Download Metabolic Engineering to Modify Flower Color

Plant Cell Physiol. 39(11): 1119-1126 (1998)
JSPP © 1998
Mini Review
Metabolic Engineering to Modify Flower Color
Yoshikazu Tanaka, Shinzo Tsuda and Takaaki Kusumi
Institute for Fundamental Research, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503 Japan
Thanks to the rapid progress in molecular biology of
flavonoid biosynthesis and plant transformation, it has
become feasible to modify the pathway and flower color
through genetic engineering. One of the advantages of
molecular breeding is that flower color can be specifically
modified without changing the other characteristics of the
targeted variety. Novel flower color varieties such as brickred petunias and violet carnations have been successfully
made by expression of heterologous flavonoid genes. Flavonoid metabolic engineering has and will give new perspectives in plant molecular biology besides its industrial application.
Key words: Anthocyanin — Flavonoid — Flower color —
Metabolic engineering — Transgenic plants.
Flower colors are mainly produced by flavonoids,
carotenoids and betalains. Carotenoids are largely responsible for the production of yellow and orange flowers such
as sun flower and tomato (Bartley and Scolnik 1995).
Betalains are yellow to red nitrogenous compounds derived
from tyrosine and are distributed only in Caryophyllales
(Stafford 1994). Flavonoids have a wide range of colors
from pale yellow to red, purple and blue (Goto 1987, Goto
and Kondo 1991). The flavonoid biosynthetic pathway has
been the choice to genetically engineer flower color to obtain novel colors. Such engineering has also helped us to understand transgene expression in plants. This review summarizes the achievements made in engineering flavonoid
biosynthesis to modify flower color. The field has been
reviewed several times (Mol et al. 1995, Davies and
Schwinn 1997, Tanaka et al. 1998).
Flavonoids and flower color—Anthocyanins are a colored class of flavonoids and accumulate in vacuoles. There
are six major anthocyanidins (chromophores of anthocyanins); pelargonidin, cyanidin, peonidin (3' O-methyl cyanidin), delphinidin, petunidin (3' O-methy delphinidin) and
malvidin (3',5' O-dimethy delphinidin). As the number of
hydroxyl groups in the B-ring is increased, the visible absorption maximum is shifted longer. The maxima in 0.01%
Abbreviations: CaMV, cauliflower
cultivar.
mosaic virus; cv.,
HCl-methanol solution of pelargonidin, cyanidin and
delphinidin are 520, 535 and 546 nm, respectively (Goto
1987).
Modification of the anthocyanidins mainly with glycosylation and acylation results in a wide variety of anthocyanins. Hundreds of anthocyanins have been purified
and their structures determined (Strack and Wray 1994).
Besides the structures of anthocyanins, changes of vacuolar pH, intermolecular stackings (self-association of anthocyanins and co-pigmentation of anthocyanins with polyphenols), intramolecular stacking of aromatically modified
anthocyanins, metal complexation and cell shapes give
almost infinite flower colors (Goto 1987, Goto and Kondo
1991, Brouillard and Dangles 1994, Mol et al. 1998). Anthocyanins are red and stable at a low pH. They are bluer but
often unstable at a vacuolar pH which is usually weakly
acidic. The stackings and metal complexation contribute to
stabilize anthocyanins and flower color, especially blue color (Goto 1987, Goto and Kondo 1991, Brouillard and
Dangles 1994). The major role of anthocyanin production
in petals is to attract pollinators. Flavonoids and anthocyanins also play important roles in photoprotection, reproduction, pathogenesis and symbiosis (Shirley 1996).
Flavonoid biosynthesis—The flavonoid biosynthetic
pathway shown in figure 1 has been extensively reviewed
(Heller and Forkmann 1994, Forkmann 1994, Holton and
Cornish 1995). The pathway leading to anthocyanidin 3glucosides is generally conserved among plant species and
is well understood. Anthocyanidin 3-glucosides are further
modified with sugars and aliphatic and aromatic acids.
