The Plant Journal (2008) 54, 733–749 doi: 10.1111/j.1365-313X.2008.03447.

HARNESSING PLANT BIOMASS FOR BIOFUELS AND BIOMATERIALS

Biosynthesis of plant pigments: anthocyanins, betalains and

carotenoids
Yoshikazu Tanaka1,*, Nobuhiro Sasaki2 and Akemi Ohmiya3
1
Institute for Plant Science, Suntory Ltd, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan,
2
Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,
Koganei, Tokyo 184-8588, Japan, and
3
National Institute of Floricultural Science, Fujimoto 2-1, Tsukuba, Ibaraki 305-8519, Japan

Received 13 October 2007; revised 16 January 2008; accepted 23 January 2008.

*
For correspondence (fax +81 75 962 3791; e-mail yoshikazu_tanaka@suntory.co.jp).

Summary

Plant compounds that are perceived by humans to have color are generally referred to as ‘pigments’. Their
varied structures and colors have long fascinated chemists and biologists, who have examined their chemical
and physical properties, their mode of synthesis, and their physiological and ecological roles. Plant pigments
also have a long history of use by humans. The major classes of plant pigments, with the exception of the
chlorophylls, are reviewed here. Anthocyanins, a class of flavonoids derived ultimately from phenylalanine, are
water-soluble, synthesized in the cytosol, and localized in vacuoles. They provide a wide range of colors
ranging from orange/red to violet/blue. In addition to various modifications to their structures, their specific
color also depends on co-pigments, metal ions and pH. They are widely distributed in the plant kingdom. The
lipid-soluble, yellow-to-red carotenoids, a subclass of terpenoids, are also distributed ubiquitously in plants.
They are synthesized in chloroplasts and are essential to the integrity of the photosynthetic apparatus.
Betalains, also conferring yellow-to-red colors, are nitrogen-containing water-soluble compounds derived
from tyrosine that are found only in a limited number of plant lineages. In contrast to anthocyanins and
carotenoids, the biosynthetic pathway of betalains is only partially understood. All three classes of pigments
act as visible signals to attract insects, birds and animals for pollination and seed dispersal. They also protect
plants from damage caused by UV and visible light.

Keywords: pigments, anthocyanin, betalain, carotenoid, flavonoid, flower.

Introduction
Plants produce more than 200 000 different types of com-
pounds (Fiehn, 2002), including many colored (pigmented)
ones. Humans recognize the color of a compound by
perceiving reflected or transmitted light of wavelengths
between 380 and 730 nm, while insects recognize light of
shorter wavelengths (Davies, 2004). This review will focus
on recent progress in understanding the biosynthesis of
three main classes of pigments for coloration in plants:
flavonoids/anthocyanins, betalains and carotenoids. Engi-
neering of flavonoid and carotenoid biosynthetic pathways
will be discussed separately (Tanaka and Ohmiya, 2008). The
genetics and biochemistry of these pigments have recently
been reviewed (Grotewold, 2006a).
Flavonoids, a group of secondary metabolites belonging
to the class of phenylpropanoids, have the widest color
range, from pale-yellow to blue. In particular, anthocya-
nins, a class of flavonoids, are responsible for the orange-
to-blue colors found in many flowers, leaves, fruits, seeds
and other tissues (Figure 1a–d). They are widely distrib-
uted in seed plants, are water-soluble, and are stored in
vacuoles. Betalains, yellow-to-red nitrogen-containing
compounds, are derived from tyrosine. They are also

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734 Yoshikazu Tanaka et al.

(a) (b)

(e)
(c) (d)

(f) (g) (h) (i)

(j)

(l)
(k)

water-soluble and stored in vacuoles, but are found only in confer yellow-to-red coloration to flowers and fruits.
the order Caryophyllalles. Carotenoids, which are isopre- Flavonoids/anthocyanins and carotenoids are often present
noids and are found ubiquitously in plants and micro- in the same organs, and their combination increases color
organisms, are essential components of photosystems and variety.

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 Plant pigments 735

Figure 1. Plant colors derived from anthocyanins.

(a) Gentian flowers containing diacylated delphinidin-based anthocyanins, such as gentiodelphin (right). Glucosylation (3¢GT, 5GT, 3¢GT) and acylation (5,3¢AT)
reactions are indicated by arrows.
(b) Chrysanthemum petals contain cyanidin 3-(6¢¢-malonyl or 3¢¢,6¢¢-dimalonyl)-glucoside. 3MAT1, malonyl CoA:anthocyanin 3-malonyltransferase; 3MAT2, malonyl
CoA: anthocyanin 3-dimalonyltransferase.
(c) A blue poppy, Meconopsis horridula. The sky-blue color of Meconopsis petals depends on cyanidin-based anthocyanin (Tanaka et al., 2001), flavonol and Fe3+.
(d) Strongylodon macrobotrys, a bat-pollinated plant of the Philippines, with unusual bluish-green flowers. They contain malvidin 3,5-diglucoside and flavones. An
uncharacterized factor seems to yield the greenish color (Iwashina et al., 1984).
(e) The leaves of red Perilla are a source of a natural colorant containing cyanidin. A Perilla anthocyanin is shown. 3AT, hydroxycinnamoyl CoA:anthocyanin
3-hydroxycinnamoyltransferase 5MAT, malonyl CoA: anthocyanin 5-malonyltransferase.
(f) A variegated portulaca (Portulaca grandiflora) flower.
(g) Globe amaranth (Gomphrena globosa) flowers
(h) Christmas cactus flowers.
(i) Variegated flowers of Mirabilis jalapa (‘four o’clocks’). The variegation is likely to be caused by transposons. These plants should be useful to clone betalain-
related genes by transposon tagging.
(j) Marigold flowers with different petal colors caused by different quantities of lutein.
(k) Flowers of Adonis aestivalis have a blood-red color derived from astaxanthin. The structure of astaxanthin is shown.
(l) Yellow and orange petals of calendula. Differences in carotenoid composition result in color differences.

position. Glycosylation at C7¢, C3¢ or C5¢ is often found.

