The Plant Journal (2007) 49, 641654

doi: 10.1111/j.1365-313X.2006.02988.x

A bHLH regulatory gene in the common morning glory,

Ipomoea purpurea, controls anthocyanin biosynthesis in
flowers, proanthocyanidin and phytomelanin pigmentation
in seeds, and seed trichome formation
Kyeung-Il Park, Naoko Ishikawa, Yasumasa Morita, Jeong-Doo Choi, Atsushi Hoshino and Shigeru Iida*
National Institute for Basic Biology, Okazaki 444-8585, Japan
Received 17 August 2006; accepted 4 October 2006.
*For correspondence (fax 81 564 55 7685; e-mail shigiida@nibb.ac.jp).

Present address: Gyeongbuk Institute for Bio-Industry, Andong-si, Gyeongbuk 760-380, Korea

Summary
The transcriptional regulators for anthocyanin pigmentation include proteins containing R2R3-MYB domains,
bHLH domains and conserved WD40 repeats, and their interactions determine the set of genes to be expressed.
Spontaneous ivory seed (ivs) mutants of Ipomoea purpurea displaying pale pigmented flowers and ivory seeds
are caused by insertions of DNA transposons into the bHLH2 gene that encodes a bHLH transcriptional
regulator. A partial reduction in the expression of all structural genes encoding enzymes for anthocyanin
biosynthesis was observed in the young flower buds of these ivs mutants. The DFR-B and ANS transcripts were
completely abolished in the ivs seed coats, whereas the early biosynthetic genes for flavonol biosynthesis
remained active. The production and accumulation of both proanthocyanidin and phytomelanin pigments in
the ivory seed coats were drastically reduced. Moreover, the unbranched trichomes in the ivory seeds were
smaller in size and fewer in number than those in the wild-type dark-brown seeds, and the surface of the
epidermis without trichomes in the dark-brown seeds looked rougher, due to the protruding tangential walls,
than that in the ivory seeds. Although the I. purpurea bHLH2 gene is the most closely related to the petunia
AN1 gene, whose mutation is known to confer white flowers and to be deficient in acidification of their
vacuoles, the vacuolar alkalization in the epidermal flower limbs of I. purpurea ivs mutants appears to occur
normally. These results are discussed with regard to the function of bHLH transcriptional regulators controlling
flower and seed pigmentation as well as other epidermal traits.
Keywords: bHLH transcriptional regulator, Ipomoea purpurea, anthocyanin pigmentation, proanthocyanidin
biosynthesis, phytomelanin accumulation, trichome formation.

Introduction
The genus Ipomoea includes about 600 species distributed
on a worldwide scale that exhibit various flower morphologies and pigmentation patterns; a large number of Ipomoea
species can be found in the Americas, particularly in Mexico
(Austin and Huaman, 1996; Clegg and Durbin, 2003). Among
them, three morning glories, Ipomoea nil (the Japanese
morning glory), Ipomoea purpurea (the common morning
glory) and Ipomoea tricolor, have been successfully
domesticated as floricultural plants, and many mutants
conferring various flower pigmentation phenotypes have
been isolated; spontaneous mutants of I. nil and I. purpurea
with various flower colors have been isolated and cultivated
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since the 17th century in Japan and Europe, respectively,

and such mutants of I. tricolor were isolated in the middle of
the 20th century in North America (Iida et al., 1999; White,
1981). Both I. nil and I. tricolor display blue flowers, whereas
I. purpurea produces dark-purple flowers (Figure 1a), and all
of them contain polyacylated and polyglycosylated cyanidin-based anthocyanins (Kondo et al., 1987; Lu et al., 1992;
Saito et al., 1995). All structural genes that encode enzymes
to produce anthocyanidin 3-O-sophorosides have been
characterized in these morning glories (Figure 2), and the
majority of their spontaneous mutations have been shown
to be caused by insertions of DNA transposons, which
641

642 Kyeung-Il Park et al.

(a)

(b)

Figure 2. Simplified flavonoid biosynthesis pathways.

The enzymes catalyzing each step in the pathways are indicated by uppercase
letters. The pathways for pelargonidin- and cyanidin-based anthocyanins are
combined after DFR, and the dotted lines with an arrowhead indicate steps not
yet identified in Ipomoea. CHS, chalcone synthase; CHI, chalcone isomerase;
F3H, flavanone 3-hydroxylase; F3H, flavonoid 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin
synthase; ANR, anthocyanidin reductase; 3GT, UDP-glucose:flavonoid 3-Oglucosyltransferase; 3GGT, UDP-glucose:anthocyanidin 3-O-glucoside-2-Oglucosyltransferase.

Figure 1. Flower and seed phenotypes.

(a) I. purpurea lines used. The black and white arrowheads indicate pigmented spots in flowers and seeds, respectively. Completely dried mature seeds
were used for the scanning electron microscopic observation.
(b) Small dark-brown reversion spot of a mature ivs-m2 seed. PS and NPB
indicate a pigmented spot and non-pigmented background on the seed coat,
respectively.
Scale bars 50 lm.

include Tpn1 and its relatives in the CACTA superfamily in

I. nil, and Tip100 as well as Tip201 of the hAT superfamily in
I. purpurea (Chopra et al., 2006; Durbin et al., 2000, 2001;
Fukada-Tanaka et al., 1997; Habu et al., 1998; Hisatomi et al.,
1997; Hoshino et al., 1997, 2001, 2003; Iida et al., 2004;
Ishikawa et al., 2002; Johzuka-Hisatomi et al., 1999; Morita

et al., 2005; A. Hoshino and Y. Morita, unpublished data). In

I. purpurea, insertions of Tip100 into the CHS-D gene often
resulted in white flowers with colored flakes (Durbin et al.,
2001; Habu et al., 1998), whereas integration of Tip201 into
the F3H gene conferred stable reddish flowers (Hoshino
et al., 2003). Although InMu1 of the Mutator family was also
found within intron 2 of the CHI gene in I. nil, it appears to
have no phenotypic effect (Hoshino et al., 2001). The structural genes for flower pigmentation in dicotyledonous plants
can be divided into early biosynthetic genes (EBGs) for
flavone and/or flavonol biosynthesis, and late biosynthetic
genes (LBGs), including DFR and ANS, for anthocyanin
biosynthesis (Figure 2), while anthocyanin pigmentation in
monocotyledonous maize (Zea mays) appears to be
coordinately regulated as a single module without division
of EBGs and LBGs (Martin and Gerats, 1993; Mol et al., 1998;
Schwinn and Davies, 2004).
The transcriptional regulators for anthocyanin biosynthesis, which activate the structural genes, are known to
include members of protein families containing R2R3-MYB

