Download Japanese morning glory dusky mutants displaying reddish

The Plant Journal (2005) 42, 353–363
doi: 10.1111/j.1365-313X.2005.02383.x
Japanese morning glory dusky mutants displaying
reddish-brown or purplish-gray flowers are deficient in a
novel glycosylation enzyme for anthocyanin biosynthesis,
UDP-glucose:anthocyanidin 3-O-glucoside-2¢¢-Oglucosyltransferase, due to 4-bp insertions in the gene
Yasumasa Morita1, Atsushi Hoshino1, Yasumasa Kikuchi2, Hiroaki Okuhara3, Eiichiro Ono3, Yoshikazu Tanaka3, Yuko Fukui4,
Norio Saito5, Eiji Nitasaka6, Hiroshi Noguchi2 and Shigeru Iida1,*
1
National Institute for Basic Biology, Okazaki 444-8585, Japan,
2
School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
3
Institute for Advanced Technology, Suntory Ltd, Mishima, Osaka 618-8503, Japan,
4
Institute for Health Care Science, Suntory Ltd, Mishima, Osaka 618-8503, Japan,
5
Chemical Laboratory, Meiji-Gakuin University, Yokohama 244-8539, Japan, and
6
Department of Biological Science, Graduate School of Science, Kyusyu University, Fukuoka 812-8581, Japan
Received 28 October 2004; revised 13 January 2005; accepted 21 January 2005.
*
For correspondence (fax þ81 564 55 7685; e-mail [email protected]).
Summary
Bright blue or red flowers in the Japanese morning glory (Ipomoea nil) contain anthocyanidin 3-O-sophoroside
derivatives, whereas the reddish-brown or purplish-gray petals in its dusky mutants accumulate anthocyanidin 3-O-glucoside derivatives. The Dusky gene was found to encode a novel glucosyltransferase,
UDP-glucose:anthocyanidin 3-O-glucoside-2¢¢-O-glucosyltransferase (3GGT), which mediates the glucosylation of anthocyanidin 3-O-glucosides to yield anthocyanidin 3-O-sophorosides. Ipomoea nil carries one copy of
the 3GGT gene that contains no intron and produces 1.6-kbp transcripts mainly in the petals and tubes of
flower buds at around 24 h before flower opening. The gene products of both In3GGT in I. nil and Ip3GGT in the
common morning glory (Ipomoea purpurea) comprise 459 amino acids and showed a close relationship to
the petunia UDP-rhamnose:anthocyanidin 3-O-glucoside-6¢¢-O-rhamnosyltransferase (3RT), which controls the
addition of a rhamnose molecule to anthocyanidin 3-O-glucosides for conversion into anthocyanidin
3-O-rutinosides. All of the 30 dusky mutants tested were found to carry the 4-bp insertion mutations GGAT
or CGAT at an identical position near the 3¢ end of the gene, and the insertions caused frameshift mutations.
The expected 3GGT enzymatic activities were found in the crude extracts of Escherichia coli, in which the 3GGT
cDNA of I. nil or I. purpurea was expressed, while no such activity was detected in the extracts expressed with
the dusky mutant cDNAs containing 4-bp insertions. Moreover, the introduced Ip3GGT cDNA efficiently
produced 3GGT that converted cyanidin 3-O-glucoside into cyanidin 3-O-sophoroside in transgenic petunia
plants.
Keywords: Japanese morning glory, anthocyanin biosynthesis gene, flower coloration, glucosyltransferase,
metabolic engineering.
Introduction
Flower color is an important floricultural trait in ornamental plants, and various flower colors, such as orange,
red, purple, blue, and blue-black, are mainly the result of
the accumulation of diverse structures of anthocyanins in
ª 2005 Blackwell Publishing Ltd
the petal vacuoles, although additional factors, including
co-pigmentation, vacuolar pH, and cell shape, also affect
flower hue (Brouillard and Dangles, 1994; Davies and
Schwinn, 1997; Honda and Saito, 2002; Mol et al., 1998).
353
354 Yasumasa Morita et al.
Nearly all structural genes that encode enzymes to produce anthocyanidin 3-O-glucosides (Figure 1), which are
the first major stable colored pigments in the anthocyanin
biosynthesis pathway, and many regulatory genes for the
transcriptional activation of the structural genes have been
identified and characterized (Forkmann and Martens, 2001;
Mol et al., 1998; Tanaka et al., 1998; Winkel-Shirley, 2001).
Further modifications of anthocyanidin 3-O-glucosides,
such as glycosylation, acylation, and methylation, which
are involved in the fine adjustment and stabilization
of flower pigmentation, can occur in a species-specific
manner (Heller and Forkmann, 1994). However, only limited
information about the genes responsible for these modifications of anthocyanidin 3-O-glucosides or their derivatives
is available. For example, the structure of mutations in the
petunia Rt gene encoding UDP-rhamnose:anthocyanidin
Figure 1. Simplified anthocyanin biosynthesis pathway, flower phenotypes, and the structures of anthocyanins in the petal of the Japanese and common morning
glories.
(a) Anthocyanin biosynthesis pathway. The enzymes catalyzing each step in the pathway are represented in upper-case letters, and the corresponding genetic loci
are represented in italics. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3¢H, flavonoid 3¢-hydroxylase; DFR, dihydroflavonol
4-reductase; ANS, anthocyanidin synthase; 3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase; 3GGT, UDP-glucose:anthocyanidin 3-O-glucoside-2¢¢-O-glucosyltransferase encoded by the Dusky gene.
(b–e) Flower phenotypes (above) and microscopic photographs of adaxial epidermal cells of flower petals (below). (b) The wild-type line TKS bearing the Magenta,
Purple, and Dusky alleles. (c) The line Q188 bearing the Magenta, Purple, and dusky-1 (dy-1) allele. (d) The line Scarlet O’Hara carrying the magenta, purple, and
Dusky alleles. (e) The line Q856 carrying the magenta, purple, and dusky-1 (dy-1) allele. Scale bars in microscopic photographs indicate 30 lm.