Flavonoids/anthocyanins
Flavonoids are among the best-characterized plant second-
ary metabolites in terms of chemistry, coloration mecha-
nism, biochemistry, genetics and molecular biology
(Grotewold, 2006b; Harborne, 1988, 1994; Stafford, 1990).
The functions of flavonoids/anthocyanins, including their
potential health benefits, have been reviewed (Harborne and
Williams, 2000). Anthocyanins are widely used as natural
food colorants, for example, the cyanidin acylglucosides
from red cabbage (Brassica oleracea var. capitata) and
Perilla frutescence (Figure 1e).
Structures and colors
Flavonoids, with a basic structure of C6–C3–C6, are widely
distributed among land plants. Depending on their struc-
tures, flavonoids may be classified into about a dozen
groups, such as chalcones, flavones, flavonols and antho-
cyanins. Modification of flavonoids with hydroxyl, methyl,
glycosyl and acyl groups results in several thousand struc-
tures. An excellent flavonoid database is available (http://
www.metabolome.jp/software/FlavonoidViewer/).
Orange to blue: anthocyanins
A total of 19 types of anthocyanidins, aglycons or chro-
mophores of anthocyanins are known, but there are only
six major ones: pelargonidin, cyanidin, peonidin, delphini-
din, petunidin and malvidin (Figure 2a). Their color greatly
depends on the number of hydroxyl groups on the B-ring;
the larger the number of groups, the bluer the color.
O-Methylation of anthocyanins has a slight reddening
effect.
Anthocyanidins are modified by glycosyl moieties in
versatile ways in a family- or species-specific manner.
Anthocyanins are most frequently O-glycosylated (usually
glucosylated) at the C3-position, followed by the C5-
Glycosylation of anthocyanins results in slight reddening.
The glycosyl moieties of anthocyanins are commonly
modified by aromatic (hydroxycinnamic or hydroxybenzoic)
and/or aliphatic (malonic, acetic, or succinic) acyl moieties.
Aromatic acylation causes a blue shift and stabilizes antho-
cyanins. Anthocyanins modified with multiple aromatic acyl
moieties (poly-acylated anthocyanins; Honda and Saito,
2002; Figure 1a) often show a stable blue color via intermo-
lecular stacking. Aliphatic acylation does not change the
color but increases the stability and solubility. 3-Deoxyan-
thocyanins, which are found in a limited number of plant
species, such as maize (Zea mays), and 6-hydroxy anthocy-
anins, which are found in Alstroemeria, show a redder color
than the corresponding anthocyanins.
The color of anthocyanins changes depending on the pH,
co-existing colorless compounds (co-pigments, typically
flavones and flavonols), and metal ions. In vitro, anthocy-
anidins are redder and more stable as the flavilium cation
form at lower pH (pH <3), colorless under mildly acidic
conditions (pH 3–6), and bluer and unstable as the quino-
noidal base form at pH 6 and above. Intermolecular stacking
by self-association or with flavones or flavonols (often called
co-pigments) stabilizes anthocyanins and causes a batho-
chromic shift (blueing and intensifying of color). Metal ions,
such as Al3+ and Fe3+, play a critical role in the generation of
blue flowers in hydrangea (Hydrangea macrophylla; Yosh-
ida et al., 2003) and tulip (Tulipa gesneriana; Shoji et al.,
2007), respectively. A subtle stoichiometric balance of a
cyanidin-based anthocyanin, a flavonol, Fe3+ and Mg2+ is
critical to generation of the sky-blue petals of Meconopsis
grandis (Himalayan poppy; Figure 1c; Yoshida et al.,
2006). In cornflower (Centaurea cyanus), a super-molecular
complex of six molecules each of cyanidin 3-(6-succinyl-
glucoside)-5-glucoside and apigenin 7-glucuronide-4-(6-
malonylglucoside), one ferric ion, one magnesium ion and
two calcium ions is responsible for the blue flower color
(Shiono et al., 2005). It is yet to be determined, however,

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 Plant pigments 737

how plants achieve such subtle balances of each component
to generate appropriate colors.
Yellow: chalcones, aurones, flavonols and flavones
Many pale-yellow flowers, such as carnations (Dianthus
caryophyllus) and cyclamens (Cyclamen persicum), contain
2¢,4,4¢,6¢-tetrahydroxy chalcone (THC) 2¢-glucoside (isosali-
purposide). Safflower (Cathamus tinctorius) produces a rare
yellow chalcone glucoside, safflomin A, which is used as a
colorant. Some Asteraceae (the chrysanthemum family)
produce pale-yellow 6¢-deoxy chalcones. Aurones, a class of
rare flavonoids, give brighter yellow flowers than chal-
cones, and are found in a limited number of species, such
as snapdragon (Antirrhinum majus) cosmos (Cosmos
bipinnatus) and Limonium. Flavonols and flavones are very
pale-yellow and are mostly invisible to the human eye. As
they absorb UV, which insects recognize, they give color and
patterns to flowers to attract insects.
Biosynthesis of flavonoids
Main pathway. The flavonoid biosynthetic pathway is
shown in Figure 2(b). The pathway is well understood and is
conserved among seed plants. Flavonoids are synthesized in
the cytosol. It has been proposed that the biosynthetic en-
zymes form a super-molecular complex (metabolon) via
protein–protein interaction and are anchored in the endo-
plasmic reticulum (ER) membrane (Grotewold, 2006a; Win-
kel, 2004). The biosynthetic enzymes belong to various
enzyme families, such as 2-oxoglutarate-dependent dioxy-
genases (OGD), cytochromes P450 (P450) and glucos-
yltransferases (GT), which suggests that plants recruited
these enzymes from pre-existing metabolic pathways. Phy-
logenetic analysis indicates that genes encoding enzymes
with the same or similar activity had diverged before the
speciation of seed plants (Tanaka and Brugliera, 2006), with
some exceptions (see below).
Chalcone synthase (CHS), a polyketide synthase, is the
first committed enzyme in the pathway, and it catalyzes
the synthesis of THC from one molecule of 4-couma-
royl CoA and three molecules of malonyl CoA. THC is
rapidly and stereospecifically isomerized to the colorless
(2S)-naringenin by chalcone isomerase (CHI). (2S)-Naringe-
nin is hydroxylated at the 3-position by flavanone
3-hydroxylase (F3H) to yield (2R,3R)-dihydrokaempferol,
a hydroflavonol. F3H belongs to the OGD family. F3H
also catalyzes the hydroxylation of eryodictyol and penta-
hydroxyl flavanones to dihydroquercetin and dihydro-
myricetin, respectively.
Flavonoid 3¢-hydroxylase (F3¢H) and flavonoid 3¢,5¢-
hydroxylase (F3¢5¢H), which are P450 enzymes, catalyze the
hydroxylation of dihydrokaempferol (DHK) to form (2R,3R)-
dihydroquercetin and dihydromyricetin, respectively. F3¢H
and F3¢5¢H determine the hydroxylation pattern of the B-ring
of flavonoids and anthocyanins, and are necessary for
cyanidin and delphinidin production, respectively. They are
the key enzymes that determine the structures of anthocy-
anins and thus their color (Figure 2b; Tanaka, 2006). F3¢H
and F3¢5¢H catalyze the hydroxylation of flavanones, flavo-
nols and flavones. Many important floricultural crops, such
as roses (Rosa hybrida), chrysanthemums (Chrysanthemum
morifolium) and carnations, do not produce delphinidin and
thus lack violet/blue color varieties. This is attributed to the
fact that they do not possess the F3 ¢5 ¢H gene, probably
because they lost it during their evolution. Interestingly,
some species of Asteraceae have re-acquired the F3¢5¢H
function from their F3 ¢H gene by convergent evolution (Seitz
et al., 2006). Transgenic blue/violet carnations and roses
have been developed by expressing a heterologous F3 ¢5 ¢H
gene (Chandler and Tanaka, 2007; Katsumoto et al., 2007).
Dihydroflavonols are reduced to corresponding 3,4-cis-
leucoanthocyanidins by the action of dihydroflavonol 4-
reductase (DFR). In some plant species, such as petunia
(Petunia hybrida) and cymbidium (Cymbidium hybrida),
DFR has strict substrate specificity and cannot utilize
dihydrokaempferol. This is the reason that these species
lack pelargonidin-based anthocyanins and thus lack flowers
of an orange/brick red color.
Anthocyanidin synthase (ANS, also called leucoanthocy-
anidin dioxygenase), which belongs to the OGD family,
catalyzes the synthesis of corresponding colored anthocy-
anidins. The genes encoding the enzymes mentioned above
have been isolated and characterized from flowers of many
plants, including petunia, snapdragon (Antirrinum majus),
gentian (Gentiana triflora), torenia (Torenia hybrida),
morning glories, and other tissues of maize, Perilla and
Arabidopsis.