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Epidermal traits controlled by bHLH2 643

domains, bHLH (basic helix-loop-helix) domains and
conserved WDR (WD40 repeats); combinations of the
R2R3-MYB, bHLH and WDR factors and their interactions
determine the set of genes to be expressed (Broun, 2005;
Irani et al., 2003; Koes et al., 2005; Mol et al., 1998; Ramsay
and Glover, 2005). A recessive white mutant of I. purpurea
exhibiting white flowers with a pigmented spot in each ray
(Ennos and Clegg, 1983), which was also designated as a
spotted flower by Imai (1927), carries small deletions in a
gene for R2R3-MYB, IpMYB1/W (Chang et al., 2005), which
is closely related to the petunia (Petunia hybrida) AN2
gene (Quattrocchio et al., 1999). In I. nil, a recessive c-1
mutant displaying white flowers bears a frameshift mutation in a gene for R2R3-MYB, InMYB1/C-1, which is the
most closely related to IpMYB1/W (Morita et al., 2006).
Mutations in the bHLH genes, petunia an1 and Arabidopsis
(Arabidopsis thaliana) gl3 and egl3, affect not only anthocyanin pigmentation but also other epidermal traits, such
as cell morphology in seed coat cells and trichome
formation in leaves, and those in the WDR genes, petunia
an11 and Arabidopsis ttg1, confer similar phenotypes
(Spelt et al., 2002; Walker et al., 1999; Zhang et al., 2003).
Interaction of the R2R3-MYB, bHLH and WDR factors is
necessary for the development of epidermal cell types,
including trichome formation (Larkin et al., 2003). In I. nil,
recessive ca mutants bearing snow-white flowers with
whitish seeds carry 7 bp insertion mutations in a gene for
WDR, InWDR1, and show reduced trichome formation in
seeds (Morita et al., 2006). As not only AN1 but also JAF13
in petunia encode a bHLH transcriptional regulator for
anthocyanin biosynthesis, the AN1 and JAF13 proteins
have been named bHLH2 and bHLH1, respectively (Koes
et al., 2005; Mol et al., 1998). Both bHLH2 and bHLH1
cDNAs from I. nil and I. purpurea were also characterized
(Morita et al., 2006). The mutable ivs allele in I. tricolor,
conferring pale-blue flowers with a few fine blue spots and
ivory seeds with tiny dark-brown spots, is caused by an
intragenic tandem duplication of the bHLH2 gene (Park
et al., 2004).
Here, we show that spontaneous I. purpurea mutants
displaying pale pigmented flowers and ivory seeds are
deficient in bHLH2 as a result of insertions of Tip100 or
IpMu elements of the Mutator family, and that an I. purpurea mutant exhibiting snow-white flowers and ivory
seeds contains mutations in both bHLH2 and MYB1 genes.
In these mutants, anthocyanin biosynthesis for flower
coloration is reduced, the expression of LBGs for proanthocyanidin biosynthesis in seed pigmentation is abolished,
and biosynthesis and/or accumulation of phytomelanins
for seed pigmentation as well as seed trichome formation
are reduced. We also discuss the function of transcriptional regulators controlling various epidermal traits in
morning glories, which can serve as model plants offering
experimental versatility for the study of gene regulation.

Results
Ipomoea purpurea mutants displaying pale flowers and
ivory seeds
While the wild-type I. purpurea plant FP39 produces fully
purple flowers and dark-brown seeds (Figure 1a), recessive
mutations in two loci of I. purpurea exhibiting whitish
flowers and normal-colored seed have been identified: two
copies of Tip100 inserted in the CHS-D intron result in white
flowers (Habu et al., 1998), and two 6 and 19 bp deletions
causing a frameshift in MYB1 (the white or myb1-1 allele)
confer white flowers with a colored spot in each ray (Chang
et al., 2005). We confirmed that plants of the SP1 line in our
collection showing similar white flowers with reddish spots
(Figure 1a) contain the identical myb1-1 allele (data not
shown). To characterize spontaneous mutations of I. purpurea conferring ivory seeds, which we designated as ivory
seed (ivs) because their phenotypes are similar to those of
the I. tricolor mutant with the mutable ivs allele in the bHLH2
gene (Park et al., 2004), we employed five ivs mutants, PR42,
PR43, PP45, PR46 and WR4. Of these, plants of the mutable
ivs (ivs-m) lines PR42 and PR43 are descendants of a single
plant (see Experimental procedures) and bear pale-reddish
flowers with fine red spots or sectors and ivory seeds with
tiny dark-brown spots, whereas the stable ivs (ivs-stb) mutants PP45 and PR46 exhibit pale-purplish and pale-reddish
flowers, respectively, and ivory seeds (Figure 1a). These ivsstb mutants and FP44 (conferring an apparently wild-type
phenotype) were segregants of a single plant producing
purple flowers and dark-brown seeds (see Experimental
procedures), and the purplish and reddish flower colorations
of PP45 and PR46 were due to the F3H gene bearing the Pink
and pink (due to Tip201 insertion; Hoshino et al., 2003)
alleles, respectively (Figure 1a; data not shown). Although
WR4 contains the myb1-1 allele (data not shown), its phenotypes of stable snow-white flowers and ivory seeds cannot be explained by the myb1-1 allele alone because myb1-1
is known to confer white flowers with a pigmented spot in
each ray and normal-colored seeds, as is shown in SP1
(Figure 1a). We thus hypothesized that WR4 might also carry
an additional ivs mutation.
As it is known that Arabidopsis and petunia mutations
in the bHLH genes affect not only anthocyanin pigmentation but also other epidermal traits, such as trichome
formation in Arabidopsis leaves and cell morphology of
petunia seed coats (Spelt et al., 2002; Zhang et al., 2003),
we examined whether the ivs mutations confer similar
deficiencies. In I. purpurea, trichomes that form in leaves
and stems are indistinguishable between the wild-type and
ivs mutants (data not shown). However, like the whitish
seeds in the I. nil ca mutants deficient in IpWDR1 (Morita
et al., 2006), the trichomes of the ivs seeds are smaller in
size and remarkably fewer in number than those of the

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644 Kyeung-Il Park et al.