(f) Bright blue flowers of the wild-type Ipomoea nil plant contain Heavenly Blue Anthocyanin (HBA), and reddish flowers of I. nil magenta mutants contain Wedding
Bells Anthocyanin (WBA) described by Hoshino et al. (2003). The anthocyanin pigment in dark-purple flowers of the wild-type I. purpurea plant is also shown. To
distinguish the three caffeic acid molecules in HBA and WBA, we arbitrarily designate them as caffeic acid (I), (II), and (III).
(g) Major anthocyanin pigments in the flowers of the dusky mutants.
(h) Reaction mediated by the Dusky gene products, 3GGT. Although delphinidin 3-O-glucoside is not produced in I. nil and I. purpurea, it serves as a good substrate
for the 3GGT enzyme (see Figure 4).
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
A novel glucosyltransferase gene and its mutants 355
3-O-glucoside-6¢¢-O-rhamnosyltransferase (3RT), which
controls the conversion of anthocyanidin 3-O-glucosides
into anthocyanidin 3-O-rutinosides, and its cDNA have been
characterized (Brugliera et al., 1994; Kroon et al., 1994).
Other cDNAs cloned and characterized include the cDNAs
encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) from perilla, verbena, and petunia (Yamazaki
et al., 1999, 2002), UDP-glucose:anthocyanin 3¢-O-glucosyltransferase (3¢GT) from gentian (Fukuchi-Mizutani
et al., 2003), malonyl-CoA:anthocyanin 3-O-glucoside-6¢¢-Omalonyltransferase (3MaT) from dahlia (Suzuki et al.,
2001), malonyl-CoA:anthocyanin 5-O-glucoside-6¢¢¢-O-malonyltransferase (5MaT) and malonyl-CoA:anthocyanin
5-O-glucoside-4¢¢¢-O-malonyltransferase (5MaT2) from salvia (Suzuki et al., 2002, 2004), hydroxylcinnamoylCoA:anthocyanin 5-O-glucoside-6¢¢-O-acyltransferase (5AT)
and hydroxylcinnamoyl-CoA:anthocyanin 3-O-glucoside-6¢¢O-acyltransferase (3AT) from gentian and perilla, respectively (Fujiwara et al., 1998; Yonekura-Sakakibara et al.,
2000). Apart from those on the petunia Rt gene, no other
reports have focused on the description of the genomic
structures of the genes for the modifications of anthocyanidin 3-O-glucosides, the identification of mutations in
these genes, and the phenotypic characterization of their
mutants.
The Japanese morning glory (Ipomoea nil) displays bright
blue flowers (Figure 1b) that contain the peonidin (3¢-methylcyanidin) 3-O-sophoroside derivative named Heavenly
Blue Anthocyanin (HBA) (Figure 1f; Kondo et al., 1987; Lu
et al., 1992), and a number of spontaneous mutants exhibiting various flower colors have been isolated since the 17th
century (Iida et al., 1999, 2004; Imai, 1927). Genetic studies on
the color of I. nil have shown that blue flower coloration was
mainly controlled by two genetic loci, Magenta and Purple
(Hagiwara, 1931; Imai, 1931). Recessive magenta and purple
mutants bloom magenta and purple flowers, respectively,
and double mutants carrying both magenta and purple
alleles display red flowers (Figure 1d). The Magenta gene
was shown to encode flavonoid 3¢-hydorxylase, which
hydroxylates the 3¢ position of the B-ring of anthocyanidin
precursors (Figure 1a), and all the magenta mutants tested
were found to carry a single nonsense mutation (Hoshino
et al., 2003). The Purple gene was identified to encode a
vacuolar Naþ/Hþ exchanger called InNHX1 that increases the
vacuolar pH during flower opening, causing a shift towards
blue coloration, and all the purple mutants tested contained
the same allele caused by the integration of the DNA
transposon Tpn4 into the InNHX1 gene (Fukada-Tanaka
et al., 2000, 2001; Yamaguchi et al., 2001). Among the
various colors of I. nil flowers, the most favorite hue for
Japanese floriculturists has been reddish-brown or purplishgray petals (Figure 1c,e) since the early 19th century, and the
flower coloration is caused by recessive dusky mutations
(Hagiwara, 1956; Imai, 1931). In the petals of the dusky
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
mutants, major anthocyanins identified were anthocyanidin
3-O-glucosylcaffeoylglucosides and their 5-O-glucoside
derivatives (Figure 1g; Saito et al., 1994, 1996), suggesting
that the activity of UDP-glucose:anthocyanidin 3-O-glucoside2¢¢-O-glucosyltransferase (3GGT), which catalyzes the
production of anthocyanidin 3-O-sophorosides, must be low.
Here, we report the isolation of In3GGT and Ip3GGT cDNA
clones from the Japanese morning glory and the common
morning glory (Ipomoea purpurea), respectively, and demonstrate the conversion of anthocyanidin 3-O-glucosides
into anthocyanidin 3-O-sophorosides (Figure 1h) in the
extracts of Escherichia coli, in which the 3GGT cDNA is
expressed. We have also identified the dusky mutations of
I. nil in the gene encoding the In3GGT enzyme and discussed
the possible origin of the spontaneous dusky mutations. We
subsequently examined the mode of expression of the
In3GGT gene in I. nil plants and showed the conversion of
cyanidin 3-O-glucoside into cyanidin 3-O-sophoroside in
transgenic petunia plants in which the Ip3GGT cDNA is
expressed.
Results
Flower coloration of dusky mutants
Table 1 shows the flower color phenotypes of the I. nil
lines used, most of which carry the dusky mutations.