There are both species and variety differences in the extent
of modification and the types of glycosyl and acyl groups attached (Fig. 1).
The first enzyme committed to flavonoid production is
chalcone synthase (CHS). This enzyme catalyzes the stepwise condensation of three acetate units starting from
malonyl-CoA with p-coumaroyl-CoA to yield 4,2',4',6tetrahydroxychalcone. CHS shares homology with some
condensing enzymes such as arabidopsis FA El and /?ketoacyl-acyl carrier protein synthase (James et al. 1995).
Flavanone 3-hydroxylase (F3H), a kind of 2-oxoglutarate
dependent dioxygenases, catalyzes hydroxylation of naringenin to dihydrokaempferol (DHK). DHK is hydroxylated to dihydroquercetin (DHQ) and to dihydromyricetin
(DHM) by flavonoid 3-hydroxylase (F3'H) and flavonoid
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Metabolic engineering to modify flower color
1120
COOH
COS-CoA
PAL
OH
OH
" • OH
Phenylalanine Cinnamic acid 4-Coumaric acid 4-Coumaroyl-CoA
3 x Malonyj^CoA
4-Caffeoyl-CoA
JCHS
OH 0
4,2',4',6'-Tetrahydroxychalcone
1 CHI
FNS
OH
OH O
FLS
Kaempferol
o
Naringenin
F3H
OH
OH O
Dihydroauercetin
OH
OH O
OH b
Dihydromyricetin
Dihydrokaempferol
I D F R , ANS, 3GT
"
Hl
I DFR, ANS, 3GT
DFR, ANS, 3GT
PH
-OH
OH
OGIc
OGIc
OH
Delphinidin 3-Glucoside
OH
Cyanidin 3-Glucoside
OGIc
OGIc-Malonate
OH
A chrysanthemum
anthocyanin
R2
OGIc
Rose anthocyanins
OH
R1 = H or OH or OCH3
R2 = OH or OGIc
OGIc-Caf
OCH3
A gentian anthocyanin
(gentiodelphin)
I
OH
HO.
OCH3
Carnation anthocyanins
R1 = H or OH
R2 = OH or OGIc
OGIc
A petunia anthocyanin
Fig. 1 Generalized pathway leading to anthocyanidin 3-glucosides (Holton and Cornish 1995). Anthocyanins of non-blue flowers
(rose, chrysanthemum and carnation) and blue flowers (gentian and petunia) are also shown. Blue flowers usually have delphinidin modified with one or more aromatic acyl group(s) such as caffeic acid (Caf) and coumaric acid. PAL, phenylalanine ammonia-lyase; C4H,
cinnamate4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; Fill, flavonoid-3'-hydroxylase; F3'5"H, flavonoid 3\5'-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; 3GT, flavonoid 3-glucosyl transferase; CC3H, 4-coumaroyl-CoA 3-hydroxylase; FNS, flavone synthase; FLS, flavonol synthase;
Glc, glucose; Rha, rhamnose.
Metabolic engineering to modify flower color
3',5'-hydroxylase (F3'5'H), respectively (Fig. 1). These two
hydroxylases belong to the same cytochrome P-450 family
(CYP 75, Holton et al. 1993a, Brugliera et al. 1997). Both
are key enzymes in determining flower color because they
determine the hydroxylation patterns of the B-ring of the
dihydroflavonols and this hydroxylation eventually determines the structure of the anthocyanidin leading to flower
color (Holton and Tanaka 1994). F3'5"H can also convert
DHQ to DHM. Dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to leucoanthocyanidins and is proposed to belong to the 3/?-hydroxysteroid
dehydrogenase/DFR superfamily (Baker and Blasco 1992).
Leucoanthocyanidins are converted to anthocyanidins by
another 2-oxoglutarate-dependent dioxygenase, anthocyanidin synthase (ANS), and then to the anthocyanidin 3-glucosides by UDP-glucose: flavonoid 3-O-glucosyltransferase
(3GT). UDP-glucose: anthocyanin 5-O-glucosyltransferase
(5GT) genes have been recently cloned (Yamazaki et al.
