biosynthesis.52)
VvMYB5a and VvMYB5b activate the transcription of
avonoid biosynthetic enzyme genes (F30 50 H, ANS,
LAR) but not the F3GT or the ANR genes.53,54) They
are more closely related to petunia PH455) than AN2.
PH4 encodes an R2R3MYB that interacts with AN1 (a
bLHL). The AN1/PH4 complex activates genes such as
PH5, which encodes a P3A -H -ATPase36) required for
vacuolar acidication via PH3, probably a transcriptional
factor.40,55) Overexpression of VvMYB5b in tobacco
results in the accumulation of anthocyanin- and proanthocyanidin-derived compounds.54) VvMYB12 might
regulate avonol biosynthesis by controlling the expression of the FLS gene,56) and its expression and avonol
biosynthesis is suppressed by post-veraison sun exposure. Flavonol synthesis is regulated dierently from
anthocyanin biosynthesis in Arabidopsis by AtMYB12.57)
In addition to the R2R3-Myb and bHLH transcriptional factors, other transcriptional factors also regulate
avonoid and anthocyanin biosynthesis. A small MYB

protein, R3-MYB (MYBL2), suppresses avonoid biosynthesis by interacting with a MBW complex. The loss
of MYBL2 resulted in a dramatic increase in anthocyanins in Arabidopsis seedlings.58,59) Overexpression of
MYBL2 inhibited the biosynthesis of proanthocyanidins
in seeds.58) Plant-specic NAC transcriptional factor has
been found to regulate anthocyanin biosynthesis. An
Arabidopsis ANAC078 induced anthocyanin accumulation by activating anthocyanin biosynthetic genes via
transcriptional factors regulating biosynthesis under
strong-light conditions.60)
5. Unsolved problems in avonoid biosynthesis and
metabolism
The avonoid biosynthetic pathway is probably the
most thoroughly studied plant secondary metabolism
pathway. Except for the glucosyltransferases and ATs
that are necessary for polyacyl anthocyanins, most of the
enzymatic genes have been characterized. Isolation of
the genes of these enzymes should provide more
molecular tools to engineer anthocyanin structure and
ower color. It is not clear whether these modications
occur in the cytosol or in the vacuole or on membranes.
The enzymes involved in some plant metabolic
pathways, including avonoid biosynthesis, perhaps
form a macromolecular complex (metabolon) for metabolic channeling and ecient biosynthesis.61) Metabolons are thought to be anchored onto the ER membrane
utilizing cytochromes P450 such as F30 H.61) More
experimental data are necessary to conrm this. If a
metabolon is necessary for ecient synthesis of anthocyanins, an enzyme from an exogenous gene must be
compatible with the endogenous metabolon.
The petals of some plants fade or lose color during
development. Anthocyanin degradation and disappearance have been reviewed,62) and also is important in
terms of engineering ower color. Further understanding
of these problems should contribute to engineering of
the pathway.

II. Color Modication by Genetic Engineering

1. Color modication towards blue
Carnations
Blue owers exhibit color through a combination of
the various factors mentioned above. Although many
genes regulating these factors have been isolated, only
delphinidin production is a proven genetic modication
tactic to alter ower color towards blue. Here we refer to
carnations and roses, species in which this tactic has
been used and new varieties have been commercialized.
Naturally, both species accumulate pelargonidin and
cyanidin-based anthocyanins, but they do not produce
delphinidin-based anthocyanins due to a lack of F30 50 H.
In order to achieve signicant color change, it is
necessary to elevate the delphinidin content to more
than 90%, and ideally close to 100%. This cannot be
achieved by a simple overexpression of a F30 50 H gene.
Avoiding competition with endogenous enzymes (F30 H,
DFR, FLS) or downregulation of endogenous enzymes
is necessary to obtain a high content of delphinidin.
Since ower color depends on vacuolar pH and copigment, choosing a host with a proper genetic background is also important.

Flower Color Modication

1765

Fig. 2. Color Modied Transgenic Flowers.