Figure 2. Anthocyanidin structures and flavonoid biosynthetic pathways relevant to color.

(a) Structures of major anthocyanidins (R groups listed on the left) and some minor anthocyanidins (R groups listed on the right). The basic structure is shown on
the far left.
(b) The biosynthetic pathway leading to the biosynthesis of anthocyanidins. Anthocyanidin is further modified with glycosyl, acyl or methyl groups, catalyzed by
glucosyltransferases (GT), acyltransferases (AT) and methyltransferases (MT). Enzymes and flavonoid classes are indicated by blue and red letters, respectively. The
typical colors of compounds are also shown, but the actual color depends on various factors as described in the text. CHS, chalcone synthase; THC2¢GT, UDP-
glucose:tetrahydroxychalcone 2¢GT; CHI, chalcone isomerase; THC4¢GT, UDP-glucose:tetrahydroxychalcone 4¢GT; AS, aureusidin synthase; F3H, flavanone
3-hydroxylase; F3¢H, flavonoid 3¢-hydroxylase; F3¢5¢H, flavonoid 3¢,5¢-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FNS, flavone
synthase; FLS, flavonol synthase; Glc, glucose.

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738 Yoshikazu Tanaka et al.
Modification of anthocyanidins. In contrast to the
well-conserved main pathway of flavonoid biosynthesis
mentioned above, the modification of anthocyanidins is
family- or species-dependent and full of diversity. Various
UDP-glycose-dependent glycosyltransferases belonging to
glycosyltransferase family 1 (http://www.cazy.org/fam/acc_
GT.html), acyltransferases (ATs) mainly belonging to the
BAHD family (D’Auria, 2006) and S-adenosylmethionine-
dependent methyltransferases are responsible for such
diversity. These enzymes whose genes have been isolated
are summarized in Figures S1 and S2, and only recent
interesting results are shown here. In general, these en-
zymes are specific to the position of modification on the
anthocyanin and the donor substrates, but less specific to
the anthocyanin structure, i.e. the enzymes catalyzing
modification of A- and C-rings can modify anthocyanins
irrespective of the hydroxylation pattern on the B-ring.
Anthocyanidins are initially 3-glucosylated by the action
of UDP-glucose:flavonoid (or anthocyanidin) 3GT. How-
ever, in roses accumulating anthocyanidin 3,5-diglucoside,
5-glucosylation precedes 3-glucosylation, and both glucosy-
lation reactions are catalyzed by a single enzyme, UDP-
glucose:anthocyanidin 5,3GT (Ogata et al., 2005). Roses also
have the conventional 3-glucosylation pathway catalyzed by
3GT, which functions in stressed cultured cells (Hennayake
et al., 2006) and mature petals in some cultivars (Mizutani
et al., 2007). While the phylogenetic analysis of GTs indi-
cates that 3GT, 5GT and 3¢/7GT form separate clusters
(Figure S1), the amino acid sequence of 3¢,5¢GT from
butterfly pea (Clitoria ternatea) is related to 3GTs rather
than to gentian 3¢GT (Figure S1), indicating that divergent
evolution occurred after speciation of the pea (Noda et al.,
2004).
Unlike other enzymes in the flavonoid biosynthetic
pathway, BAHD-type anthocyanin AT genes diverted after
speciation of seed plant families (Figure S2; Luo et al.,
2007; Tanaka and Brugliera, 2006). Furthermore, the
anthocyanin 3¢,5¢AT of butterfly pea is an acylglucose-
dependent acyltransferase and a member of the serine
carboxypeptidase-like (SCPL) family that localizes in the
vacuole (Noda et al., 2006, 2007). Acyl glucose-dependent
AT activity has also been reported in carrot (Daucus carota;
Glassgen et al., 1998; Okuda et al., 2007). Elucidation of
non-characterized biosynthetic enzymes and the mecha-
nism for production of poly-acylated anthocyanins from
plants such as cineraria (Senecio cruenta) and delphinium
(Delphinium ajacis) may indicate more diversity.
The crystal structure of a chrysanthemum anthocyanin
manolonytransferase and subsequent mutational study
identified the binding sites of acyl CoA and the acyl acceptor
(Unno et al., 2007). Salvia (Salvia splendens) anthocyanin
5-malonyltransferase was successfully modified to show
hydroxycinnamoyltransferase activity towards the 3-gluco-
syl and 5-glucosyl moieties of anthocyanin in vitro by
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 733–749
substituting only three amino acid residues (Suzuki et al.,
2007). This study exemplifies the possible engineering of
enzymes with desirable substrate specificity and kinetic
properties.
Biosynthesis of aurones, flavonols and flavones. THC is
unstable and requires stabilization by glucosylation or
methylation in order to show its yellow color. Mutations of
the CHI gene have been shown to be necessary for accu-
mulation of isosalipurposide in carnation (Forkmann and
Dangelmayr, 1980) and barley (Marinova et al., 2007a).
Additional mutation of the DFR gene confers better yellow
coloration in carnation (Itoh et al., 2002). The existence of
several genes encoding THC 2¢GT activity from carnation
(Figure S1; Ogata et al., 2004; Okuhara et al., 2004) indicates
that several non-specific enzymes may catalyze the reaction
in vivo, unlike the case of anthocyanin GTs. In snapdragon,
THC 4¢-glucoside is synthesized by the action of THC 4¢GT,
presumably in the cytosol (Ono et al., 2006), transported to
the vacuoles, and further converted to aureusidin 6-gluco-
side by aureusidin synthase [AS, a polyphenol oxidase
(PPO)] in the vacuoles (Nakayama et al., 2000). As aurones
are found in a limited number of unrelated species, sev-
eral types of enzymes may be able to catalyze aurone
biosynthesis.
Flavonols and flavones are synthesized from correspond-
ing dihydroflavonols and flavanones by the action of the
OGD flavonol synthase (FLS) and flavone synthase (FNS).
Interestingly, there are two types of FNS: OGD (FNSI) and
cytochrome P450 (FNSII). FNSI has been found in parsley
(Petroselium crispum), and is suggested to have evolved
from F3H (Martens et al., 2003). FNSII is more common, and
is related to flavanone 2-hydroxylase (F2H) in legumes
(Akashi et al., 1999), in which flavones are synthesized in
two steps (F2H and dehydratase) from flavanones.
Transport to the vacuole and vacuolar pH regulation
Flavonoid glycosides, including anthocyanins, are usually
transported into the vacuole. The transport mechanism is
less well understood than the biosynthesis. Transport me-
chanisms may be redundant or depend on plant species and