(a)

The ivs mutant lines contain insertions of DNA transposons

in the bHLH2 gene

(b)

(c)

Figure 3. Northern blot analyses of regulatory genes for anthocyanin biosynthesis (a, b) and Southern blot analysis of the bHLH2 gene (c).
Total RNAs (10 lg) prepared from flower limbs at 36 h before flower opening
were separated and hybridized with cDNA probes as indicated, and the
constitutively expressed gene for the mitochondrial F1F0 ATP synthase csubunit (c-sub) was used as an internal control (Park et al., 2004). We noted
that both bHLH1 and bHLH2 produce small and minor transcripts, whose
structures remain to be characterized. For Southern blot analysis, genomic
DNAs (10 lg) cleaved with XbaI were separated and hybridized with the
bHLH2 cDNA probe.

wild-type line FP39, an apparently wild-type plant, FP44,

and the myb1-1 mutant SP1 (Figure 1a). In the mutable ivs
seeds of PR43, the difference in trichome formation can be
clearly seen at the boundary of a pigmented reversion spot
(Figure 1b). Moreover, the surface of the epidermis without trichomes in the dark-brown somatic reversion spot
looks rougher than that in the mutant ivory background;
the former tangential wall appears to be thicker and
protrudes more than the latter, even though the cell sizes
are similar.

To test whether the ivs mutations of I. purpurea also fall into

the bHLH2 gene, we first chose three ivory seed mutants,
PR42, PR43 and WR4, and two control plants, FP39 and SP1,
and examined their bHLH2 transcripts as well as those of
bHLH1, MYB1 and WDR1. The mRNA accumulation of
bHLH2 was drastically reduced in PR42 and WR4, and PR43
produced slightly shorter bHLH2 transcripts than the control
plants, whereas no such obvious differences were detectable in other bHLH1, MYB1 and WDR1 genes (Figure 3a). The
bHLH2 transcripts in the stable ivs-stb mutants, PP45 and
PR46, were subsequently found to be longer than those in
FP39 with the wild-type (Ivs-wt) allele (Figure 3b). Southern
blot analysis of bHLH2 indicated that all five ivs mutants and
FP44 with the phenotypically wild-type (ivs-pwt) allele carry
noticeable DNA rearrangements, and that the three ivs-stb
mutants, WR4, PP45 and PR46, exhibit restriction fragments
of the same size, suggesting the same rearrangement in
bHLH2, named the ivs-stb1 allele, whereas the two mutable
ivs-m lines, PP42 and PP43, carry obviously different alleles,
designated ivs-m1 and ivs-m2, respectively (Figure 3c).
We cloned and sequenced the bHLH2 regions from the
Ivs-wt line FP39, the ivs-m1 plant PR42, and the ivs-stb1
mutant WR4. Because the bHLH2 fragment cloned from WR4
lacked the promoter region due to the presence of an XbaI
site within exon 1, we sequenced the PCR fragment
containing the promoter amplified with the primers IpMF27 and IpM-R6. A comparison of the genomic sequence of
FP39 with its bHLH2 cDNA (AB154369) revealed that bHLH2
consists of eight exons, and that exon 7 contains the bHLH
domain (Figure 4a), which conformed to the bHLH2 gene of
I. tricolor with identical intron positions (Park et al., 2004).
The ivs-m1 line PR42 carries two copies of the 3.9 kbp
transposon Tip100 integrated into bHLH2 exon 7 and
intron 2, whereas the ivs-stb1 line WR4 bears two insertions
of new Mu-related elements, 838 bp IpMu1 and 828 bp

Figure 4. Characterization of the bHLH2 gene and its mutations.

(a) Structure of the wild-type and mutant bHLH2 genes in I. purpurea. The white, gray and black segments within exon boxes indicate the untranslated, coding and
bHLH domain regions, respectively, and the vertical lines marked X represent the XbaI restriction sites. The insertion sites of Tip100, IpMu1, IpMu2 and IpMu3 are
shown above the maps, and the horizontal arrows within the Tip100 boxes represent the transposase gene. The brackets below the maps represent the regions
where PCR-amplified fragments were used for sequencing (see text), and the arrowheads indicate the PCR primers (Table S1).
(b) Semi-quantitative RT-PCR analysis for bHLH2 expression in the Ivs-wt and ivs-pwt lines. The entire coding region of bHLH2 was amplified using primers IpMYC35 and IpM-R1 (Table S1). The constitutively expressed GAPDH2 gene in I. purpurea was used as an internal control, and the numerals in parentheses indicate the
number of cycles of PCR amplification.
(c) Reversion of ivs-m2 in PR43 caused by a somatic excision of Tip100 from bHLH2 exon 7. The upper panel shows the 0.5 kbp fragments amplified with the primers
IpM-F5 and IpMYC3-R1 (Table S1) from a wild-type flower (WT), a pale pigmented background (BG) and a red spot (RS) from the same flower of PR43. The 0.5 kbp
fragments from RS and BG were cloned and sequenced, and their sequences are shown below. The wild-type, somatic reversion and somatic excision alleles are
indicated by Ivs-wt, Ivs-sr and ivs-sx, respectively, and the target site sequence used for Tip100 integration is underlined. The occurrences of the given sequences are
shown in parentheses. The internal control c-sub used is as described in Figure 3.
(d) A phylogenetic tree for bHLH transcription regulators. The tree was constructed as described previously (Fukada-Tanaka et al., 1997). The bootstrap values out of
100 retrials are indicated on each branch, and the scale shows 0.1 amino acid substitutions per site. The abbreviation in front of each protein name indicates the plant
species: Ip, Ipomoea purpurea; In, Ipomoea nil; It, Ipomoea tricolor; Ph, Petunia hybrida; At, Arabidopsis thaliana; Zm, Zea mays; Am, Antirrhinum majus. The
accession numbers for the protein sequences are given here in parentheses: AmDELILA (AAA32663), AtEGL3 (Q9CAD0), AtGL3 (NP_680372), AtTT8 (Q9FT81),
IpbHLH1 (AB252664), IpbHLH2/IpIVS (BAD18982), InbHLH1 (AB232774), InbHLH2/InIVS (AB232775), InbHLH3 (AB232776), ItbHLH2/ItIVS (BAD18984), PhAN1
(AAG25928), PhJAF13 (AAC39455), ZmIN1 (AAB03841), ZmLC (AAA33504) and ZmB-Peru (CAA405449).

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Epidermal traits controlled by bHLH2 645

IpMu2, in exon 2 and intron 5, respectively (Figure 4a).
IpMu1 and IpMu2 contain 150 and 136 bp terminal inverted
repeats flanked by 9 and 10 bp target site duplications,
respectively (Figure S1).