A, Transgenic carnations, Moon Series, that accumulate anthocyanins derived from delphinidin and their production farm in Colombia.
B, Transgenic roses, Suntory Blue Rose APPLAUSE. C, Schematic of the T-DNA designed to deviate the anthocyanin pathway from
delphinidin to pelargonidin in Nierembergia. The host ower is shown on the left and a transgenic ower right. P1, Mac1 promoter;78) P2,
enhanced cauliower mosaic virus 35S promoter;79) T, mannopine synthase terminator, NF30 50 H contains the inverted repeat of the
Nierembergia F30 50 H sequence, and NFLS contains an inverted repeat of the Nierembergia FLS sequence. D, Outdoor eld trial of color
modied transgenic torenia plants in Australia. Half-shade trial is shown above and full-sun trial below. Strong growth retardation was observed
for SWB/805-27. These photos were taken December 2007. E, Comparison of owers of torenia plants grown indoors (rst row of each photo)
and outdoors (second row of each photo). Some lines showed unstable phenotypes, especially outside. These photos were taken in March 2008.
See the text for details.

Almost exclusive accumulation of delphinidin was

achieved in carnations, by expressing a F30 50 H gene and
a petunia DFR gene in white carnation cultivars that
were specically decient in the DFR gene (Fig. 2A).
The nal color hue depends on the genetic backgrounds
of the host cultivars. Since the activity of transgenes
varies between transgenic lines, the color and the
amount of anthocyanins also vary. It is important to
establish ecient transformation systems and to generate a large number of transgenic lines in order to
obtain plants with the desirable phenotype. Twelve, six,
and three lines are now sold in the USA, Japan, and EU,
assuming permits for commercialization. The permit is
necessary for every country or area, and regulatory
procedures depend on the country. Commercial and
regulatory issues relating to carnations have been
discussed in the literature.1,4)

Rose
The cultivated rose (Rosa hybrida) was bred from
about eight wild species, including R. chinensis,
R. galica, R. gigantia, R. moschata, R. multiora,
R. wichuraiana, and R. foetida after extensive interspecic hybridization (http://www.gifu-u.ac.jp/
fukui/
02-1-2-2.htm). Pink and red colors are derived from
pelargonidin or cyanidin-based anthocyanins. The expression of a pansy (Viola spp) F30 50 H gene resulted in
signicant amounts of delphinidin-derived anthocyanins accumulating in the petals of the transgenic plants.
Expression of the pansy F30 50 H gene in rose cultivars
that have higher vacuolar pH, relatively large amounts
of avonols (co-pigments), and weak or no F30 H
activity resulted in transgenic lines in which 95% of
the anthocyanidins was delphinidin. The colors of the
owers in these lines were of a signicantly bluer hue