organs. The first and most established mechanism involves
transport of anthocyanins via a glutathione S-transferase
(GST)-like protein and a multi-drug resistance-like protein
(a type of ABC transporter). Involvement of the former has
been shown in maize (Marrs et al., 1995), petunia (Alfenito
et al., 1998) and Arabidopsis (Kitamura et al., 2004), and the
latter has been identified in maize (Goodman et al., 2004).
The molecular mechanism whereby these proteins, espe-
cially GSTs, achieve the transport has not yet been clarified.
A second mechanism involves vesicle-mediated mass
transport of anthocyanins to vacuoles, as has been observed
in, for example, lisianthus (Eustoma grandiflorum; Zhang
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et al., 2006). Anthocyanins are targeted directly to the pro-
tein storage vacuole via ER-derived vesicles in Arabidopsis
seedlings, and this process does not depend on GST activity
or an ATP-dependent transport mechanism (Poustka et al.,
2007). A third mechanism may involve an Arabidopsis multi-
drug and toxic compound extrusion (MATE) transporter
(TT12), which is a vacuolar flavonoid/H+-antiporter that is
necessary for vacuolar accumulation of proanthocyanidins
and has been shown to mediate anthocyanin transport
in vitro (Marinova et al., 2007b). Interestingly, intact flavo-
noid biosynthesis exerts control over the activity of the
vacuolar flavonoid/H+-antiporter in barley (Marinova et al.,
2007a). The molecular mechanism whereby a plant cell
transports many kinds of flavonoids into a vacuole remains
to be elucidated.
Regulation of the vacuolar pH, which greatly affects
anthocyanin color, is partly understood. The only known
structural gene that regulates vacuolar pH with relevance to
color is the Japanese morning glory (Ipomea nil) Na+/H+-
antiporter that is specifically expressed before flower open-
ing and increases the vacuolar pH to generate blue flowers
(Fukada-Tanaka et al., 2000). Petunia loci that regulate
vacuolar pH have also been identified (Koes et al., 2005),
and the petunia PH4 gene, which activates vacuolar acidifi-
cation, has been shown to be R2R3 Myb (Quattrocchio et al.,
2006).
Transcriptional regulation
The spatial and temporal expression of structural genes in
anthocyanin biosynthesis is determined by a combination of
R2R3 Myb, basic helix–loop–helix (bHLH) and WD40-type
transcriptional factors and their interaction. This has been
well established in maize, Arabidopsis, petunia and some
other plants (reviewed by Koes et al., 2005), and in Japanese
morning glory (Morita et al., 2006). The WD40 and bHLH
proteins are pleiotropic and are involved in multiple pro-
cesses in addition to anthocyanin synthesis, such as the
control of vacuolar pH in petunia flowers and the formation
of trichomes and root hairs in Arabidopsis. It is believed that
they affect these processes via their interactions with
specific MYB proteins, such as PH4 in petunia and GL1/Wer
in Arabidopsis (Koes et al., 2005).
Intimate functional analysis of maize R (a bHLH) has
provided new insight into its regulatory mechanism. R has a
novel dimerization domain for its regulatory activity, and
this domain was evolved from the ACT domain that is
involved in the allosteric regulation of many amino acid
metabolic enzymes (Feller et al., 2006). The bHLH region of R
recruits an EMSY-like maize nuclear factor to flavonoid
biosynthetic gene promoters, and this recruitment is asso-
ciated with histone acetylation (Hernandez et al., 2007).
R2R3 Myb contributes to speciation of some plant species. A
small family of R2R3 Myb genes controls floral pigmentation
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Plant pigments 739
intensity and patterning in the genus Antirrhinum, and their
activity causes natural variation in anthocyanin pigmenta-
tion (Schwinn et al., 2006). Mutations in AN2 (an R2R3 Myb
gene) result in acyanic corolla limbs in the wild petunia,
P. axillaris, which has white moth-pollinated flowers, while
P. integrifolia, which has a functional AN2 allele, has
magenta bee- and butterfly-pollinated flowers (Hoballah
et al., 2007).
Betalains
Betalains show brilliant color in flowers (Figure 1f–i) or fruits
of species belonging to the families of Caryophyllales,
except for Caryophyllaceae and Molluginaceae. The mutual
exclusiveness of anthocyanins and betalains in the Caryo-
phyllales has given rise to considerable taxonomic debate
(Clement and Mabry, 1996). Poor understanding of betalain
biosynthesis in terms of biochemistry and molecular biology
has prevented researchers from interpreting this taxonomic
mystery. Betalains from red beet (Beta vulgaris) are used as
a natural colorant. The advantage of betalain color is that the
color does not depend on the pH and is more stable than that
from anthocyanins.
Structures and colors
Betalains are classified into red (crimson) betacyanins and
yellow betaxanthins. They are immonium conjugates of
betalamic acid with cyclo-dihydroxyphenylalanine (cDOPA)
glucoside and amino acids or amines, respectively (Figure 3;
Strack et al., 2003). Only betacyanins are modified by glyco-
syl or acyl moieties. More than 50 molecular species of
betacyanins and several betaxanthins have been isolated and
identified, and novel betalain molecules are being reported in
accordance with the progress in development of analytical
equipment (Kugler et al., 2007; Wybraniec et al., 2007).
The earliest biological research on betalains was on
genetic inheritance of the flower colors red (crimson), yellow
and white in ‘four o’clocks’ (Mirabilis jalapa) by Mendel
(Mendel, 1950). Later it was discovered that the C (Color)
gene is required to synthesize both the yellow and red
pigments, and that the R (Red) gene acts downstream of the
C gene to yield the red pigment from the yellow pigment in
the flowers of portulaca (Portulaca grandiflora; Trezzini and
Zryd, 1990). A similar result was obtained in studies on
flower coloring of M. jalapa (K. Kasahara, Yokohama, Japan,
unpublished results).
Biosynthesis of betalains
The biosynthetic pathways of betalains and the enzymes
and genes involved in the pathway are much less well
understood than those of flavonoids and carotenoids. The
biosynthetic steps were first proposed a quarter of a
740 Yoshikazu Tanaka et al.

Figure 3. Proposed biosynthetic pathway for betalain pigment.