Although their flower coloration may subtly vary due to
various genetic backgrounds, the dusky mutants carrying
the wild-type Magenta allele generally exhibit bluish or
purplish-gray flowers that contain cyanidin derivatives,
whereas those with the magenta mutation tend to display
reddish-brown flowers that contain pelargonidin derivatives, as represented in Figure 1(c,e). Among them, Magenta plants with the wild-type Purple or Purple-revertant
allele show a tendency to produce more bluish-gray
flowers than those with the purple mutation. Similarly,
magenta mutants with the Purple or Purple-revertant allele
generally exhibit darker reddish-brown flowers than those
with the purple mutation. We noticed that the petals in all
of the dusky mutations examined often contained
intensely pigmented globules, also called anthocyanic
vacuolar inclusions (Markham et al., 2000), in the vacuoles
of their epidermal cells (Figure 1c,e), whereas petals in the
Dusky plants showed no such globules (Figure 1b,d).
As the globules were found in both Purple and purple
flowers, they formed even in the vacuoles at around pH 7
(Yamaguchi et al., 2001).
Isolation of novel cDNAs encoding glycosyltransferases
The dark purple flowers of I. purpurea contain a cyanidin
3-O-sophoroside derivative that lacks one glucose molecule and a methyl group from HBA (Figure 1f; Saito et al.,
356 Yasumasa Morita et al.
Table 1 Flower color phenotypes and genotypes of Ipomoea nil mutants
Alleles
Line
Flower color
Mg
Pr
Dy
Y
Reference and source
TKS
KK/ZSK-2
Q188
Q813
Q111
Q231
Q1221
Benkei (Tak)
Unzen-no-maki (Tak)
Q105
Q1209
Q1231
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Pr
Pr
Pr
Pr
Pr-r11
Pr-r11
Pr-r11
Pr-r12
Pr-r12
Pr-r12
Pr-r12
Pr-r12
Dy
Dy
dy-1
dy-1
dy-1
dy-1
dy-1
dy-2
dy-2
dy-2
dy-2
dy-2
þ
þ
þ
þ
þ
þ
þ
)
)
)
)
)
Kawasaki and Nitasaka (2004)
Hoshino et al. (2003)
Our collection
Our collection
Our collection
Our collection
Our collection
Saito et al. (1996)
Saito et al. (1996)
Our collection
Our collection
Our collection
YI/PRF
Q163
Q810
Q822
Q1203
Q1210/Benkei
Mg
Mg
Mg
Mg
Mg
Mg
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
Dy
dy-1
dy-1
dy-1
dy-1
dy-2
)
þ
þ
þ
þ
)
Fukada-Tanaka et al. (2000)
Our collection
Our collection
Our collection
Our collection
Our collection
Violet
Q830
Q114/Danjuro
Q1215/Danjuro
Q1233
Q1239
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
Pr-r11
Pr
Pr-r12
Pr-r12
Pr-r12
Pr-r12
Dy
dy-1
dy-2
dy-2
dy-2
dy-2
þ
þ
þ
)
)
)
Hoshino et al. (2003)
Our collection
Our collection
Our collection
Our collection
Our collection
Scarlet O’Hara
Akatsuki-no-yume
Danjuro (Tak)
Q113
Q116
Q213
Q353
Q856
Q1225
Q1229
Q1235
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
mg-1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
pr-m1
Dy
dy-1
dy-1
dy-1
dy-1
dy-1
dy-1
dy-1
dy-1
dy-1
dy-1
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Hoshino et al. (2003)
Saito et al. (1994)
Hoshino et al. (2003)
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
The genetic symbols Mg, Pr, Dy, and Y represent Magenta, Purple, Dusky and Yellow, respectively. The I. nil lines in our collection are part of the
collection at Kyushu University (http://mg.biology.kyushu-u.ac.jp/index.html). Each flower color was taken from the adaxial petal part of individual
flowers. Two dusky mutant alleles, dy-1 and dy-2, are shown in Figure 3(c). All magenta mutants tested carry the single nonsense mutation mg-1
(Hoshino et al., 2003). Likewise, all purple mutants tested are caused by the identical insertion of Tpn4 in the Tpn1 family into the untranslated exon
1 of the Purple gene (Fukada-Tanaka et al., 2000, 2001; Yamaguchi et al., 2001). Although some of the purple mutants display variegated flowers
and others exhibit rather stable and uniformly colored flowers with occasional fine spots, we designated the mutation as pr-m1. The two Purplerevertant alleles, Pr-r11 and Pr-r12, contain 3-bp insertions, CAG and CTG, at theTpn4 insertion site, respectively, and they are footprints generated
by independent Tpn4 excisions (Fukada-Tanaka et al., 2000). The wild-type and recessive-mutant alleles at the Yellow locus, which confer darkergreen- and yellow-green-leaf phenotypes, are denoted here with a þ and ), respectively. Three lines named Danjuro from different sources used
here appear to be of at least two different origins because the dusky alleles are different. The line Benkei (Tak) is probably a Pr-r revertant of the line
Q1210/Benkei.
1995). When isolating cDNA clones coding for enzymes to
modify anthocyanidin 3-O-glucosides from young flower
buds of I. purpurea, we obtained a novel cDNA clone
apparently encoding an enzyme for glycosylation (see
Experimental procedures), the deduced amino acid
sequence of which showed about 43% identity to that of
the petunia Rt cDNA (Figure 2a; Brugliera et al., 1994;
Kroon et al., 1994). Subsequently, the I. nil cDNA corresponding to the obtained I. purpurea cDNA was isolated,
and the coding sequences in both cDNA clones and their
deduced amino acid sequences exhibited 97 and 99%
identities in relation to each other. Both of them encode
polypeptides of 459 amino acids with a characteristic
C-terminal 42 amino acid consensus motif (Figure 2a),
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
A novel glucosyltransferase gene and its mutants 357
Figure 2(b) shows a phylogenetic tree containing nine
UDP-glycosyltransferases that are involved in the glycosylation of anthocyanidins and anthocyanins among various glycosyltransferases in higher plants (Ross et al.,
2001). The most closely related UDP-glycosyltranseferase
to the putative gene products of the newly isolated cDNAs
is the petunia 3RT, which adds a rhamnose molecule
to anthocyanidin 3-O-glucosides forming anthocyanidin
3-O-rutinosides (Brugliera et al., 1994; Kroon et al., 1994).