1998). Both 3GT and 5GT belong to the distinct families of
glycosyltransferase superfamily. Petunia UDP-rhamnose:
anthocyanin rhamnosyltransferase also has homology to
glycosyltransferases (Brugliera et al. 1994, Kroon et al.
1994). Genes of aromatic acyl transferase (AAT) catalyzing
the transfer of aromatic group to 3- or 5-glucose of anthocyanins have been cloned (Tanaka et al. 1997). Many of
these enzymes have been biochemically characterized by expressing cloned cDNAs in heterologous systems (Holton et
al. 1993b, Tanaka et al. 1996, 1997, Yamazaki et al. 1998).
Peonidin-type anthocyanins are synthesized from the cyanidin-type anthocyanins and petunidin- and malvidin-type
anthocyanins are from the delphinidin-type anthocyanins
by the transfer of a methyl group from S-adenosylmethionine catalyzed by anthocyanin methyltransferases (Heller
and Forkmann 1994).
Anthocyanins are reversibly modified with glutathione
(Marrs et al. 1995, Alfenito et al. 1998) and postulated to
be transported into vacuoles by ATP-binding cassette
(ABC) transporters (Lu et al. 1997). A 24 kDa vacuolar protein has been suggested to be involved in intravacuolar pigmented structures in sweet potato (Nozue et al. 1997).
These processes are not clearly understood.
Flavonols are derived from dihydroflavonols by a
2-oxoglutarate-dependent dioxygenase, flavonol synthase
(FLS, Holton et al. 1993b). Apigenin (a flavone) is synthesized from naringenin by flavone synthase (FNS). Interestingly there are two kinds of FNS, a cytochrome P-450 and
a dioxygenase (Heller and Forkmann 1994).
Regulation of flavonoid biosynthetic pathway—Spatial and/or temporal expressions of structural genes or
enzymes of flavonoid biosynthesis in flowers, leaves and
seedlings have been well studied in many plants such as
petunia (Brugliera et al. 1994), snapdragon (Jackson et al.
1992), gerbera (Helariutta et al. 1993), carnation (Stich et
al. 1992), rose (Tanaka et al. 1995), eggplant (Toguri et al.
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1993), Arabidopsis (Pelletier et al. 1997), grape (Boss et al.
1996), perilla (Gong et al. 1997) and lisianthus (Nielsen
and Podivinsky 1997). Transcription of these genes is spatially and developmentally regulated in a well-coordinated
way paralleling flavonoid synthesis.
In maize, two gene families, R (Myc type) and Cl
(Myb type) encoding transcriptional factors, regulate the
expression of the structural genes in the pathway. Snapdragon and petunia have similar transcriptional factors
that regulate the structural genes (Holton and Cornish
1995, Mol et al. 1998). Regulatory genes controlling anthocyanin biosynthesis are functionally conserved among
plant species and some of them function in heterologous
species. They have distinct sets of target genes (Quattroccio
et al. 1993, Martin 1996), which allows regulatory diversity
in the pathway depending on species. In petunia flowers,
Anl (a Myc gene), An2 (a Myb gene) and Anil are regulatory genes of anthocyanin biosynthesis in the flower. Anil
encodes a novel Trp-Asp repeat protein and is a component
of a signal transduction cascade that modulates An2 function (de Vetten et al. 1997).
Molecular tools for flower color modification—The
first step in metabolic engineering is to clone the structural
genes. Cloning structural genes in the flavonoid pathway is
not difficult because all but a few structural genes in the
pathway (Fig. 1) have been cloned. The homologous genes
can be isolated by the hybridization approach at least from
closely related species. This approach is very effective for
conserved genes such as CHS and is less effective for
variable genes such as CHI (Gutterson 1995). In our experience, most structural genes can be readily cloned by
low stringent screening with heterologous counterpart
genes within dicots. For less conserved genes such as CHI
and sometimes DFR, a polymerase chain reaction approach using conserved amino acid sequences has been successfully used (Gutterson 1995, Tanaka et al. 1996). Cloned
genes in the pathway from various flowers are listed
(Davies and Schwinn 1997).