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century ago, but mainly on the basis of phytochemical
evidence, such as feeding experiments. Advances in
molecular biological techniques will permit elucidation of
the pathway in terms of biochemistry and molecular biol-
ogy. Here, recent significant progress regarding the beta-
lain biosynthetic pathway is reviewed, with particular focus
on DOPA 4,5-dioxygenase (DOD) and cDOPA 5-O-GT
(cDOPA5GT).
The biosynthetic pathways consist of several enzymatic
reaction steps and spontaneous chemical reaction steps
(Figure 3). DOPA formation is catalyzed by tyrosine hydrox-
ylase (enzyme I in Figure 3), betalamic acid formation by
DOPA 4,5-dioxygenase (DOD; enzyme II), cDOPA formation
by plant PPO or DOPA oxidase (enzyme III), conjugation of
betalamic acid and amino acid, amine or cDOPA by
enzyme VIII, and modification with sugar molecules and
aliphatic or aromatic compounds by enzymes IV–VII.
Although DOPA is an important precursor not only of
betalains but also of various secondary metabolites in
plants, there have only been a few reports about the partial
purification of tyrosine hydroxylase from P. grandiflora
(Steiner et al., 1996; Yamamoto et al., 2001). cDOPA is
presumably synthesized from DOPA by the action of PPO,
but there is no direct evidence to prove it in vitro except
correlation of the accumulation of PPO mRNA and betacy-
anin contents in tissues of pokeweed (Phytolacca americana;
Joy et al., 1995). Recently, some betalains have been
suggested to be synthesized by tyrosinase or PPO after the
condensation of betalamic acid and amino acid (Gandia-
Herrero et al., 2005a,b); however, the tyrosinase has not
been successfully purified, nor has the cDNA encoding it
been identified from betalain-producing plants. The con-
densation step of betalamic acid and amino acid, amine or
cDOPA probably occurs as a spontaneous chemical reaction
in vivo, as suggested by feeding experiments (Hempel and
Boem, 1997; Schliemann et al., 1999).
As shown in Figure 3, betalains are synthesized through
two independent pathways from DOPA, i.e. the betalamic
acid biosynthetic pathway (enzyme II and onwards) and the
cDOPA synthetic pathway (enzyme III and onwards). Beta-
lamic acid is the chromophore molecule of both betacyanins
and betaxanthins, and cDOPA and its derivatives are essen-
tial to produce betacyanin. DOD and cDOPA synthase are
crucial enzymes for betacyanin synthesis.
DOPA 4,5-dioxygenase (DOD). Purification of the DOD
enzyme and isolation of the gene encoding DOD were first
achieved in fungi (Giord and Zryd, 1991). The fungal DOD
converts DOPA to both betalamic acid and muscaflavin. As
muscaflavin has not been detected in plants (Mueller et al.,
1997), the biochemical properties of fungal DOD are likely to
be different from those of plant DOD. Subsequent attempts
to identify plant DOD protein homologs by immunoscreen-
ing using an antibody to the fungal DOD have not been
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 733–749
Plant pigments 741
successful, further indicating that fungal and plant DODs are
structurally divergent (Christinet et al., 2004). However,
Christinet et al. (2004) identified a candidate DOD cDNA
from P. grandiflora using a cDNA subtraction method. The
sequence of P. grandiflora DOD (PgDOD) is homologous to
that of bacterial extradiol 4,5-dixoygenase. The activity
encoded by the candidate cDNA was assayed in a P. gran-
diflora plant of the ccR) genotype, bearing whitish petals,
using a particle bombardment method, and betacyanin was
synthesized to generate red spots by transient expression.
However, recombinant PgDOD expressed in Escherichia coli
did not display DOD activity in vitro. DOD homologs have
subsequently been found in many plant species irrespective
of the production of betalains, even Arabidopsis (Christinet
et al., 2004).
The DOD cDNA from M. jalapa has been successfully
expressed in yeast and E. coli, and DOD activity has been
detected in vitro. Some genes encoding DOD homologs
derived from non-betalain-synthesizing plant species have
also shown DOD activity in vitro, although the in vivo
functions of the homologs are not known. DOD genes in
betalain-synthesizing species may have evolved from these
DOD homologs (Sasaki et al., 2005c, 2007).
Glucosylation steps. Like anthocyanins, many other plant
secondary compounds are synthesized first as a basic skel-
eton and then modified with sugar moieties by the action of
GTs and with aliphatic or aromatic acyl moieties by ATs. In
the case of betalains, feeding experiments and early phyto-
chemical studies suggested two routes for the biosynthetic
pathways: one involves glucosylation at the betanidin step
(enzyme IV, route A, blue arrow in Figure 3), which matches
the biosynthetic processes of other plant secondary prod-
ucts, and the other involves glucosylation at the cDOPA step,
in which modified cDOPA molecules with sugar and acyl
moieties are condensed with betalamic acid (enzyme V,
route B, magenta arrow in Figure 3 ). Which pathway is
the main route for producing betacyanins in vivo remains
controversial.
Evidence for route A was provided by experiments in
which exogenously supplied radiolabeled-betanidin was
incorporated into betanin in fruits of erect prickly pear
(Opuntia dillenii; Sciuto et al., 1972). Biochemical data
supporting route A include the identification of glucosyl-
transferase activity towards betanidin in crude enzyme
prepared from cultured cells of livingstone daisy (Doroth-
enthus bellidiformis; Heuer and Strack, 1992). In addition,
two region-specific enzymes that catalyze glucosylation of
betanidin [UDP-glucose:betanidin 5-O-GT (B5GT) and UDP-
glucose:betanidin 6-O-GT (B6GT)] were purified from living-
stone daisy cell cultures, and their enzymatic properties
were characterized in detail (Vogt et al., 1997). Isolation of
their cDNAs and the subsequent enzymatic characterization
of recombinant B5GT and B6GT revealed that they can utilize
742 Yoshikazu Tanaka et al.
flavonoids as well as betanidin as substrates (Vogt, 2002;
Vogt et al., 1999). B5GT and gentian 3¢GT belong to the same
cluster (Figure S1).
On the other hand, Sciuto et al. (1974) also reported that
cDOPA 5-O-glucoside rather than betanidin or betanin was
an efficient precursor of amaranthin (betanidin 5-O-b-glu-
curonosylglucoside) in feather cockscomb (Celosia cristata),
which supports route B. The accumulation of cDOPA 5-O-
glucoside in young beet plants (Wyler et al., 1984) and root
peels of red beet (Kujala et al., 2001) also supports route B.
In addition, cDOPA accumulation has been shown in the
petals of mutant M. jalapa plants using phytochemical
complementation (Figure S3). Crossing of a yellow flower
line with a whitish flower line (W1, shown in Figure S3) gave
progeny with yellow flowers. On the other hand, crossing of
the same yellow line with another white flower line (W2,
shown in Figure S3) produced progeny with red flower
(K. Kasahara, Yokohama, Japan, unpublished results). Mix-
ing betalamic acid with the petal extracts derived from W1
and W2 flowers produced yellow and red colors, respec-
tively (Figure S3), which was consistent with the results of
the cross. The red mixture contained mainly betanin and
traces of betanidin. These results indicate that the extract
from the W2 petal contained mainly cDOPA 5-O-glucoside
not cDOPA, and that W2 contained cDOPA GT activity.
Activity of cDOPA GT has been detected in crude extracts
prepared from red petals of M. jalapa and several beta-
cyanin-producing plants, e.g. portulaca (P. grandiflora),
pokeweed (Phytolacca americana), feather cockscomb
(C. cristata), bougainvillea (Bougainvillea glabra) and Christ-
mas cactus (Schlumbergera buckleyi), but not from antho-
cyanin-producing plants, e.g. carnation, chrysanthemum,
tulip and anise-scented sage (Salvia guaranitica). In contrast
to cDOPA GT activity, anthocyanidin 3GT activity was not
detected in betacyanin-producing plants, but was detected
in anthocyanin-producing plants (Sasaki et al., 2004). Fur-
thermore, cDNAs encoding cDOPA5GT were isolated from
the petals of M. jalapa and the inflorescences of C. cristata
(Figure S1; Sasaki et al., 2005b). The profile of the transcripts
of the cDOPA5GT gene agreed with the cDOPA5GT activity
during development of red petals of M. jalapa, which closely
paralleled the synthesis and accumulation of betacyanin.
These results support the presence of route B via enzyme V
shown in Figure 3, at least in M. jalapa.
Other modification of betalains with sugar and acyl
moieties might occur at the cDOPA or glycosylated cDOPA
stages rather than the aglycon (betanidin) stage. Recently,
UDP-glucuronic acid:cDOPA 5-glucoside glucuronosyltrans-
ferase activity (enzyme VI) was detected in C. cristata (Sasaki
et al., 2005a), indicating that modification with the glucu-
ronic acid moiety occurs at cDOPA. The modification by acyl
moieties on betanidin glucoside, such as a malonyl moiety
of phyllocactin (malonylbetanin) and a 4-coumaroyl or
ferulic moiety of lampranthins I (4-coumaroylbetanin) or II
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 733–749
(feruloylbetanin), may be catalyzed by acyl CoA-dependent
BAHD acyltransferase or acyl glucose-dependent acyltrans-
ferase belonging to the SCPL enzyme family, as in the case
of anthocyanin acylation. As acyl-glucose-dependent acyl-
transferase activities towards betanidin glucosides (enzy-
me VII) have been reported (Bokern and Strack, 1988; Bokern
et al., 1992), isolation of cDNAs of the SCPL protein family
followed by their functional analysis may be a promising
way to understand acylation in the betacyanin pathways.
The step at which glucosylation and acylation occurs may
depend on the species, and both routes may be possible.
Obviously, further study is necessary for clarification.
Further characterization of the pathway
Mirabilis jalapa has many kinds of variegated flowers, and
Mendel reported many of its intriguing patterns (Mendel,
1950). Such variegated patterns may be caused by trans-
posons (Figure 1i; N. Sasaki and Yoshihiro Ozeki, Tokyo
University of Agriculture and Technology, unpublished
results). Transposon tagging, which has been successfully
used to identify relevant genes in the flavonoid biosyn-
thetic pathways in maize, petunia and morning glories,
may be the most efficient way to identify the genes
involved in betalain biosynthesis. Characterization of the
genes encoding the flavonoid/anthocyanidin biosynthetic
enzymes in betacyanin-producing plants has begun
(Shimada et al., 2004, 2005, 2007). This may help clarify
the taxonomic mystery of Caryophyllales.
Carotenoids
Function of carotenoids
Carotenoids are isoprenoid compounds (mostly C40) with
polyene chains that may contain up to 15 conjugated double
bonds (Figure 4). More than 700 naturally occurring carote-
noids have been identified (Britton et al., 1995, 2004). Car-
otenoids differ from anthocyanins and betalains in that they
play essential roles in plant life, for example, photoprotec-
tive functions during photosynthesis (Green and Durnford,
1996; Niyogi, 2000) and provision of substrates for biosyn-
thesis of the plant growth regulator abscisic acid (ABA;
Nambara and Marion-Poll, 2005) and perhaps other hor-
mones as well (Auldridge et al., 2006). Carotenoids also play
an important role in human nutrition and health, providing
provitamin A and having anti-cancer activities (Mayne,
1996). Some carotenoids are used as food colorants,
cosmetics or pharmaceuticals.
Carotenoid composition
Carotenoids show qualitative differences depending on the
plant organs and species. For example, the green tissues
ª 2008 The Authors
 Plant pigments 743