As flower pigments of I. nil and I. purpurea are anthocyanidin 3-O-sophoroside derivatives (Figure 1f), we hypothesized that the newly isolated cDNAs encode 3GGT,
which adds a glucose molecule to anthocyanidin
3-O-glucosides forming anthocyanidin 3-O-sophorosides
(Figure 1h), because the substrates of both 3RT and 3GGT
are the same anthocyanidin 3-O-glucosides and these
enzymes add the respective sugar molecule to their
glucoside residues.
Genomic structures of the novel gene for glycosyltransferase
in the wild type and dusky mutants of I. nil
Figure 2. Alignment of amino acid sequences and a phylogenetic tree of
3GGT and its related proteins.
(a) Multiple alignment of deduced amino acid sequences of the In3GGT and
Ip3GGT proteins with the petunia UDP-rhamnose:anthocyanidin 3-O-glucoside-6¢¢-O-rhamnosyltransferase, Ph3RT. Asterisks and dashes indicate identical amino acids and gaps in the sequences for the best alignment,
respectively. The underline indicates a putative C-terminal UDP binding motif
for glycosyltransferases. The arrowhead indicates the position of the 4-bp
insertion sequences in dusky mutants.
(b) A phylogenetic tree of known plant UDP-glycosyltransferases for anthocyanin biosynthesis. The entire amino acids were aligned with CLUSTALW, and
the tree was constructed as described before (Fukada-Tanaka et al., 1997). The
bootstrap values out of 100 retrials are indicated at each branch, and the scale
shows 0.1-amino acid substitution per site. Petunia 3RT (CAA50377), petunia
3GT (BAA89008), gentian 3GT (BAA12737), grape 3GT (AAB81683), maize 3GT
(CAA31856), perilla 5GT (BAA36421), verbena 5GT (BAA36423), petunia 5GT
(BAA89009), and gentian 3¢GT (BAC54092).
which is thought to be involved in binding of the protein
to the UDP moiety of the sugar nucleotide (Ross et al.,
2001), suggesting that the newly isolated cDNA clones
encode UDP-glycosyltransferases.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
Southern blot analysis using the newly isolated I. nil cDNA
as a probe revealed that the wild-type lines of I. nil, TKS, and
KK/ZSK-2 carry one copy of the putative In3GGT gene in their
genomes (Figure 3a), even though the genes for UDP-glycosyltransferase constitute one of the largest multigene
families in plants (Ross et al., 2001). We also analyzed four
dusky mutants of I. nil, Q856, Danjuro (Tak), Benkei (Tak),
and Unzen-no-Maki (Tak), in which the In3GGT activity was
suggested to be low (Saito et al., 1994, 1996; Toki et al.,
2001), and found no apparent DNA rearrangements in their
putative In3GGT genes (Figure 3a). We cloned and sequenced the 10.2-kbp XbaI fragment from the wild-type
plant KK/ZSK-2 and found that the putative In3GGT gene
carries no intron and resides near the 5¢ region within the
XbaI fragment (Figure 3b). Subsequently, 4.5-kbp fragments
containing the putative In3GGT gene were amplified by
polymerase chain reaction (PCR) from the wild-type line
TKS as well as eight different dusk mutants with various
genetic backgrounds, Danjuro (Tak), Akatsuki-no-yume,
Unzen-no-maki (Tak), Q105, Q114, Q188, Q856, and Q1210,
with the primers Dy-1F and Dy-1R (Figure 3b), and the
amplified fragments were sequenced. No sequence alterations was detected among these fragments except for the
presence of 4-bp insertions, GGAT (dy-1) or CGAT (dy-2), at
the 3¢ coding region of the putative In3GGT gene in all the
dusky mutants tested (Figure 3c; Table 1). These insertions
result in frameshift mutations that produce polypeptides
that are five amino acids longer than the wild type (Figure 3c). We have further examined additional Dusky lines,
Scarlet O’Hara, YI/PRF, and Violet, and 22 dusky mutant lines
by sequencing the 1.5-kbp coding segments obtained by
PCR amplification with the primers Dy-2F and Dy-2R; our
358 Yasumasa Morita et al.
Figure 3. Structure and expression of the Dusky gene.
(a) Southern blot analysis of the In3GGT gene. DNA (10 lg) was digested with restriction enzymes NheI and XbaI and hybridized with the In3GGT cDNA as a probe.
Lane 1, TKS; lane 2, KK/ZSK-2; lane 3, Q856; lane 4, Danjuro (Tak); lane 5, Benkei (Tak); lane 6, Unzen-no-maki (Tak).
(b) A structure of the 10.2-kbp XbaI fragment containing the In3GGT gene. The 3GGT gene contains no intron. The gray and black boxes indicate the coding and
putative C-terminal UDP binding motif regions, respectively. The large vertical arrow indicates the site of 4-bp insertions, and small horizontal arrows represent the
site and orientation of the primers.
(c) The 4-bp insertions indicated by the upper lines in the dusky mutants result in frameshift mutations. The underlines with GAT indicate reminiscent sequences of
hypothetical target site duplications. Asterisks indicate the ends of proteins.
(d) RT-PCR analysis for spatial expression of the In3GGT gene in the wild-type line TKS. The I. nil DFR-B gene for anthocyanin biosynthesis (Inagaki et al., 1999) and
the constitutively expressed c-sub gene for mitochondrial F1F0 ATP synthase c-subunit in I. nil (A. Hoshino, unpublished data) were used for comparison. The
numerals in parenthesis indicate cycles of PCR amplification.