1766

Y. TANAKA et al.
63)

than any conventionally bred cultivar. The torenia

anthocyanin 5-AT or perilla anthocyanin 3-AT gene
was co-expressed with the pansy F30 50 H but this did
not signicantly enhance bluing. Two lines, WKS82130-4-1 and WKS82-130-9-1, were granted commercial release in Japan after proof that their release would
be most unlikely to aect biodiversity in Japan (https:
//ch.biodic.go.jp/bch/OpenSearch.do) under the Law
Concerning the Conservation and Sustainable Use of
Biological Diversity through Regulations on the Use
of Living Modied Organisms (Cartagena Protocol
Domestic Law). One line, WKS82-130-4-1, was commercially propagated and launched in 2009 (Fig. 2B).
This was the rst and currently is the only commercial
production of a genetically modied plant in Japan.
In order to achieve exclusive delphinidin accumulation irrespective of the endogenous pathway leading to
pelargonidin or to cyanidin, a genetic construct designed
to downregulate the endogenous DFR and overexpress
both a viola F30 50 H and a Dutch iris DFR genes was
introduced into rose cultivars. The petals of the resulting
plants accumulated delphinidin-based anthocyanins exclusively. When a selected line (LA919-4-10) was crossed
with a deep red rose (a cyanidin producer), progeny
harboring the transgenes produced delphinidin exclusively. The amount of delphinidin varied between lines.
The ower color was generally magenta, presumably
because of a low vacuolar pH.63) However, this modication of the pathway also caused some growth retardation and hence is not suitable for commercialization.
As previously mentioned, blue ower color is usually
the additive result of various factors, such as anthocyanin modication, co-pigment concentration, vacuolar
pH, and metal ion type and concentration.6) Targeting of
these factors in addition to delphinidin production in
transgenic carnation and rose is expected to yield bluer
ower colors.
2. Color modication towards red
To cause an accumulation of cyanidin or pelargonidin
is a proven way to generate pink or red owers.
Downregulation of F30 50 H in petunia, torenia, osteospermum, and gentian (as reviewed by Tanaka et al.1))
and also cyclamen64) has been shown to redirect the
delphinidin pathway to cyanidin.
Some species lack red or orange colored owers due
to a deciency of pelargonidin. Eorts to produce
pelargonidin started with the petunia.65) The petunia
does not accumulate pelargonidin since its DFR does not
catalyze DHK. Expression of the maize DFR gene and
those of a few other species in a petunia mutant,
decient in FLS, F30 H and F30 50 H, resulted in transgenics with orange owers, as a result of an accumulation of pelargonidin. It is interesting that the expression of a gerbera DFR gene resulted in the production of
more pelargonidin than in the maize DFR gene. Gene
expression appeared to be more stable.66) The choice of
gene source is important even when enzyme itself has
the same activity. Similarly, campanula F30 50 H was
shown to be more ecient than petunia or lisianthus
F30 50 H in tobacco,67) and buttery F30 50 H more ecient
than verbena F30 50 H in verbena.68)
Red tobacco accumulating pelargonidin was produced
by downregulating the F30 H and FLS genes and over-

expressing a gerbera DFR gene.69) Downregulation of

the F30 50 H gene and the gene encoding anthocyanin
5,30 -AT in gentian changed the ower color from blue to
pink, and was associated with an accumulation of nonacylated cyanidin glucosides.70)
Nierembergia, a popular bedding Solanaceae plant,
has only violet and white varieties. Downregulation of
the F30 50 H gene and additional expression of a rose DFR
gene in Nierembergia resulted in white owers only.71)
This was probably due to its weak F30 H activity and
strong FLS activity. Downregulation of both the F30 50 H
and the FLS gene and overexpression of the rose DFR
gene (Fig. 2C) in blue-owered Nierembergia cultivar
NB18 (in which 99.7% of total anthocyanins are
delphinidin-based) resulted in transgenic plants with
pink owers accumulating pelargonidin (Fig. 2C). In the
petals of the transgenic line with the strongest color
change 74% of total anthocyanins were pelargonidinbased and 26% were cyanidin-based. The amount of
avonols decreased from 2.05 mg/g fresh petal in NB18
to 0.58 mg/g fresh petal in the owers of the transgenic
line, but the total amount of anthocyanidin did not
increase. Enhancement of the ux to pelargonidin-based
anthocyanins and more complete suppression of FLS
gene might lead to transgenic lines with redder owers.
3. Outdoor trial of color-modied transgenic torenia
Transgenic carnations have been produced under
greenhouse conditions for more than 15 years, and the
color-modied phenotypes of the selected transgenic
lines have been stable throughout this continuous period
of vegetative propagation. In ower color-altered transgenic petunia harboring the maize DFR gene under the
control of cauliower mosaic virus 35S promoter,65)
however, the phenotype was not stable and variegated
owers were observed due to methylation of the
promoter when the plants were grown outdoors.72) It is
not clear whether the dierences in phenotypic stability
of the carnation and petunia are due to dierences in the
promoter or coding sequences of the transgene or to
growing conditions. A related question is whether
downregulation of a target gene by the transcription of
double-strand RNA (RNAi technology73)) is stable or
deleterious in transgenic plants grown under outdoor
conditions (which are more extreme). This prompted us
to carry out eld trial of color-modied transgenic
torenia by RNAi.
Two cultivars of torenia (Torenia hybrida) we have
used for genetic modication are Summerwave Blue
(SWB) and Summerwave Violet (SWV). In terms of
assessing the commercial potential of transgenic plants,
two critical factors are the possible eects on biodiversity of the gene ow from the transgenic plants to other
related species and the possibility of the transgenic lines
becoming weeds. SWB and SWV are benign in this
respect as they are both male and female sterile.
The petals of both SWB and SWV mainly accumulate
anthocyanins derived from delphinidin. Cultivar SWV
has darker-colored owers, but both contain a larger
concentration of avones than anthocyanins in the
petals. In the course of a program to modify ower
color in these two varieties, we generated about 100
independent transgenic plants of each cultivar. The lines
with the most stable ower-color phenotype over several