Based on the bHLH2 sequences obtained, we characterized the bHLH2 regions of PR43, FP44, PP45 and PR46
(Figure 4a) using relatively short overlapping PCR fragments
with appropriate primers (Table S1). The ivs-m2 line PR43

(a)

(b)

(d)

(c)

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646 Kyeung-Il Park et al.

contains only one copy of Tip100 in exon 7 and no apparent
footprint of Tip100 in intron 2, suggesting that ivs-m2 might
be a precursor of ivs-m1. Other than the Tip100 insertions,
the only differences detected among the Ivs-wt, ivs-m1 and
ivs-m2 alleles were the lengths of the AT arrays in introns 1
and 2 (AB252659, AB252660 and AB252661); their lengths in
ivs-m1 and ivs-m2 were identical, supporting the notion that
ivs-m1 was derived from ivs-m2. As predicted from Southern blot analysis in Figure 3(c), two other mutants, PP45 and
PR46, also carry the ivs-stb1 allele with IpMu1 and IpMu2
insertions (Figure 4a). Although FP44 with the ivs-pwt
(phenotypically wild-type) allele produces normal purple
flowers and dark-brown seeds (Figure 1a), it carries not only
IpMu2 in intron 5 but also a 1303 bp Mu-related element,
IpMu3, at 66 bp upstream of the transcription start site,
instead of IpMu1 in exon 2. We examined the effects of
these IpMu3 and IpMu2 insertions on bHLH2 expression
using semi-quantitative RT-PCR analysis (Figure 4b). The
bHLH2 mRNA accumulation in FP44 ivs-pwt was less than
that in FP39 Ivs-wt, suggesting that the IpMu3 insertion in
the promoter region affects bHLH2 transcription. In addition
to the transposon insertions, 311 and 31 sequence alterations in the 10.1 kbp genomic and predicted amino acid
sequences, respectively, could be detected between FP39
(AB252659) and FP44 (AB252663). The bHLH2 sequences in
FP39, PR42 and PR43 were very similar, while those in FP44,
PP45, PR46 and WR4 were closely related, suggesting that
FP39 and FP44 (or its progenitor without insertions) are
probably derived from different progenitors.
The ivs phenotypes are due to insertions of DNA transposons into the bHLH2 gene
As we were unable to obtain a germinal revertant among
more than 3000 selfed progeny of PR43, we examined
whether the flower variegation of PR43 ivs-m2 is due to the
somatic excision of Tip100 from bHLH2 exon 7 by analyzing
a somatic reversion event. DNA from a rare but relatively
large spot and the pale-pigmented background of the same
flower were amplified using the primers IpM-F5 and IpMYC3-R1 (Figure 4a). The 0.5 kbp fragments that did not
contain Tip100 were much more easily amplified from the
red spot than from the background, and all fragments
examined from the red spot contained the precisely excised
sequence, whereas both frameshifts and the precisely excised sequence could be found in the rarely amplified fragments from the background (Figure 4c). We conclude that
flower variegation in the ivs-m2 mutant is due to the somatic
excision of Tip100 from bHLH2 exon 7. The bHLH2 transcripts were drastically reduced in PR42, whereas those in
PR43 were slightly shorter than the wild-type transcripts
(Figure 3a) and could encode a truncated bHLH2 protein.
Northern blot hybridization using various exon regions as
probes confirmed that the Tip100 insertion within intron 2 in

PR42 affects mRNA accumulation, and that virtually no

transcripts hybridizable with the 3 half of exon 7 are produced in PR42 (Figure S1). The flower and seed variegations
in PR43 with a single Tip100 insertion in bHLH2 tend to be
greater than those in PR42 with double Tip100 insertions in
bHLH2, whereas the flower pigmentation in PR43 is paler
than that in PR42 (Figure 1a).
The notion that I. purpurea mutants displaying pale pigmented flowers and ivory seeds are deficient in bHLH2 was
further supported by the fact that the bHLH2 transcripts in
both PP45 and PR46, which contained IpMu1 and IpMu2
insertions (Figure 4a) and showed longer but slightly
reduced bHLH2 transcripts with additional discrete bands
(Figure 3b), were found to comprise at least four differently
spliced mRNAs in the IpMu1intron 2 region and that the
major transcripts contained the entire IpMu1 sequence
(Figure S1). As these four differently spliced transcripts all
contain the TGA stop codon near the 5 terminus of the
inserted IpMu1, the IpMu1 insertion results in a null mutation;
the slight reduction of the bHLH2 transcripts in PP45 and PR46
may be due to nonsense-mediated mRNA decay. (Belostotsky and Rose, 2005). While bHLH2 expression decreased only
partially in the myb1-1 mutant SP1, a drastic reduction was
observed in the ivs-stb1 myb1-1 double-mutant WR4
(Figure 3a); the results were further confirmed by Northern
blot analysis using various exon regions as probes
(Figure S1). Based on these results, we conclude that I. purpurea null mutations in bHLH2 affect pigmentation more
severely in seeds than in flowers (Figure 1a). A phylogenetic
tree indicated that bHLH2 (or IpbHLH2/IpIVS) in I. purpurea
and the corresponding bHLH2 proteins in I. nil and I. tricolor
are most closely related to AN1 in petunia (Figure 4d).
Expression of the genes for anthocyanin pigmentation in
flower buds
To assess the effects of the ivs and myb1-1 mutations on
expression of the structural genes encoding enzymes for
anthocyanin biosynthesis (Figure 2), Northern blot analysis
was performed in young flower buds of FP39 Ivs-wt, PR42
ivs-m1, PR43 ivs-m2, WR4 ivs-stb1 myb1-1 and SP1 myb1-1
plants. The analysis included CHS-E, which is responsible
for pigmentation mainly in flower tubes (Durbin et al., 2000;
Fukada-Tanaka et al., 1997; Johzuka-Hisatomi et al., 1999),
and GST, which encodes glutathione S-transferase, a
homologue of petunia AN9 that is believed to be involved in
escorting anthocyanin pigments into the vacuole (Alfenito
et al., 1998). As shown in Figure 5, only a partial reduction of
all structural genes tested was observed in PR42 and PR43
plants exhibiting pale flowers (Figure 1a). However, no
apparent reduction of GST was found in these ivs mutants,
whereas virtually no GST expression was detectable in SP1
myb1-1 (Figure 5). In the myb1-1 mutant, we also noticed a
modest reduction of CHS-D and CHI for flavone biosynthe-

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Epidermal traits controlled by bHLH2 647

(a)

(b)

Figure 5. Northern blot analysis of genes for anthocyanin pigmentation.

Total RNAs (10 lg) prepared from petals of flower buds at 36 h before flower
opening were hybridized with appropriate cDNA probes as indicated.

sis, a slightly greater reduction of F3H for flavonol synthesis,

and a much more drastic reduction of the remaining LBGs
for anthocyanin biosynthesis. The transcripts of each corresponding structural gene in the ivs-stb1 myb1-1 double
mutant were reduced compared with those of the myb1-1
mutant.