(e) Northern blot analysis for the temporal expression of the 3GGT gene in petals of young buds of the line TKS. The numerals above indicate the time (h) of the RNA
preparation before flower opening. Total RNA (10 lg) was separated and hybridized with an appropriate probe, the I. nil 3GGT cDNA or I. nil DFR-B cDNA. The
ethidium bromide-stained rRNA bands are shown as a loading control.
(f) Northern blot analysis of the In3GGT gene expression in dusky and c-1 mutants. Total RNAs were prepared from petals of flower buds at 36 h before flower
opening, and the I. nil 3GGT cDNA was used as a probe. Lane 1, TSK; lane 2, KK/ZSK-2; lane 3, Q856; lane 4, Benkei (Tak); lane 5, 78WWc-1.
results agree with previous results, which show that these
dusky lines carry either the dy-1 or the dy-2 mutation
(Table 1). These results are consistent with the notion that
the Dusky gene encodes the putative In3GGT enzyme and
that the dusky mutations are 4-bp insertions. The notion was
further supported by the fact that the expression of the
putative 3GGT gene of I. nil was mainly expressed in the
petals and tubes of flower buds, where the genes for
anthocyanin biosynthesis, represented by the DFR-B gene,
were expressed and anthocyanin pigments were consequently accumulated (Figure 3d). Northern blot analysis also
showed that its transcripts accumulated most at approximately 24 h before flower opening, which appeared to be
slightly later than DFR-B transcript accumulation (Figure 3e).
Moreover, the expression of the putative In3GGT gene was
significantly reduced in the c-1 mutant (Figure 3f), which is
deficient in the function of the MYB transcriptional activator
for anthocyanin biosynthesis, and the mRNA accumulation
of its structural genes was known to be reduced considerably
(Fukada-Tanaka et al., 1997; Hoshino et al., 1997, 2003;
Y. Morita, unpublished data), indicating that the putative
In3GGT gene is regulated by the C-1 protein.
Enzymatic characterization of the Dusky gene product
expressed in E. coli
To examine whether the Dusky gene indeed encodes the
3GGT enzyme, we first cloned the I. nil cDNA for the
putative In3GGT enzyme into the E. coli expression vector
pQE30, transformed it into E. coli, and analyzed the
enzymatic activity of the gene product in the E. coli crude
extracts (see Experimental procedures). Figure 4 shows
typical examples of such experiments. The crude extracts
containing the expected protein of 51.2 kDa (data not
shown) were able to convert cyanidin 3-O-glucoside into
cyanidin 3-O-sophoroside (Figures 1h and 4a), which
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
A novel glucosyltransferase gene and its mutants 359
(a)
(b)
(c)
Figure 4. HPLC analysis of the reaction products of the In3GGT recombinant
proteins.
(a) Cyanidin 3-O-glucoside treated with the In3GGT protein.
(b) Cyanidin 3-O-glucoside treated with the mutated In3GGT protein produced
from the dusky-2 gene.
(c) Cyanidin 3,5-O-diglucoside treated with the In3GGT protein.
(d) Cyanidin 3,5-O-diglucoside treated with the mutated In3GGT protein
produced from the dusky-2 gene.
(e) Delphinidin 3-O-glucoside treated with the Ip3GGT protein.
(f) Delphinidin 3,5-O-diglucoside treated with the Ip3GGT protein.
(g) Structure of anthocyanidin 3,5-O-diglucoside.
The elution profiles of each substrate and its product are shown, and the
symbols C3G, C3S, D3G, D3S, C3,5G, and D3,5G are abbreviations for
cyanidin 3-O-glucoside, cynidin 3-O-sophoroside, delphinidin 3-O-glucoside,
delphinidin 3-O-sophoroside, cyanidin 3,5-O-diglucoside, and delphinidin 3,5O-diglucoside, respectively.
comigrated with the authentic sample in a reverse-phase
column (Fukuchi-Mizutani et al., 2003). In addition, the
peak m/z 611 [M]þ was detected by mass spectrometry
(MS) analysis, also supporting the production of cyanidin
3-sophoroside. Similarly, pelargonidin 3-O-glucoside was
converted into pelargonidin 3-O-sophoroside (data not
shown). However, cyanidin 3,5-O-diglucoside could not
serve as a substrate, and no product could be detected
(Figure 4c). Likewise, neither pelargonidin 3,5-O-glucoside
nor delphinidin 3,5-O-diglucoside could serve as substrates (data not shown). The same results were also
obtained with the putative Ip3GGT cDNA of I. purpurea
expressed in E. coli using the expression vector pQE61
(Figure 4f). Interestingly, delphinidin 3-O-glucoside, which
is not present in I. nil and I. purpurea, was also converted
into delphinidin 3-O-sophoroside (Figure 4e), which was
further supported by MS analysis by detecting the peak
m/z 627 [M]þ that matched with the expected molecular
weight. We also examined the dusky mutant genes by
cloning the coding regions of the genomic sequences into
the E. coli expression vector pQE30 and characterized the
activities of their gene products in the same way as the
wild-type Dusky cDNA. No 3GGT activities could be
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
Figure 5. Flower phenotypes and expression of the Ip3GGT gene in transgenic petunia plants.
Flower phenotypes of the host plant, cultivar Vakara Red (a), and one of the
transgenic plants, PT102-10 (b).
(c) RT-PCR analysis of the overexpression of the Ip3GGT transcripts and the
content of cyanidin 3-O-sophoroside (C3S) in the transgenic petunia PT102
plants. Total RNAs were prepared from young leaves of eight independently
prepared transgenic plants and a non-transformed host plant (NT), and the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; accession number:
X60346) gene of petunia was used as an internal control. PT102-10 indicates
plant 10 and the C3S content in PT102-8 was not determined.
detected in the dusky genes with the CGAT insertion
(Figure 4b) or with the GGAT insertion (data not shown).