IPP IPI GGPS GGPP carrot (Baranska et al., 2006) and sweet potato (Hageni-
PPO PPO
mana et al., 1999) are exceptions. They accumulate a
Phytoene PSY high concentration of b-carotene, and are an important
source of vitamin A in the human diet. The majority of
PDS carotenoids in the petals of sandersonia (Sandersonia
ζ-Carotene Z-ISO aurantiaca) are b,b-carotenoids, such as b-cryptoxanthin,
zeaxanthin and b-carotene (Nielsen et al., 2003). On the
ZDS other hand, more than 90% of the carotenoids in the petals
Lycopene CRTISO of marigold (Tagetes sp.; Figure 1j; Moehs et al., 2001) and
chrysanthemum (Kishimoto et al., 2004) are lutein and/or
lutein derivatives (b,-carotenoids).
The main carotenoids of the flower petals of most plants
LCYE LCYB
α-Carotene LCYB β-Carotene are yellowish xanthophylls, which are pale to deep yellow
in color (Table S1). The petals of some plants have a
CHYB Zeaxanthin CHYB modified carotenoid biosynthetic capacity, accumulate
Lutein CHYE
OH
OH
unique carotenoids associated with their respective genus
HO or even species, and are orange to red in color. Astaxan-
HO ZEP VDE
Lutein-5,6-epoxide
? Antheraxanthin thin (3,3¢-dihydroxy-4,4¢-diketo-b,b-carotene) is a ketocaro-
OH
OH
tenoid that is produced in a number of bacteria, fungi and
O

HO
O
HO algae; it furnishes an attractive orange–red color. Only a
ZEP VDE few plant species are known to produce astaxanthin. The
Violaxanthin
O
OH
petals of Adonis aestivalis and A. annua anomalously
O
HO accumulate a large amount of astaxanthin, resulting in
Neoxanthin NSY NCED their blood-red color (Figure 1k; Cunningham and Gantt,
OH
O

NCED ABA 2005). The orange petals of calendula (Calendula officinal-

HO
OH
Figure 4. Carotenoid biosynthesis pathway in plants.
For the sake of simplicity, only all-trans-configurations are shown. DXPS,
1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phos-
phate reductoisomerase; IPI, isopentenyl pyrophosphate isomerase; GGDP,
geranylgeranyl diphosphate synthase; PSY, phytoene synthase; PDS, phyto-
ene desaturase; ZDS, f-carotene desaturase; LCYB, lycopene b-cyclase; LCYE,
lycopene -cyclase; CHYB, b-ring hydroxylase; CHYE, -ring hydroxylase; ZEP,
zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; CRTISO, carotenoid
isomerase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxy-
genase.
is) contain reddish carotenoids that are absent in yellow
petals (Figure 1l). Some have a cis-structure at C5 or C5¢,
which is very rare in plants (Kishimoto et al., 2005). The
red style branches of crocus (Crocus sativus), from which
the spice saffron is derived, accumulate the unique apo-
carotenoids, crocetin glycosides, picrocrocin and safranal.
They are produced by the cleavage of zeaxanthin and are
responsible for the color, taste and aroma of saffron
(Bouvier et al., 2003b).