(c)

Expression profiles of the genes for proanthocyanidin

synthesis in seed coats
Proanthocyanidins, or condensed tannins, are colorless flavan-3-ol polymers that oxidize upon desiccation to yield
brown pigments in the seed coats of many plants (Marles
et al., 2003; Tanner, 2004). To examine whether the darkbrown Ivs-wt and myb1-1 seeds as well as the ivory ivs-m1
and ivs-stb1 myb1-1 seeds contain proanthocyanidins, we
used a vanillin-HCl assay, which produces reddish vanillin
proanthocyanidin adducts (Debeaujon et al., 2000; Downie
et al., 2003), and a p-dimethylaminocinnamaldehyde
(DMACA) treatment, which results in blue staining of proanthocyanidins (Abrahams et al., 2002; Marles et al., 2003).
Green immature seeds at 18 days after pollination (DAP)
were removed from the carpels and treated for staining. The
vanillin-HCl and DMACA solutions immersed with the FP39
and SP1 seeds became pink and blue, whereas the solutions
with the PR42 and WR4 seeds did not show any change
(Figure 6a), indicating that proanthocyanidins were present
in the dark-brown Ivs-wt and myb1-1 seeds but absent in the
ivory ivs-m1 and ivs-stb1 myb1-1 seeds. Similarly to the
Arabidopsis TT8 gene encoding a bHLH protein (Nesi et al.,
2000), the bHLH2 gene is apparently required for proanthocyanidin accumulation in the seed coats. The proanthocyanidins are presumed to have eluted into the solutions due

Figure 6. Detection of proanthocyanidins and RT-PCR analysis of genes for

proanthocyanidin biosynthesis in seed coats.
(a) Vanillin and DMACA assays for immature seeds at 18 DAP.
(b) Histochemical observation of proanthocyanidins at 15 DAP in the seed
coats. OE, outer epidermis; OH, outer hypodermis; PL, palisade layers; M,
mesophyll.
(c) RT-PCR analysis. Seed coats were harvested at the indicated days after
pollination, and F below FP39 indicates a sample from flowers. The GAPDH2
gene (Figure 4b) was used as an internal control, and 30 cycles of PCR
amplification were performed for RT-PCR analysis.

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648 Kyeung-Il Park et al.

to accumulation of proanthocyanidins in the two outer layers, the outer epidermis and the outer hypodermis (Figure 6b), which must be susceptible to elution. In contrast,
proanthocyanidins accumulate in the innermost integument
layers of Arabidopsis and tomato (Lycopersicon
esculentum), which show distinctive vanillin-stained layers
(Debeaujon et al., 2003; Downie et al., 2003).
As most structural genes in the biosynthesis pathway of
anthocyanins are in common with that of proanthocyanidins
(Figure 2), we analyzed the expression of the structural
genes and their regulatory genes in the seed coats using RTPCR (Figure 6c). The temporal expression profiles of the
genes in FP39 Ivs-wt showed that the accumulated transcripts of bHLH2, bHLH1, MYB1 and WDR1 regulatory genes
are the highest at 12, 6, 18 and 24 DAP, respectively, whereas
those of most structural genes examined are the highest at
18 DAP. Thus, the bHLH2 expression appears to precede the
expression of these structural genes. The results also
showed that CHS-E rather than CHS-D is predominantly
expressed in the seed coats. As both CHS-D and CHS-E of
I. purpurea were shown to produce active enzymes
(Shiokawa et al., 2000), CHS-E must be involved in the first
committed step of the proanthocyanidin biosynthesis
pathway. Of the LBGs, only DFR-B and ANS were completely
diminished in the ivs mutants, while no apparent reduction
of the EBGS for flavonal biosynthesis, CHS-E, CHI and F3H,
were observed (Figure 6c). The results demonstrate that
bHLH2 is absolutely required for proanthocyanidin
biosynthesis in the seed coats, while bHLH2 only partially
controls flower anthocyanin pigmentation.
Phytomelanin pigment accumulation in seed coats
The black pigments of I. purpurea seed coats have been
reported to be phytomelanins (Nicolaus and Piattelli, 1965).
Indeed, the pigment was highly stable in acidic solutions,
slightly soluble in dimethyl sulfoxide (DMSO), bleachable
with H2O2 or NaOCl and extractable in alkaline solution, and
the crude dark-brown materials were precipitated when the
alkaline solution was adjusted to pH 2 by adding HCL (data
not shown), which are characteristics of melanins in fungi
and plants (Downie et al., 2003; Fogarty and Tobin, 1996;
Harki et al., 1997; Sava et al., 2001). For further characterization, the pigments from the wild-type dark-brown seeds were
extracted with NaOH and subjected to ultraviolet (UV) and
infrared (IR) spectrometric analyses. The UV spectrum of the
pigments showed the characteristics of phytomelanins, with
a typical shoulder at about 280 nm (Harki et al., 1997; Sava
et al., 2001) (data not shown), and the IR spectrum was nearly
identical to that from sunflower (Helianthus annuus)
(Figure 7a), which was used as an authentic phytomelanin by
Downie et al. (2003). Although phytomelanins were also
present in the ivory ivs seed coats, their levels were only
about 8% of those of the wild-type seeds (Figure 7b),

indicating that the bHLH2 gene also controls the biosynthesis

and/or accumulation of phytomelanins in the seed coats.
Phytomelanins accumulated mostly in the outer epidermis
and palisade layers of the Ivs-wt seeds, and the light-brown
pigment in the outer epidermis of the PR43 ivs-m2 seeds
might be residual phytomelanins (Figure 7c). The outermost
epidermal layer, in which phytomelanins accumulated
extensively, appeared to overlap with the proanthocyanidinaccumulating layers (Figure 6b). At the boundary of a large
pigmented reversion spot, generated by the somatic excision
of Tip100 from bHLH2 exon 7 in a PR43 ivs-m2 seed
(Figure 7d, upper panel), phytomelanin pigments appear to
diffuse from a somatically reverted dark-brown area to a nonreverted ivory area, whereas trichome formation coincides
with the dark-brown reversion spot (data not shown). Such a
view is strengthened by a close inspection of the boundary,
which clearly shows a gradation of phytomelanin deposition
in the outer epidermis and palisade layers (Figure 7d, lower
panel), probably because the precursors of phytomelanins
can migrate from synthesized cells into the space where
phytomelanins are polymerized (Pandey et al., 1989).
Therefore, in addition to trichome formation and proanthocyanidin pigmentation, phytomelanin production is another
epidermal trait that is controlled by the transcriptional regulator bHLH2 in I. purpurea seed coats.
Development of epidermal cells and trichomes in seed coats
We examined the temporal development of epidermal cells
and trichomes in the wild-type FP39 seed coats (Figure 8).
While neither apparent trichome development nor thickening
of tangential walls could be detected at 6 DAP, both trichome
formation and protruding of thickened tangential walls were
observed at around 12 DAP. The development of unbranched
trichomes was clearly seen at 18 DAP, and their pigmentation
was detectable at 24 DAP. As in the case of the trichomes of
cotton (Gossypium hirsutum) seeds (Kim and Triplett, 2001),
DAPI staining of I. purpurea seed coats at 18 DAP showed
that all trichomes are unicellular. While the dark-brown
pigments detected at 24 DAP look like phytomelanins, they
must also contain proanthocyanidin pigments because
proanthocyanidin accumulation has been detected at
18 DAP, when the structural genes for proanthocyanidin
biosynthesis are most abundantly expressed (Figure 6).
Discussion
We show here that a number of spontaneous ivs mutations
of I. purpurea conferring pale flowers and ivory seeds are
caused by insertions of DNA transposons into bHLH2; the
mutable ivs-m and stable ivs-stb alleles contain Tip100 and
IpMu elements, respectively. The expression of all
structural genes for anthocyanin biosynthesis is partially
reduced in the ivs mutants, whereas no such reduction of