The results confirmed that the Dusky gene encodes
3GGT, which mediates the conversion of anthocyanidin
3-O-glucosides into anthocyanidin 3-O-sophorosides.
Expression of the I. purpurea Ip3GGT cDNA in transgenic
petunia
As the products of the obtained 3GGT genes promote the
conversion of cyanidin 3-O-glucoside into cyanidin
3-O-sophoroside, we asked whether the same conversion
could occur in heterologous transgenic plants having the
introduced Ip3GGT cDNA expressed. For this purpose, we
chose the petunia plant cultivar Vacara Red (Ando et al.,
2000), which exhibits red flowers (Figure 5a) and contains
cyanidin 3-O-glucoside and cyanidin 3-O-sophoroside as
major anthocyanins (38 and 32% of total anthocyanins,
respectively). Of 14 independent transgenic plants generated, seven accumulated the introduced Ip3GGT mRNA
(Figure 5c), and the peak profiles of high performance liquid
chromatography (HPLC) analysis indicated that these
transgenic petunia flowers expressing the Ip3GGT gene
showed varied amounts of cyanidin 3-O-sophoroside from
30 to 80% of total anthocyanins generally depending on the
360 Yasumasa Morita et al.
degree of expression level of the Ip3GGT gene. In the
transgenic petunia plant PT102-10, which accumulated the
highest amount of cyanidin 3-O-sophoroside and displayed
reddish flowers (Figure 5b), virtually no cyanidin 3-O-glucoside was present in the petals. The production of cyanidin
3-sophoroside in the extract of PT102-10 flowers was further
supported by liquid chromatography-quadrupole liquid
chromatography-mass spectrometry (LC-Q LC-MS) analysis,
in which the peak m/z 611 [M]þ was detected as the major
peak. Therefore, the introduced Ip3GGT cDNA of I. purpurea
could efficiently produce UDP-glucose:anthocyanidin 3-Oglucoside glucosyltransferase, which can glucosylate
cyanidin 3-O-glucoside to cyanidin 3-O-sophoroside as
a single major constituent in the transgenic petunia
flower, although its hue is not drastically changed.
Discussion
We showed that the I. nil Dusky gene and its I. purpurea
orthologous gene encode 3GGT enzymes that add a glucose
molecule to anthocyanidin 3-O-glucosides forming anthocyanidin 3-O-sophorosides (Figure 1h). Apart from the
petunia Rt gene, the 3GGT gene described here is the only
isolated gene encoding the UDP-glycosyltransferase that
adds a sugar residue to the glucose moiety of anthocyanin.
Not only the pelargonidin and cyanidin derivatives found in
I. nil and I. purpurea but also delphinidin 3-O-glucoside,
which is absent in these Ipomoea flowers, can serve as a
good substrate (Figure 4e). However, anthocyanidin 3,5-Odiglucosides could not serve as a substrate (Figure 4c,f)
even though they are found in the petals of dusky mutants
(Saito et al., 1994, 1996). Major anthocyanins identified in
the petals of the dusky mutants conferring reddish-brown
or purplish-gray flowers were anthocyanidin 3-O-glucosylcaffeoylglucosides and their 5-O-glucoside derivatives
(Figure 1g; Saito et al., 1994, 1996), and intensely pigmented
globules were observed in the vacuoles of their epidermal
cells (Figure 1c,e). The pigmented globules, or anthocyanic
vacuolar inclusions, are known to have a certain profound
effect on both flower color and intensity (Markham et al.,
2000). It is thus likely that reddish-brown or purplish-gray
flower coloration in dusky mutants is due not only to the
accumulation of anthocyanidin 3-O-glucosylcaffeoylglucosides and their 5-O-glucoside derivatives in their petals
(Figure 1g; Saito et al., 1994, 1996) but also to the appearance of intensely pigmented globules in the vacuoles
(Figure 1c,e).
We also showed that dusky mutations in I. nil conferring
reddish-brown or purplish-gray flowers are insertion mutations of 4 bp, GGAT or CGAT, at the same position near the
3¢ terminus of the Dusky gene (Figure 3b,c). Close inspection
of the insertions revealed that their 3¢ common trinucleotides GAT can also be found 5¢ adjacent to these 4-bp
insertions: the wild-type sequence GAT has changed into
GAT(G/C)GAT. We speculate that these 4-bp insertion
sequences are footprints generated by excision of a DNA
transposon in the Tpn1 family. The transposons in the Tpn1
family, which belong to the En/Spm superfamily and are
thought to have acted as major mutagens for the generation
of various floricultural traits in I. nil, generate 3-bp target site
duplications upon their integrations and can generate 4-bp
insertions as footprints upon their excisions (Hoshino et al.,
2001; Iida et al., 1999, 2004; Inagaki et al., 1996; Kawasaki
and Nitasaka, 2004). It is thus likely that two dusky alleles,
insertions of GGAT or CGAT, were generated by two
independent excision events of an integrated Tpn1-related
element that must be flanked by 3-bp direct repeats of GAT
generated as target duplications upon integration near the 3¢
end of the In3GGT gene. We also examined the dusky
mutations in the yellow mutants (Table 1), which confer
yellow-green leaves and are tightly linked to the dusky allele
(Hagiwara, 1956; Imai, 1929). Only the dy-2 allele with the
insertion of CGAT was found in all the yellow mutants
tested. As Japanese floriculturists who favor reddish-brown
or purplish-gray flowers of I. nil have taken advantage of the
tight linkage between dusky and yellow to select the mutants
with the recessive dusky allele in the homozygous condition
over a century, our results indicate that the dusky allele in
I. nil mutants with yellow-green leaves was probably derived
from a single progenitor, in which a putative Tpn1-relative
had been excised from the In3GGT gene characterized here.