of most plants show similar carotenoid profiles, accumu-
lating both b,-carotenoids (with one b- and one -ring) and
b,b-carotenoids (with two b-rings) (Goodwin and Britton,
1988). Carotenoids such as zeaxanthin, violaxanthin,
antherxanthin and lutein are invariably found in leaves and
stems. In contrast, carotenoids in non-green tissues show
distinctive compositions that depend on the plant species.
For example, tomato (Solanum lycopersicum) fruit accu-
mulates a large amount of lycopene (Fraser et al., 1994).
Capsanthin and capsorbin, ketocarotenoids that contain
one and two acyl-cyclo-pentanol rings, respectively, are
the typical carotenoids of red pepper (Capsicum annuum)
(Hornero-Méndez et al., 2000). Bixa orellana is the only
plant that accumulates bixin in its seeds (Bouvier et al.,
2003a). Bixin is a dicarboxyl monomethyl ester apocarot-
enoid, also known as annatto, and is used in food and
cosmetics as a red color additive. In general, plants do not
accumulate carotenoids in their roots. The storage roots of
Biosynthesis of carotenoids
In the past decade, the genes encoding nearly all the
enzymes for carotenoid biosynthesis in plants have been
identified, and their enzymatic activities have been charac-
terized (see reviews by Cunningham and Gantt, 1998;
Hirschberg, 2001; Howitt and Pogson, 2006). In plants, the
entire pathway starting from isopentenyl pyrophosphate
(IPP) occurs in the plastids, and it is there that the product
accumulates. It is hypothesized that carotenogenic enzymes
exist in complex with and associated with the plastid
membranes (Cunningham and Gantt, 1998).
Figure 4 summarizes the carotenoid biosynthesis path-
way in plants. Carotenoid biosynthesis starts from a C5
isoprene unit, IPP. Four IPPs are condensed to form C20
geranylgeranylpyrophosphate (GGPP). A head-to-head
coupling of two GGPP molecules, catalyzed by phytoene
synthase (PSY), yields the first C40 carotenoid, phytoene.
In tomato, two different types of PSYs (Psy-1 and Psy-2)

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744 Yoshikazu Tanaka et al.
are expressed in an organ-specific manner (Fraser et al.,
1999). Psy-1 encodes a fruit- and flower-specific isoform
and is responsible for carotenogenesis in chromoplasts. In
green tissues, Psy-2, which is homologous to Psy-1 but
highly divergent from it, is predominantly expressed,
and makes a major contribution to carotenogenesis in
chloroplasts.
Conjugated double bonds are subsequently added by
two structurally similar enzymes, phytoene desaturase
(PDS) and f-carotene desaturase (ZDS). These desaturation
reactions yield the intermediates phytofluene, f-carotene,
neurosporene and lycopene, containing 5, 7, 9 and 11
conjugated double bonds, respectively. Increasing the
number of conjugated double bonds shifts the absorption
towards longer wavelengths, resulting in colorless phyto-
ene and phytofluene, pale-yellow f-carotene, orange–
yellow neurosporene and red lycopene. During the
desaturation steps, several reaction intermediates with a
cis-configuration are produced. Conversion of a cis- to a
trans-configuration to form all-trans-lycopene is carried out
by carotenoid isomerase (CRTISO), which has been iden-
tified in tomato (Isaacson et al., 2002) and Arabidopsis
(Park et al., 2002). CRTISO is specific for adjacent double
bonds at the 7,9 and 7¢,9¢-positions, and converts 7,9,9¢-tri-
cis-neurosporene and 7¢,9¢-di-cis-lycopene, the products of
ZDS, to 9¢-cis-neurosporene and all-trans-lycopene, respec-
tively. Recently, Li et al. (2007) reported a second caroten-
oid isomerase (termed Z-ISO) in maize that converts the
15-cis-bond in 9,15,9¢-tri-cis-f-carotene, the product of PDS,
to 9,9¢-di-cis-f-carotene, the substrate of ZDS.
The cyclization of lycopene is a branch point in the
pathway, catalyzed by lycopene b-cyclase (LCYB) and
lycopene -cyclase (LCYE). Because LCYE in most plants
adds only one -ring to lycopene (Cunningham and Gantt,
2001; Cunningham et al., 1996), the pathway in plants
typically proceeds only along branches, leading to carot-
enoids with one b- and one -ring (a-carotene and its
derivatives) or two b-rings (b-carotene and its derivatives).
Lycopene -cyclase in romaine lettuce (Lactuca sativa) has
the ability to add two -rings to lycopene and yields a
bicyclic -carotene, lactucaxanthin (Cunningham and Gantt,
2001). A single amino acid residue (457th Histidine) is
important to form bicyclic -carotene.
b- and a-carotenes are further modified by hydroxylation
or epoxidation, providing a variety of structural features.
The oxygenated derivatives of carotene are called xantho-
phylls. Hydroxylation of the b- and -rings is catalyzed by
b-hydroxylase (CHYB) and -hydroxylase (CHYE), respec-
tively. CHYB is a non-heme di-iron mono-oxygenase, while
CHYE is a P450, CYP97C1 (Tian et al., 2004). CHYB is a well-
studied enzyme that has been cloned and characterized
from many organisms, while CHYE has been identified only
in Arabidopsis. A flower-specific CHYB (CrtR-b2) was
recently identified in tomato (Galpaz et al., 2006). Taken
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 733–749
together with the existence of flower- and fruit-specific
PSY, GGPS and LCYB (tomato expression database, http://
ted.bti.cornell.edu/), this finding supports the hypothesis
that there is a chromoplast-specific carotenoid biosynthesis
pathway.
Epoxidation at positions C5,6 and C5¢,6¢ of the b-ring of
zeaxanthin, catalyzed by zeaxanthin epoxidase (ZEP), yields
violaxanthin. Violaxanthin is converted to neoxanthin by
neoxanthin synthase (NSY). Both 9-cis-violaxanthin and
9-cis-neoxanthin are cleaved to xanthoxin (C15) by 9-cis-
epoxycarotenoid dioxygenase (NCED), and then converted
to ABA via the ABA aldehyde intermediate (Nambara and
Marion-Poll, 2005).
Regulation of carotenoid biosynthesis and
accumulation of carotenoids
Plant tissues, in particular flower petals and fruits, have a
wide variety of carotenoid contents, ranging from little or
none to large amounts even within the same plant species.
There is increasing evidence that carotenogenesis in plant
tissues is predominantly regulated at the transcriptional
level (see review by Sandmann et al., 2006). In marigold, the
differences in petal color from pale-yellow to orange–red are
caused by the different levels of accumulation of yellow
carotenoid lutein (Figure 1j). Moehs et al. (2001) demon-
strated that a higher level of PSY and 1-deoxy-D-xylulose-5-
phosphate synthase might be responsible for the color
development from pale-yellow to orange. It has also been
demonstrated that PSY is a rate-limiting enzyme of carot-
enoid biosynthesis in canola (Brassica napus) seeds
(Shewmaker et al., 1999) and tomato fruits (Fraser et al.,
1994).
The successful isolation of genes for carotenoid biosyn-
thesis will allow identification of the key regulatory steps of
carotenoid biosynthesis. Nevertheless, knowledge on the
molecular aspects that regulate the pathway is still limited.
Recently, the genes responsible for hp1 and hp2 (mutations
conferring a high level of carotenoids) have been shown
to encode the proteins UV-DAMAGED DNA-BINDING
PROTEIN 1 (DDB1) and DEETIOLATED 1 (DET1), compo-
nents that are involved in the light-signal transduction
pathway (Liu et al., 2004). In addition, other light-signaling
components, such as HY5 and COP1, have been shown to
antagonistically regulate the carotenoid level in tomato
fruits (Davuluri et al., 2005; Liu et al., 2004). In petals of the
Mimulus species, a single QTL at the YUP locus controls the
presence and absence of carotenoids (Bradshaw and
Schemske, 2003). It is interesting to note that the observed
changes of pollinator preference associated with YUP alleles
in Mimulus are comparable to those associated with AN2
alleles in petunia (Hoballah et al., 2007) as described above,
although the identity of the YUP locus remains to be
elucidated.
ª 2008 The Authors