Constitutive promoters including cawliflower mosaic
virus (CaMV) 35S promoter, an enhanced CaMV 35S promoter (Mistuhara et al. 1996) and Mac promoter (Comai et
al. 1990) have been successfully used to express genes in
petals. Constitutive expression of the structural genes does
not have deleterious effect on the transgenic plant. Promoters derived from genes in the flavonoid pathway should be
useful for temporal and spatial expression.
Suppression of a gene is more challenging than expression of a gene in transgenic plants. Plant gene expression
can be suppressed by introducing either an antisense (Mol
et al. 1990) or sense gene of interest or its close homologs.
The mechanism of sense suppression (cosuppression) is not
yet fully understood (Taylor 1997, Gallie 1998) although
the phenomenon is a post-transcriptional event. Doublestranded RNA is proposed to be a mediator in sequence-
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Metabolic engineering to modify flower color
specific genetic silencing and cosuppression (Montgomery
and Fire 1998). The frequency and degree of cosuppression
depend on transgene promoter strength and an intact full
length cDNA may be preferable for efficient suppression
(Que et al. 1997) although it is not essential to use full
length cDNA to obtain phenotypes of antisense or sense
suppression (Gutterson 1995). The frequency and degree
of antisense and sense suppression also depend on the
targeted gene and species. The frequency varies from less
than 1% to 40% in our laboratory.
Transformation of floricultural crops—Plant cells
have can detect the transgenes in the genome and suppress
the expression unless the transgenes are integrated in "correct positions" (Kumpatla et al. 1998, Matzke and Matzke
1998, Gallie 1998). It is, however, not possible to integrate
a transgene into the correct position artificially. It is
necessary to obtain many (dozens to hundreds) transgenic
plants and select a few transgenic lines whose phenotypes
are desirable and stable enough to be commercialized. Therfore, a highly efficient transformation system for the targeted species or variety is essential. Interestingly, a high
frequency of shutdown of transgene expression during
growth is often observed in chrysanthemum (Deroles et al.
1997).
Transformation of floricultural crops has been reviewed (Deroles et al. 1997, Tanaka et al. 1998). Rose, carnation, tulip and chrysanthemum that occupy more than 50%
of the cut flower market are claimed to be routinely transformed (Mol et al. 1995) but there is much variation in extent of success among cultivars within a species. Problems
and strategies of plant transformation have already been
discussed (Birch 1997).
Plant transformation generally consists of three steps.
The first step is to transfer foreign DNA into plant cells (infection), the second step is to select transgenic cells (selection) and the third step is to regenerate them into complete
plants (regeneration). Each step needs careful optimization
of various procedures and parameters.
To deliver foreign DNA into plant cells, Agrobacte/•/wm-mediated gene transfer has been widely used especially for dicots and some monocots. Particle bombardment is
often used for monocots resistant to Agrobacterium
(Deroles et al. 1997).
For selection, the neomycin phosophotransferase gene
that confers kanamycin resistance has been most widely
and successfully used as a selection marker. Herbicide-resistant genes encoding enzymes which are insensitive to the
herbicide or detoxify it can be used as selectable markers. It
is important to select only the cells with the introduced
transgenes. Weak selection often results in chimerical problems.
Most dicot species regenerate well via organogenesis
on media containing balanced hormone supplementation,
usually cytokinin and auxin. An efficient transformation
system has been established for petunias and torenias and
50 to 100 independent transgenic plants per gene construct
are routinely obtained in our laboratory. Embryogenesis is
the preferred route to transform roses in our laboratory
(Fig. 2A, Katsumoto et al. 1995). A rose cultivar (cv.)