Likewise, the Purple allele in the dusky and yellow double
mutants tested was not the wild-type allele but the identical
revertant allele, Pr-r12, which probably originated from a
single progenitor, in which an excision of Tpn4 in the Tpn1
family from the InNHX1 gene had occurred (Table 1; FukadaTanaka et al., 2000).
The major anthocyanins that accumulated in the petals
of the dusky mutants were anthocyanidin 3-O-glucosylcaffeoylglucosides and their 5-O-glucoside derivatives
(Figure 1g; Saito et al., 1994, 1996; Toki et al., 2001), indicating that the addition of the caffeoyl molecule (I) to anthocyanidin 3-O-glucosides could occur in the absence of 3GGT
activity. In these accumulated anthocyanins, the position of
the outer glucosyl residue is curiously attached at the 3
position of the caffeoyl moiety (I) in Figure 1(g), whereas the
corresponding glucose molecule in HBA is attached at the 4
position of the same caffeoyl moiety (I) in Figure 1(f).
Interestingly, the outmost two glucose molecules in HBA
are attached at the 3 position of the caffeoyl moieties (II) and
(III) in Figure 1(f). Perhaps, the glucosyltransferase that
mediates the addition of a glucose molecule to the caffeoyl
moiety (I) requires anthocyanidin 3-O-caffeoylsophorosides
and fails to transfer the glucose moiety to the 4 position of
the caffeoyl moiety (I) of anthocyanidin 3-O-caffeoylglucosides that are accumulated in the dusky mutants. Under such
circumstances, another glucosyltransferase, which usually
catalyzes the addition of a glucose molecule to the 3 position
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
A novel glucosyltransferase gene and its mutants 361
of the caffeoyl moieties (II) and (III), can promote the transfer
of the glucose molecule into the 3 position of the caffeoyl
moiety (I) that results in anthocyanidin 3-O-glucosylcaffeoylglucosides and their 5-O-glucoside derivatives
(Figure 1f,g; Saito et al., 1994, 1996). Such flexibility in
anthocyanin biosynthesis is likely to be an important factor
for exhibiting diverse flower hues in various mutants of the
Japanese morning glory.
Experimental procedures
Plant materials
All I. nil lines used are listed in Table 1 except for the c-1 mutant
78WWc-1 (Abe et al., 1997; Hoshino et al., 2003). The I. purpurea line
used for the preparation of the bud cDNA library was YO/FP-39,
which bears dark-purple flowers (Habu et al., 1998; Hoshino et al.,
2003).
Nucleic acid procedures
General nucleic acid procedures, including plant DNA and RNA
preparation, Northern and Southern blot analyses, plaque hybridization, PCR amplification, and DNA sequencing analysis, were
performed as described before (Habu et al., 1998; Hisatomi et al.,
1997; Hoshino et al., 2003; Yamaguchi et al., 2001). The presence of
the purple allele in Table 1 was determined by PCR amplification
(Fukada-Tanaka et al., 2001), and the magenta allele was by PCR
amplification followed by sequence determination of the amplified
fragments (Hoshino et al., 2003). To determine the Purple-revertant
mutations, Pr-r11 and Pr-r12, the 0.5-kbp PCR fragments containing
the footprints caused by the excision of Tpn4 from the exon 1 region
of the InNHX1 gene (Fukada-Tanaka et al., 2001) were obtained with
the primers EX1FW2 (5¢-TACTGATAGGAGAGTTGTCGTTC-3¢) and
EX2RV (5¢-CAATGTCGTGGTTTCTGTTCACATA-3¢) and sequenced
directly.
For the isolation of a cDNA clone encoding Ip3GGT of I. purpurea,
first-strand cDNAs were synthesized from total RNAs isolated from
flower buds of YO/FP-39 using the SuperScript II Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with the primer Not27dT18 (5¢-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3¢), and subsequent PCR amplification with the primers
ATC1 (5¢-GAYTTYGGITGGGGIAA-3¢) and Not27 (5¢-AACTGGAAGAATTCGCGGCCGCAGGAA-3¢) yielded a probe for screening a
cDNA library prepared from the flower buds of YO/FP-39 (Hoshino
et al., 2003) under low-stringency hybridization conditions (Hisatomi
et al., 1997). One of the clones isolated apparently contained the
sequence for a glycosytransferase. Subsequently, the corresponding cDNA of I. nil was also obtained from a cDNA library from the
flower buds of KK/ZSK2 (Inagaki et al., 1999).
To determine the genomic In3GGT sequence of I. nil, the 10.2-kbp
XbaI fragment of KK/ZSK-2 containing the entire In3GGT gene was
cloned into the kDASH II vector (Stratagene, La Jolla, CA, USA) and
sequenced. To analyze the dusky mutations, the 4.5-kbp segments
containing the entire In3GGT gene from KK/ZSK-2, TKS, Danjuro
(Tak), Akatsuki-no-yume, Unzen-no-maki (Tak), Q105, Q114, Q188,
Q856, and Q1210 were obtained by PCR amplification using LA Taq
polymerase (Takara Biomedicals, Ohtsu, Japan) with the primers
Dy-1F (5¢-TCTAGAACTTCAGCAAATAAGATGC-3¢) and Dy-1R
(5¢-TGGTCTTGATCTCATACTTTCAACT-3¢) and directly sequenced.
Subsequently, the 1.5-kbp PCR fragments containing the entire
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363
In3GGT gene from 22 additional dusky mutants (Table 1) were
obtained with the primers Dy-2F (5¢-CAGAAAGCTAGCTAGCTAGGTATAGG-3¢) and Dy-2R (5¢-CTAGCTCAAGGCAGTGATACC-3¢)
and sequenced directly.