The amount of carotenoids in the tissues is not attributed
solely to the ability to synthesize carotenoids. Some plant
tissues have the capacity to synthesize carotenoids but
contain only a trace amount of carotenoids. The mechanism
that controls carotenoid accumulation is largely unknown.
Recently, two different regulatory mechanisms were postu-
lated. One is focused on carotenoid degradation, and the
other is focused on sink capacity. In the case of chrysanthe-
mum petals, there was no significant difference in the
expression levels of the carotenogenic genes between the
white and yellow petals of chrysanthemums (Kishimoto
and Ohmiya, 2006). However, a gene encoding carotenoid
cleavage dioxygenase (CmCCD4a) was specifically
expressed in white petals (Ohmiya et al., 2006). Suppression
of CmCCD4a expression resulted in a change in the petal
color from white to yellow, indicating that normally white
petals synthesize carotenoids but immediately degrade
them into colorless compounds. The importance of sink
capacity for carotenoid accumulation was first demon-
strated in cauliflower (Brassica oleracea var. botrytis)
Orange (Or) mutant. Or is a gain-of-function mutation, and
single-locus Or mutation confers a high level of b-carotene
accumulation in tissues where accumulation of carotenoid is
normally repressed (Li et al., 2001). The Or gene encodes a
plastid-associated protein with a cysteine-rich domain sim-
ilar to that found in DnaJ-like molecular chaperones (Lu
et al., 2006). This protein plays an important role in trigger-
ing differentiation of proplastids and/or other non-colored
plastids into chromoplasts, which in turn act as a metabolic
sink for carotenoids. Transformation of the Or gene into
wild-type cauliflower (or) converts the white colour curd tis-
sue into an orange color with increased levels of b-carotene.
Remaining questions and future challenges
The flavonoid/anthocyanin biosynthetic pathway is well
understood, and the relevant main enzymes and genes have
been characterized. However, it is not clear how the simple
precursors found in the cytosol of the plant cell are con-
verted into the final complicated pigment compounds in the
vacuole. The questions of whether there is protein–protein
interaction to form metabolic channels and how plant cells
transport complicated flavonoids into vacuoles need to be
answered in order to achieve efficient engineering of the
pathway. Furthermore, only limited knowledge is available
regarding regulation of the vacuolar pH, and even less is
known about the transport of metal ions from roots to petal
vacuoles.
Betalain biosynthesis has been partly characterized in a
limited number of species, and a key enzyme, tyrosine
hydroxylase, has not yet been isolated. The vacuolar trans-
port of betalains and the transcriptional regulation of
betalain biosynthesis are unknown, and engineering of its
pathway is yet to be achieved. The reason why betalains and
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 733–749
Plant pigments 745
anthocyanins do not co-exist is an interesting unresolved
question.
There are many carotenoids whose biosynthesis has not
been characterized. Understanding their biosynthesis and
their transcriptional regulation should accelerate engineer-
ing of the pathway, which has only been partly achieved.
Carotenoid engineering is expected to contribute to human
health, as carotenoids are important nutrients as well as
pigments. A good example is ‘Golden Rice’, which expresses
the bacterial desaturase gene and the daffodil or maize PSY
gene (Ye et al., 2000 and Paine et al., 2005, respectively).
The plant pigments described here are highly diverse with
regard to structures and colors, reflecting the diversity of
plants and their metabolic pathways. Obviously, further
analysis of more plant species will add novel structures and
possibly novel pathways. Characterization of relevant genes
should provide a better knowledge of plant evolution. The
study of plant pigments will also contribute to the floricul-
tural, food and chemical industries. For example, extensive
biochemical characterization, including the determination of
crystal structures and structure–function relationships, will
be useful to generate novel enzymes to synthesize versatile
compounds that cannot be synthesized by non-enzymatic
reactions. Generating ornamental plants with novel colors
by engineering plant pigment pathways has been successful
and will continue to benefit from plant pigment research.
Acknowledgements
We apologize to the authors whose work has not been cited because
of the limitations on the length of this manuscript. We thank the
editor and anonymous reviewers for their valuable suggestions to
improve this article. The authors acknowledge Dr Tsukasa Iwashina
(Tsukuba Botanical Garden, National Science Museum, Japan) for
providing the photographs of Meconopsis horridula and Strongyl-
odon macrobotrys, and Mr Masahiro Uchida (http://www9.plala.
or.jp/mosimosi) for the photograph of Adonis aestivalis for
Figure 1. We sincerely thank Dr Kichiji Kasahara (Yokohama, Japan)
for providing the Mirabilis jalapa plant materials, and for permission
to use his original photographs in Figure 1.
Supplementary material
The following supplementary material is available for this article
online:
Figure S1. Non-rooted phylogenetic tree of putative flavonoid or
betalain glycosylcosyltransferases.
Figure S2. Non-rooted phylogenetic tree of BAHD-type acyltrans-
ferases, including anthocyanin acyltransferases.
Figure S3. cDOPA 5-O-glucoside accumulation in the white petals of
Mirabilis jalapa (‘four o’clocks’).
Table S1. Carotenoids in flower petals.
This material is available as part of the online article from http://
www.blackwell-synergy.com
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials supplied
by the authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
746 Yoshikazu Tanaka et al.
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