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Epidermal traits controlled by bHLH2 649

(a)

(b)

(c)

(d)

the gene encoding GST was observed (Figure 5). In the

myb1-1 mutant, expression of the F3H gene (an EBG for
flavonol biosynthesis) appears to be reduced more than

Figure 7. Phytomelanins accumulated in Ivs-wt and ivs seed coats.

(a) IR spectra of phytomelanins. Phytomelanins extracted from sunflower and
synthetic melanin purchased from Sigma-Aldrich Co. (http://www.sigmaaldrich.com/) were used as control samples.
(b) Phytomelanin contents in I. purpurea seed coats. The phytomelanin
content was expressed as the mg g)1 fresh weight of seeds.
(c) Histological observation of seed epidermal layers. OE, outer epidermis;
OH, outer hypodermis; PL, palisade layers; ES, endosperm.
(d) Magnified view (upper panel) and histological observation (lower panel) of
the boundary region of a dark-brown reversion spot in the seed coats of the
mutable PR43 ivs-m2 line. The filled and open arrows with PS and NPB or grey
broken bars indicate a pigmented spot and non-pigmented background or a
pigment-diffused area(s) on the seed coat, respectively.
Scale bars 100 lm.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 8. Seed trichome development of the wild-type line FP39.

(a,b,d,f) Microscopic view of the surface of developing seed coats. The black
arrowheads in (b) indicate the protruding corners of thickened tangential
walls.
(c) Scanning electron micrograph.
(e) Fluorescence micrograph of (d) stained with DAPI.
Scale bars 50 lm.

other modestly reduced EBGs for flavone biosynthesis, and

LBGs for anthocyanin biosynthesis decreased more drastically. A plausible explanation is that bHLH2 controls
partially, and MYB1 more strictly, all the structural genes
for anthocyanin biosynthesis in a coordinated manner, and
that EBGs must be partially activated by another controlling system(s) for flavone and probably flavonol biosynthesis. It remains to be studied whether bHLH1, in addition
to bHLH2, has partial control of flower pigmentation. The
flower coloration of PR43, which can produce a longer
truncated bHLH2 protein, is paler than that of PR42, PR45
and PR46, all of which carry null mutations in bHLH2
(Figures 1a and 3a). A possible explanation is that the
putative longer truncated bHLH2 protein interferes partially
with the interaction of bHLH1 with R2R3-MYB and WDR
transcription factors. In the Japanese morning glory (I. nil),
almost all structural genes were found to be reduced in a
coordinated manner in white-flowered mutants deficient in
either MYB1 or WDR1. However, a slight but significant
expression of the CHS-D, CHI and F3H genes for flavonol
biosynthesis was detectable in these mutants, whereas no

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650 Kyeung-Il Park et al.

such residual expression could be observed for LBGs for
anthocyanin biosynthesis in the flowers (Morita et al.,
2006). As MYB12 in Arabidopsis has been shown to be a
flavonol-specific transcriptional regulator in seedlings
(Mehrtens et al., 2005), it is conceivable that a similar MYB
protein(s) also modestly activates EBGs in morning glories.
Because spotted pigmentation occurs in each ray of
I. purpurea flowers in the absence of MYB1 activity, as
observed in the myb1-1 plant (Figure 1a), it remains to be
examined whether another MYB protein also controls the
pigmented area.
We also show here that the production of proanthocyanidins and phytomelanin pigments is reduced in the ivs
mutant seed coats, which must be the main reason for the
production of ivory seeds. For proanthocyanidin biosynthesis, no transcripts of DFR-B and ANS (belonging to the
LBG group) were detectable in the seed coats of the ivs
mutants, while EBGs for flavonol synthesis (CHS-E, CHI
and F3H) were expressed at 18 DAP (Figure 6c). The results
for proanthocyanidin biosynthesis in seed coats clearly
contrast with those for anthocyanin production in flowers
(Figures 5 and 6c). The expression of DFR-B and ANS was
only modestly reduced in flowers, whereas their transcripts in seed coats were completely abolished. In addition, only CHS-E was expressed in seed coats, while CHS-D
and CHS-E are known to be mainly responsible for
anthocyanin pigmentation in flower limbs and tubes,
respectively (Habu et al., 1998; Johzuka-Hisatomi et al.,
1999). As Figure 2 shows, there are two alternative pathways that lead to proanthocyanidins: (a) leucoanthocyanidins are converted into 2,3-trans-flavon-3-ols by
leucoanthocyanidin reductase (LAR), and (b) anthocyanidins are converted into 2,3-cis-flavon-3-ols by anthocyanidin reductase (ANR) (Marles et al., 2003; Tanner, 2004).
Because the ANS gene was extensively expressed (Figure 6c), it is likely that flavan-3-ols are produced from
leucoanthocyanidins via two steps mediated by ANS
followed by ANR (Xie et al., 2003). The 3GT gene, which
promotes the production of anthocyanidin 3-O-glucosides,
the first major stable colored anthocyanin pigments in the
biosynthesis pathway (Figure 2), appeared not to be
expressed in the seed coats (Figure 6c). However, small
amounts of 3GT transcripts could be detected after extended PCR amplification with 35 cycles (data not shown),
suggesting that a minute amount of anthocyanins might
be present in the seed coats.
We show that phytomelanins accumulate mainly in the
outer epidermis and palisade layers of the wild-type I. purpurea seed coats, and that the level of phytomelanin pigments accumulated in the seed coats of the ivs mutants was
about 8% of the wild-type or myb1-1 level (Figure 7). The
results indicate that the biosynthesis and/or accumulation of
seed phytomelanins are controlled by bHLH2, even though
both the chemical nature and biosynthetic pathway of