Royalty was also transformed via embryogenic callus
(Firoozababy et al. 1994).
Modification of anthocyanin amount—White is not a
novel color because of abundant occurrence. Still, molecular breeding of a white variety is industrially useful because
only flower color can be modified without sacrificing the
other desirable characteristics. Such a sacrifice is often
made during hybridization breeding. It is possible to obtain
white flowers from anthocyanin producing flowers by suppressing the expression of one of many structural or regulatory genes in the pathway. The reported results are summarized in Table 1. It is interesting that novel color patterns
were obtained in petunia and lisianthus while only uniform
suppression was observed in rose, gerbera, and chrysanthemum. This may be relevant to the fact that petunia and
lisianthus have natural white-sectored patterns (Deroles et
al. 1998).
Suntory Ltd. markets Surfinia™ (a petunia) and Summerwave™ (a torenia) that are superior to conventional
petunias and torenias with their creeping and vigorous characters. We have been working on widening their flower color variation by metabolic engineering. Antisense CHS-A in
a petunia cv. Surfinia Mini Purple resulted in a few plants
with pale color or pure white flowers (Katsumoto et al. unpublished results, Figure 2B). White and blue/white (two
out of four petals are white and the other two petals are
blue) transgenic torenia plants were successfully generated
from a blue torenia cv. Summerwave Blue by sense suppression of CHS or DFR genes (Suzuki et al. 1997). In both
cases the transgenic plants retain the superior characteristics of the host and only flower color was modified.
The amount of anthocyanins was increased using the
transcriptional factors of the pathway. The flower color of
the tobacco was changed from pink to intense red by expressing the maize R gene (Lc allele) with CaMV 35S promoter. Cl driven by the same promoter had no effect.
Arabidopsis root, petal and stamen accumulate anthocyanins by constitutive expression of both Cl and R (Lloyd et
al. (1992)). Transgenic petunia plants that had a higher
anthocyanin content in floral and vegetative tissues were obtained by constitutive expression of Lc. Leaves of the transgenic petunia are purple due to accumulation of anthocyanins (Bradley et al. 1998). Delila (an R homolog controlling
flavonoid synthesis in snapdragon) increased anthocyanins
in tobacco flower and tomato flowers and vegetative tissues
(Mooney et al. 1995).
Because dihydroflavonols are the common precursors
of anthocyanins and colorless flavonols, DFR and FLS
compete with each other for dihydroflavonols. Antisense
Metabolic engineering to modify flower color
1123
B
Fig. 2 A. Embryogenic rose calli under kanamycin selection. White parts will regenerate into plantlets and brown parts are dead cells.
B. Transgenic plants with white or pale colored flower were obtained from a petunia cv. Surfinia™ Mini Purple (up) with antisense suppression of chalcone synthase gene. C. Suppression of flavonol synthase gene in a petunia cv. Surfinia Pink (right) resulted in a redder
flower (left) that hardly contained flavonols. D. A pale pink petunia accumulating dihydrokaempferol (right) was transformed with rose
dihydroflavonol 4-reductase cDNA and a novel flower color (left) derived from pelargonidin that petunias hardly produce was obtained.
E. Flowers of a torenia cv. Summerwave™ Blue (right) and its sport cv. Pinlc and White (right). Pink and White is deficient in flavonoid
3'5'-hydroxylase and aromatic acyl transferase. F. Transgenic violet carnations (left). Moondust™ (paler ones) and Moonshadow™
(darker ones). They produce delphinidin type anthocyanins in their petals and have a novel blue hue. A transgenic carnation plant in tissue culture (right). Efficient carnation transformation has been reported previously (Lu et al. 1991).