For reverse transcription-PCR (RT-PCR) analysis, total RNAs were
prepared from petal, tube, anther, stigma, and sepal of buds as well
as leaf blade, petiole, and stem of young plants. The primers used
for RT-PCR amplification were In3GGT cDNA, Dy-2F mentioned
above and Dy-3R (5¢-GAGGATGATCTCAATGTCGTTCTGG-3¢); DFRB cDNA, DFR-BFJ-5F (5¢-ACGAGGCTACCATATTCACGCCACC-3¢) and
DFR-BFJ-7R (5¢-ATGTTGAGCACTCCATTGATGGCTG-3¢); c-subunit
cDNA for mitochondrial F1F0 ATP synthase c-subunit, JMFS029C13F (5¢-ATCAAGCCGTTAAGAGCAAACCC-3¢) and JMFS029C1-3R
(5¢-AATATTCCGGCTTGTCTTGACAG-3¢). First-strand cDNAs were
synthesized using SuperScript III reverse transcriptase (Invitrogen),
and AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster
City, CA, USA) was used for subsequent PCR amplification. For
In3GGT and DFR-B cDNAs, the PCR reaction was carried out for
initial denaturation at 95C for 9 min, 21 or 23 cycles of denaturation (95C for 30 sec), annealing and extension (68C for 1 min),
and additional extension at 72C for 10 min. For the c-subunit cDNA,
we slightly modified the annealing and extension temperature to
65C.
Biochemical analysis in E. coli
The 3GGT sequences of the wild-type I. nil line, its dusky
mutants containing either CGAT or GGAT sequences, and the
wild-type I. purpurea plant were inserted into the expression
vector pQE30 or pQE61 (Qiagen, Hilden, Germany). The reaction
mixture for the 3GGT enzymatic assay consisted of 100 mM
potassium phosphate buffer (pH 7.5), 0.6 mM delphinidin
3-O-glucoside, cyanidin 3-O-glucoside or delphinidin 3,5-O-diglucoside, 1 mM UDP-glucose, and 20 ll of crude E. coli extracts
as the enzyme solution in a volume of 100 ll. The reaction was
started by adding an anthocyanin and stopped by an equal volume of 90% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid
(TFA) after incubation for 15 min at 30C. Twenty microliters of a
reaction mixture was applied on a reverse-phase column and
separated as previously described (Fukuchi-Mizutani et al., 2003).
The authentic cyanidin 3-O-sophoroside was purchased from
Polyphenoles Laboratories (Sandnes, Norway). A large-scale
reaction with 2.5 mg of either cyanidin 3-O-glucoside or delphinidin 3-O-glucoside, UDP-glucose and In3GGT proteins, which
were purified by Chelating Sepharose Fast Flow (Amersham
Biosciences, Piscataway, NJ, USA) charged with Ni, was performed, and the products were separated by reverse-phase HPLC
using a Polymer C18 column (30 cm · 2 cm; YMC Co., Ltd, Kyoto,
Japan) with a gradient elution (after 10 min isocratic elution of
10% CH3CN containing 0.5% TFA in H2O, 40 min linear gradient
elution from 10 to 25% CH3CN, and then 5 min isocratic elution
of 25% CH3CN containing 0.5% TFA in H2O at a flow rate of
6 ml min)1) and detected with photodiode array detector
SPD-M6A (Shimadzu, Kyoto, Japan). We obtained two fractions
in the reaction with cyanidin 3-O-glucoside: a fraction containing
cyanidin 3-O-glucoside eluted at 52.7 min and a new fraction
eluted at 44.3 min that was subjected to MS analysis. Similarly, a
fraction eluted at 43.2 min was obtained and subjected to MS
analysis in addition to the fraction containing delphinidin
3-O-glucoside eluted at 51.0 min in the reaction with delphinidin
3-O-glucoside. ESI-MS of these new products was obtained by
Q-TOF with z-spray ion source (Micromass, Manchester, UK) in
positive mode.
362 Yasumasa Morita et al.
Production of transgenic petunia plants
The Ip3GGT cDNA of I. purpurea was inserted between an enhanced
CaMV35S promoter and nopaline synthase terminator in a binary
vector and transformed into Petunia hybrida cultivar Vacara Red
(Sakata Seeds Ltd, Yokohama, Japan) with Agrobacterium tumefaciens strain Agl0 (Lazo et al., 1991) as described previously
(Fukuchi-Mizutani et al., 2003). Expression of the Ip3GGT gene in
the transgenic petunia plants was confirmed with RT-PCR with a
pair of the specific primers for the Ip3GGT gene. The resulted
fragments were detected by Southern blot analysis. The extracts of
petals of the host and a transgenic petunia were analyzed with the
LC-Q LC-MS system (Thermo Quest, San Jose, CA, USA) using an
ODP-40 3D column (15 cm · 3 mm; Shoko. Co., Ltd, Tokyo, Japan)
with a gradient elution (10 min linear gradient of 10–50% of acetonitrile containing 0.6 N HCl followed by 5 min of isocratic elution of
50% of acetonitrile containing 0.6 N HCl at a flow rate of
0.3 ml min)1). The detection was carried out with an ESI probe
in the positive mode and absorption at 520 nm. Under these conditions, the retention time and molecular ions of cyanidin 3-Osophoroside and cyanidin 3-O-glucoside were 7.28 min and m/z
611[M]þ and 7.77 min and m/z 449[M]þ, respectively.
Acknowledgements
We thank Miwako Matsumoto and Kyoko Ikegaya for technical
assistance, Masaatsu Yamaguchi for valuable advice, and Tokyoasagao-kenkyukai and Robert A. Ludwig for providing dusky mutant
seeds and A. tumefaciens Agl0, respectively. This work was supported in part by grants from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
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Accession numbers of sequence data: The nucleotide sequences have been deposited to DDBJ, EMBL, GenBank under the accession numbers
AB192314 (In3GGT mRNA), AB192315 (Ip3GGT mRNA) and AB192316 (the wild-type In3GGT gene), AB192317 (the dusky-1 mutant In3GGT
gene), and AB192318 (the dusky-2 mutant In3GGT gene).
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 353–363