Flower Color Modication

1767

Table 1. Summary of Phenotypes of the Transgenic Torenia in Field Trial (trial 1)

Genetic constructs

Line numbers

Color stability

Over expression of snapdragon THC 40 -GT and aureusidin synthase genes

and knock down of F3H gene in SWB74)

SWB/308-25
SWB/308-35
SWB/308-36
SWB/805-27
SWB/805-36
SWB/1322-45
SWB/1322-90
SWV/1341-17
SWV/1341-85

Unstable
Unstable
Unstable
Stable
Unstable
Stable
Stable
Stable
Unstable

Knock down of ANS gene in SWB75)

Knock down of F30 H and F30 50 H genes in SWB76)
Over expression of pelargonium DFR gene and knock down of F30 H
and F30 50 H genes in SWV76)

years of maintenance in a contained greenhouse were

selected for outdoor trials. The lines selected were as
follows: (i) SWB308-25, SWB 308-35, and SWB-30836. In these transgenic lines, the petals accumulate
aurone and are yellow due to expression of the
snapdragon THC 40 -glucosyltransferase and aureusidin
synthase genes and suppression of the endogenous F3H
gene to downregulate anthocyanin biosynthesis,74) (ii)
SWB805-27 and SWB 805-36. These transgenic lines
have white or partially white (a white cross in blue
petals) owers respectively. This is due to the knockdown of expression of the endogenous ANS gene in
SWB,75) (iii) SWB1322-45 and SWB-1322-90. These
have pink owers due to an accumulation of cyanidinbased anthocyanins as a result of knockdown of F30 H
and F30 50 H genes,76) (iv) SWV1341-17 and SWV 134185. The owers of these two lines accumulate pelargonidin and have strongly pink-colored petals as a result of
knockdown of the F30 H and F30 50 H genes, complemented by the expression of a pelargonium DFR gene.76)
All lines selected were grown in replicated outdoor trials
in Melbourne, Australia, under a license (DIR068/2006,
http://www.ogtr.gov.au/ir/dir068.htm) issued by the
Oce of the Gene Technology Regulator. Permission
for this trial, containing multiple lines and constructs,
was more readily granted than would have been possible
in Japan.
All nine transgenic torenia lines, and two unmodied
controls, SWB and SWV, were grown outdoors in
hanging baskets for two spring-summer-autumn seasons:
October 2007-July 2008 (trial 1), and October 2008-May
2009 (trial 2). In each trial, plants were hung in two
positions, one protected from the wind in partial sun and
the other in a fully exposed, the full-sun position.
Trial 1
By November in each trial, the plants were fully
grown in the shaded area but owered less vigorously
than those in the full-sun area. The results are summarized in Table 1. Several transgenic lines did not grow as
well as the control or the other transgenic lines. These
poor lines were SWB308-36, SWB308-35, and
SWB805-27. In full-sun, SWB805-27 stood out as being
extremely poor as the plants rarely owered (Fig. 2D).
The growth of line SWV1341-17 was as good as the
untransformed control, and line SWB1322-90 stood out
as a very good line, with slightly more vigorous growth
than the control. Reversion to parental ower color was
observed in some plants in some lines. This was most
pronounced in line SWB308-25, where the parental blue
ower color was observed, and in the lines SWB308-36
and SWB308-35, where the petals also partly regained