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Metabolic engineering to modify flower color
Table 1 Suppression of flavonoid pathway
Host species (colour)
Petunia (purple)
Petunia (violet)
Petunia (purple)
Chrysanthemum (pink)
Gerbera (red)
Rose (red)
Carnation (pink)
Torenia (blue)
Torenia (blue)
Carnation (red)
Lisianthus (purple)
Gene construct
Antisense CHS-A
Sense CHS-A
Sense CHS-A
Sense CHS
Antisense CHS, DFR
Sense CHS
Sense CHS
Sense CHS, DFR
Sense CHS, DFR
Antisense CHS, DFR
Antisense F3H
Antisense CHS
Phenotype
Reference
White, pattern
White, pattern
White, pattern
White, uniform
Pink, uniform
Pink, uniform
Pale pink, uniform
White, pattern
Pale blue, pattern
van der Krol et al. (1988)
Napoli et al. (1990)
van der Krol et al. (1990)
Courtney-Gutterson et al. (1994)
Elomaa et al. (1993)
Gutterson (1995)
Gutterson (1995)
Suzuki et al. (1997)
Aida et al. (1997)
White, uniform
White, pattern
Zucker et al. (1998)
Deroles et al. (1998)
Phenotypes of transgenic plants depend on each transgenic event. The strongest phenotype of each report is shown. Some species showed uniform suppression and some formed novel color patterns.
suppression of the FLS gene in petunia and tobacco
resulted in a higher content of anthocyanin and more intense flower color (Holton et al. 1993b). A similar result
was obtained with sense suppression of FLS gene in a
petunia cv. Surfinia Pink. Flower color changed from pink
to red purple and the transgenic plant flower contained
more anthocyanins and less flavonol than the host (Fig. 2C.
Suzuki et al., unpublished results).
Making brick-red flowers—Petunia flowers rarely contain pelargonidin-type anthocyanin and are not brick-red
because DHK can not be reduced by petunia DFR that has
high activity for DHM (Forkmann and Ruhau 1987) and
is suiable for delphinidin production. Transgenic brickred petunias accumulating pelargonidin-type anthocyanins
have been obtained from DHK accumulating petunia (deficient in F3'5H, F3"H and FLS) with using maize, gerbera
and rose DFR cDNAs (Meyer et al. 1987, Helariutta et al.
1993, Tanaka et al. 1995 (Fig. 2D), respectively). Stability
of the phenotype and the transgene of the petunias containing maize DFR cDNA has been extensively studied (Linn et
al. 1990. Meyer and Heidmann 1994). The gerbera DFR
gene showed a stronger and more consistent expression
than maize DFR. The choice of the gene source is a factor
to be considered for engineering plants even if the gene encodes the same enzyme activity (Elomaa et al. 1995). When
the rose DFR gene was introduced to a petunia cv. Surfinia
Purple that did not accumulate DHK due to its dominance
in F3"H and FS'S'H genes was transformed, no flower color
changes were observed (unpublished results). This indicates
that it is difficult for a transgene to compete with endogenous genes.
Molecular breeding of blue flowers—Structures of anthocyanin, presence of co-pigments and relatively high vacuolar pH (more than about 5.0, Holton and Tanaka 1994)
are key factors for flowers to have a bluish hue (Goto and
Kondo 1991, Holton and Tanaka 1994, Mol et al. 1998).
Most blue flowers contain aromatically acylated delphinidin derivatives (Fig. 1). The absorbance maximum of anthocyanin shifts towards longer wavelengths (bluer color)
by about 10 nm with one hydroxylation of B-ring, and by
about 4 nm with one aromatic acylation in \% trifluoroacetic acid solution (Fujiwara et al. 1997). Figure 2E shows the
effect of hydroxylation of the B-ring and aromatic acylation of anthocyanins to flower color. Blue petals of a
torenia cv. Summerwave Blue mainly contain malvidin
5-(p-coumaroyl)-glucoside 3-glucoside while petals of its
pink counterpart contain peonidin 3,5-diglucoside. This indicates that F3'5'H and AAT activities are necessary for the
production of blue flowers in vivo. There is little or no
change in color of anthocyanins with glycosylation and
aliphatic acylation. Rose, chrysanthemum and carnation
only have pelargonidin- and cyanidin-type anthocyanins
not modified with aromatic acyl groups.