phytomelanins remain largely unknown. The seeds of

I. purpurea are covered with unbranched and unicellular
trichomes that start to develop at around 12 DAP and turn
dark-brown at 24 DAP (Figure 8), and both proanthocyanidins and phytomelanins seem to accumulate in these
trichomes. Interestingly, neither phytomelanin accumulation nor trichome formation was completely abolished in the
ivs mutants (Figures 1a and 7b). It remains to be studied
whether such residual activities in the seed epidermal traits
are related to bHLH1 expression in the seed coats (Figure 6c). A reduction of proanthocyanidin and phytomelanin
pigments (K.-I. Park, unpublished data) as well as trichomes
(Morita et al., 2006) was also found in the whitish seeds of
I. nil mutants defective in the WDR1 gene. As most of the
structural genes for anthocyanin biosynthesis were coordinately reduced in the same I. nil mutants deficient in WDR1
and exhibiting white flowers, both bHLH2 and WDR1
transcriptional regulators in Ipomoea must control the
biosynthesis and/or accumulation of anthocyanin, proanthocyanidin and phytomelanin pigments as well as the
formation of seed trichomes.
In petunia, both an1 and an11 mutations in bHLH2 and
WDR, respectively, confer white flowers and fragile
yellowish seed coats with an increased number of smaller
cells, whereas the an2 mutation in MYB gives white
flowers and a wild-type morphology of the seed coats
(Spelt et al., 2002). For anthocyanin biosynthesis in
flowers, only InMYB1/C-1 and IpMYB1/W genes of I. nil
and I. purpurea respectively, have been identified as
R2R3-MYB factors in Ipomoea (Chang et al., 2005; Morita
et al., 2006), and these mutants produce normal darkbrown seeds covered with trichomes (Figure 1a; see also
Morita et al., 2006). Thus, these MYB1 genes exert neither
pigmentation nor trichome formation in the seeds, even
though MYB1 was found to be expressed slightly at
18 DAP in the seed coats (Figure 6c). With respect to seed
epidermal traits, Arabidopsis TT2 and cotton GaMYB2
have been shown to be responsible for proanthocyanidin
production and trichome formation, respectively (Nesi
et al., 2001; Wang et al., 2004). The identification and
characterization of Ipomoea R2R3-MYB genes for the seed
epidermal traits described here must await further investigation.
The vacuolar pH of flower limbs in I. nil and I. tricolor
increases during flower opening, and the NHX genes for
Na/H antiporters are responsible for vacuolar alkalization
(Fukada-Tanaka et al., 2000; Ohnishi et al., 2005; Yamaguchi
et al., 2001; Yoshida et al., 1995), whereas the vacuolar pH
decreases in petunia petals (Spelt et al., 2002). While AN1,
AN2 and AN11 in petunia are involved in acidification of the
vacuoles (Koes et al., 2005), neither the vacuolar pH nor the
expression of NHX1 as the major Na/H antiporter were
affected in the I. nil c-1 and ca mutants deficient in MYB1 and
WDR1, respectively (Morita et al., 2006; Yamaguchi et al.,

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Epidermal traits controlled by bHLH2 651

2001), or in the I. tricolor ivs mutant lacking bHLH2 activity
(Park et al., 2004). Likewise, the I. purpurea ivs mutations
appeared to have no effect on the alkalization of the vacuoles
(K.-I. Park, unpublished data). The ways in which the
regulation and processes for these epidermal traits are
similar or divergent at the molecular level in different
species are intriguing, and answers to these questions
would certainly shed light on the evolution of flowering
plants. The findings described here validate use of the
morning glories as model plants offering experimental
versatility with regard to gene regulation in pigmentation
and related research areas.

Experimental procedures
Plant materials
The I. purpurea lines YO/FP-39, used as a wild-type (Habu et al.,
1998), KK/WRSP-1 carrying the pink and myb1-1 (or white) alleles
(Chang et al., 2005; Hoshino et al., 2003), and YJH/WR-4 containing
the pink and stable ivs-stb1 alleles are from our collection and are
designated here as FP39, SP1 and WR4, respectively (Figure 1a). AH/
PR-42 (PR42) carrying the pink and mutable ivs-m1 alleles, and AH/
PR-43 (PR43) containing the pink and mutable ivs-m2 alleles, were
isolated from descendants of the selfed progeny of a germinal
revertant isolated from a plant with the mutable flaked allele (Habu
et al., 1998) which also bears the homozygous pink and heterozygous ivs-m1/ivs-m2 alleles. Among the selfed progeny of a phenotypically wild-type plant (Pink/pink, ivs-pwt/ivs-stb1), three different
segregants were isolated: PKI/FP-44 (FP44) carrying the wild-type
Pink and silent mutation ivs-pwt (phenotypically wild-type) alleles
and exhibiting dark-purple flowers and dark-brown seeds, PKI/PP-45
(PP45) bearing the Pink and ivs-stb1 alleles and producing palepurple flowers and ivory seeds, and PKI/PR-46 (PR46) containing the
pink and ivs-stb1 alleles and displaying pale-reddish flowers and
ivory seeds (Figure 1a).

Nucleic acid procedures

General nucleic acid procedures, including the preparation of DNAs
from young leaves and RNAs from flower buds at 36 h before flower
opening, Northern and Southern blot analyses, and PCR and
sequencing analyses, were performed as described previously
(Hoshino et al., 2003; Park et al., 2004). Ipomoea purpurea cDNAs
(CHS-D, CHS-E, DFR-B, 3GT, 3GGT, MYB1, bHLH2 and WDR1) and
I. nil cDNAs (CHI, F3H and ANS), used as hybridization probes, have
been described previously (Fukada-Tanaka et al., 1997; Hoshino
et al., 2001; Morita et al., 2006; Park et al., 2004). We obtained the
GST and bHLH1 cDNA clones of I. purpurea by screening an FP39
cDNA library with the petunia AN9 and JAF13 cDNA probes,
respectively (Alfenito et al., 1998; Morita et al., 2006; Quattrocchio
et al., 1998). The presence of Tip201 in the pink mutation was
determined as described by Hoshino et al. (2003). To examine
whether plants carry the myb1-1 allele, the PCR fragments amplified
with the primers IpMYB1-F1 and IpMYB1-R1 (Table S1) were sequenced. The mRNA accumulation quantified by Northern blot
hybridization was performed as described by Park et al. (2004). For
RT-PCR analysis, total RNAs were prepared from flower buds or
immature seed coats using a Get pureRNA Kit (Dojindo Molecular
Technologies; http://www.dojindo.com). First-strand cDNAs were

synthesized using the SuperScript III reverse transcriptase kit

(Invitrogen, http://www.invitrogen.com/), and the transcripts of
GAPDH2 for glyceraldehyde 3-phosphate dehydrogenase 2 were
used as an internal control for normalizing the levels of the transcripts to be examined. The expression level was estimated semiquantitatively by altering the number of cycles of PCR amplification
with appropriate primers (Table S1).