Growth compared
to the host
Poor
Poor
Poor

the blue parental ower color (Fig. 2E). The transgenic

phenotype was stable in lines SWB805-27, SWB132245, SWB1322-90, and SWV1341-17 (Fig. 2E). It was
found that outdoor plants had smaller owers than the
same lines grown in an adjacent greenhouse at the same
time (Fig. 2E).
Trial 2
The second trial was held from October 2008 to May
2009. In this trial the morphological characters of the
owers were measured, and it was conrmed that the
outdoor plants had smaller owers than the same lines
grown in an adjacent greenhouse at the same time. In
this trial the only lines SWB805-36, SWB1322-45,
SWB1322-90, and SWV1341-17 had both a fully stable
modied ower color phenotype and growth characteristics as good as the host variety.
In these experiments, the instability of the phenotype
was apparent only under outdoor conditions, and only in
some lines. During the trials, the plants outside were
subjected to much higher temperatures and higher light
levels than inside. The implication is that these
conditions interfered with the expression of the aurone
biosynthesis genes transformed into the 308 series of
transgenic lines, or with the method used to suppress the
endogenous F3H gene.
As far as growth of plants outside is concerned, the
results indicated that articial induction of RNAi does
not necessarily have a negative eect on plant growth
but SWB308-35, SWB308-36, and SWB805-27 grew
more slowly the host and the other transgenic lines.
Where the transgenic phenotype was lost in line
SWB308-25 and owers reverted to blue during the
trial, plant growth was comparable to the host. Torenia
petals contain avones and anthocyanins, but only the
anthocyanin biosynthetic pathway was downregulated in
the transgenic plants exhibiting poor growth outdoors.
These lines still produced avonoids, mainly avones,
which are necessary for UV-protection. This implies that
anthocyanins are necessary for normal plant growth
under outdoor conditions, playing a role that avones
cannot play. Another possible explanation is that new
compounds having negative eects on plant growth
accumulate in transgenic plants in which aurone biosynthesis has been engineered.

III. Future Perspectives

Since the beginning of ower color alteration using
genetic modication more than 20 years ago, considerable progress has been made. Though novel blue-violet
ower color carnations and roses have been successfully

1768

Y. TANAKA et al.

marketed, much remains to be done to create more

colorful or even bluer owers by genetic engineering.
For example, among many useful genes for color
modication, F30 H and F30 50 H have been the primary
focus of modication so far. The genes relevant to
avone synthesis, polyacylation of anthocyanins, vacuolar pH regulation, and metal transport have not been fully
exploited. This is partly due to the diculty of optimizing the expression of multiple genes in target species
and the provision of enough substrates for the heterologous enzymes derived from transgenes. A repeated
trial-and-error approach is inevitable to obtain transgenic
lines exhibiting stable phenotypes, and it is therefore
also desirable to develop new technologies to suppress
endogenous gene expression more eciently than current RNAi. Irreversible genome modication by zincnger nuclease77) is a candidate for such technologies.
Although avonoid biosynthesis is the most characterized plant secondary metabolism pathway, the process of avonoid transport to the vacuoles is not yet
understood well, and the proposed metabolon for
avonoid biosynthetic enzymes is still hypothetic in
our view. Specicity for vacuolar transport or metabolon
formation, if any, has to be considered in developing
strategies to produce novel ower color.
These scientic and technological diculties may be
resolved in due course, but the, costs of development
and regulation remain high. Proper permits must be
obtained in countries and areas for commercialization,
and regulatory systems vary greatly depending on the
country and area. Simple, global standards have to be set
for the application of genetic engineering to plant
breeding to ourish. This is especially true for oricultural crops, whose market is segmented and sales per
cultivar relatively small.

Acknowledgments
We wish to thank the current and former members of
Suntory and Florigene for their contributions, over many
years. In order to focus on recent progress, only a
limited number of original papers are cited here.

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