Genes encoding F3'5'H have been isolated from
petunia (Holton et al. 1993a), eggplant (Toguri et al. 1993),
gentian (Tanaka et al. 1996), lisianthus (Nielsen and
Podivinsky 1997) and many others (unpublished results).
Petunia F3'5'H cDNAs could complement their deficiency
in petunia that was also deficient in F3'H and low in pH
(see below). The flower color changed from pale pink to
reddish purple (Holton et al. 1993a).
Florigene Ltd, (Australia) and Suntory Ltd. successfully developed transgenic violet carnations by introduction
of a petunia F3'5'H and DFR genes into a DFR-deficient
white carnation (Holton et al., unpublished results). The
petals of the carnations predominantly contain delphinidin, which is not produced by the native carnations. Such
bluish flowers have not been produced by traditional
Metabolic engineering to modify flower color
breeding of carnation (Fig. 2F). The transgenic violet carnations named Moondust™ have been marketed in Australia
and Japan and became the first transgenic floricultural crop
to be sold. Darker versions, Moonshadow™, have been obtained and will be marketed soon. Application of AAT
genes (Tanaka et al. 1997) to modify flower color is interesting.
When anthocyanins make a complex with polyphenols
such as flavonol and flavone glycosides (co-pigmentation),
their absorption is increased by up to about 35 nm in wavelength (bathocromic shift, Goto 1987). Co-pigmentation
can also result in a large increase in absorptivity. The
amount of flavonols can be modified by genetic engineering
of FLS genes (Holton et al. 1993b). Although intramolecular stacking of anthocyanins may be achieved by expression
of multiple AAT and GT genes in heterologous plants,
only some of the genes have been cloned.
Anthocyanins are bluer at a higher pH (Goto 1987,
Goto and Kondo 1991). Even an peonidin derivative can
make blue flowers as in blue petals of morning glory when
it is highly modified with six glucose and three caffeic acid
molecules and its vacuolar pH is high, 7.7 (Yoshida et al.
1995). Because non-blue flowers, however, tend to have a
lower vacuolar pH, it is important to increase the pH to
produce blue flowers. Petunia is known to have seven loci
controlling vacuolar pH (phl-phl, Mol et al. 1998). When
one of them is homozygous recessive, the vacuolar pH increases in the petal. Only Ph6 has been cloned (Chuck et al.
1993). Anl and Ph6 turned out to be alleles of the same
locus (Mol et al. 1998). Mutations in An2 and Anil also
affect pH (Mol et al. 1998). Cloning of Ph genes and their
functional analysis may be useful to elevate flower vacuolar
pH (Mol et al. 1998). However, it is not certain if pH regulation by petunia Ph genes is applicable to other species. Further biochemical studies on the mechanism of pH control
in vacuoles or bluing phenomena of senescent flowers often
observed in some species may help us to engineer vacuolar
pH.
The three dimensional structure of Commelina blue
pigment has been determined. The complex is made of six
anthocyanins (malonylawobanin), six flavones (flavocommelin) and two magnesium ions (Kondo et al. 1992). The
biochemical process of formation of the complex is yet to
be revealed. A specific co-pigment and a metal ion are
necessary for a specific anthocyanin to form a stable metalloanthocyanin.
Tulip, impatiens, cyclamen and pelargonium produce
delphinidin but lack true blue colors (Davies and Schwinn
1997). A species-specific strategy is required to obtain blue
varieties.
Many genes must be introduced and properly expressed to synthesize highly modified anthocyanins or form
metal complexes and/or elevate vacuolar pH in order to
make truly blue flowers from non-blue flowers. This is a
1125
very challenging task at the moment and substantial improvement of plant biotechnology is essential. Further
chemical and biological understanding of flower color and
flavonoid pathway together with improved plant transformation systems should make modification of flower color
more feasible.
The authors thank Florigene Ltd. for providing the photo of
Figure 2 and permission of presenting unpublished results.
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