Characterization of genomic bHLH sequences

To characterize the genomic bHLH2 regions, the 12 kbp XbaI
fragment containing bHLH2 from FP39 Ivs-wt and the 8.6 kbp XbaI
fragment from WR4 ivs-stb1 myb1-1 were cloned into kDASH II
and kZAP Express vectors (Stratagene, http://www.stratagene.com/), respectively, as described by Park et al. (2004). As
bHLH2 exon 1 in WR4 carries an additional XbaI site and the
8.6 kbp XbaI fragment contains no promoter region, a 2.5 kbp
promoter segment was amplified by PCR using the primers IpMF27 and IpM-R6 (Table S1) for sequencing the promoter region
(Figure 4a). Because the inserted Tip100 element carries an XbaI
site, the bHLH gene region of PR42 produced three XbaI fragments of 8.1, 6.5 and 5.1 kbp (Figures 3c and 4a), which were
cloned into the kZAP Express vector. To determine the genomic
bHLH2 regions in PR43, PP45, PR46 and FP44 (Figure 4a), we
sequenced overlapping PCR-amplified fragments with appropriate
primers (Table S1). To compare the coding regions of FP39 Ivs-wt
and FP44 ivs-pwt, we sequenced 2.1 kbp cDNAs prepared by RTPCR amplification with the primers IpMYC3-5 and IpM-R1 (Figure 4a, Table S1), which correspond to the 5 and 3 UTRs
adjacent to the coding region. To determine footprints generated
by the excision of Tip100 from bHLH2 exon 7, 0.5 kbp PCRamplified fragments with the primers IpM-F5 and IpMYC3-R1
(Table S1) were sequenced (Figure 4c).

Characterization of proanthocyanidin and phytomelanin

pigments and microscopic observation of epidermis
traits in seeds
To detect proanthocyanidins, seeds were harvested at 18 DAP and
soaked in a 2.5% vanillin solution (MeOH:HCl 2:1 v/v) or a 0.075%
DMACA solution (MeOH:HCl 2:1 v/v) at room temperature
for 10 min (Abrahams et al., 2002; Debeaujon et al., 2000). For
histochemical observation, immature seeds at 15 DAP were sectioned on a Vibratome DSK microslicer DTK-3000W (Dousaka,
http://www.kit.gr.jp/dousaka/index.html), and 50 lm sections were
placed in 0.5 N NaOH on glass slides. The sections were then
washed with distilled water, stained using solutions with or without
vanillin for 30 min, and observed using an Olympus BH2-RFCA
microscope (http://www.olympus-global.com/).
The phytomelanin pigments were extracted from the wild-type,
myb1-1 and ivs mutants of I. purpurea as well as from sunflower in
5 ml of 0.5 N NaOH at around 25C for 1 h, and purified using the
method described by Harki et al. (1997). IR spectra of the extracts
were recorded with a HORIBA FT-730 IR spectrometer (HORIBA,
http://www.jp.horiba.com) using KBr pellets. For histological observation, seeds were harvested before complete hardening and
sectioned on a Vibratome DSK microslicer DTK-3000W. The 25 lm
sections were placed on glass slides, mounted in Entellan New
(Merck, http://www.merck.de) and observed using a Nomarski
differential interference contrast microscope DMRX E (Leica Microsystems, http://www.leica-mycrosystems.com).
To view seed epidermal development, the seed coats were
removed from seeds at 6, 12, 18 and 24 DAP, and seed coats at

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652 Kyeung-Il Park et al.

18 DAP were stained with DAPI (1 lg ml)1) in a PBST buffer (1%
Tween-20 in PBS, v/v) for 15 min after being fixed in 3% glutaraldehyde in PBST for 3 h. All seed coat samples were mounted in a
70% glycerol/PBST solution (1:1 v/v) and observed with the same
Normarski microscope. To obtain scanning electron microscopic
photographs of the seed epidermis, immature and mature seeds
were viewed using a VE-8800 digital scanning electron microscope
(Keyence http://www.keyence.com).

Acknowledgements
We thank Miwako Matsumoto and Kyoko Ikegaya for technical
assistance, Yasuyo Johzuka-Hisatomi for her initial characterization of WR4 and invaluable discussion, Eisho Nishino (Laboratory
of Plant Morphology, Faculty of Horticulture, Chiba University,
Japan) for enlightening us with regard to seed coat structures and
providing valuable comments on the manuscript, Norio Saito
(Chemical Laboratory, Meiji-gakuin University, Japan) for discussion, and an anonymous reviewer for kindly editing the previous
version of our manuscript. We also thank Mark D. Rausher
(Department of Biology, Duke University, USA), Francesca Quattrocchio and Ronald Koes (Department of Developmental Genetics, Institute for Molecular Biological Sciences, Vrije Universiteit,
The Netherlands) for providing cDNA probes. The IR spectra and
scanning electron micrographs were taken at the Center for
Analytical Instruments in the National Institute for Basic Biology,
and at the Research Center for Molecular Materials in the Institute
for Molecular Science, respectively. This work was supported by
grants from the Ministry of Education, Culture, Sports, Science
and Technology of Japan. K.-I.P. and J.-D.C. were recipients of a
fellowship awarded by the Japan Society for the Promotion of
Science for Foreign Researchers.

Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Molecular analyses of bHLH2 and its mutations.
(a) Structure of bHLH2.
(b) Northern blot analysis of bHLH2 transcripts.
(c) Schematic representation of alternative splicing patterns detected in the IpMu1-intron2 region.
(d) Alignment of the IpMu termini.
Table S1 List of primers
This material is available as part of the online article from http://
www.blackwell-synergy.com

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Accession numbers: The nucleotide sequences have been deposited to DNA Data Bank Of Japan (DDBJ) under the accession
numbers AB252659 (Ivs-wt in FP39), AB252660 (ivs-m1 in PR42), AB252661 (ivs-m2 in PR43), AB252662 (ivs-stb1 in WR4),
AB252663 (ivs-pwt in FP44), and AB252664 (IpbHLH1 cDNA from FP39).

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Journal compilation 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 641654