Isolation of a Regulatory Gene of Anthocyanin
Biosynthesis in Tuberous Roots of Purple-Fleshed
Sweet Potato[OA]
Hironori Mano*, Fumiaki Ogasawara, Kazuhito Sato, Hiromi Higo, and Yuzo Minobe
Plant Genome Center, Tsukuba, Ibaraki 305–0856, Japan
Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been
identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory
elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1
and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed
in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as
nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but
not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained
anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that
IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable
transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin
genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.
Sweet potato (Ipomoea batatas L. Lam.) ranks as the
world’s seventh most important crop after wheat
(Triticum aestivum), rice (Oryza sativa), maize (Zea
mays), potato (Solanum tuberosum), barley (Hordeum
vulgare), and cassava (Manihot esculenta Crantz.). Most
sweet potato cultivars, like the other major crops that
are grown and consumed, have white or yellow flesh.
However, there are also some sweet potato cultivars
with orange flesh that contains carotenoids or with
purple flesh that contains anthocyanins.
Anthocyanins and related compounds such as
proanthocyanidins protect the leaves from various
stressors such as strong light and heavy metals (for
review, see Gould, 2004), play a role in entomophily in
flowers, and deter pathogens and predators of seeds
(Shirley, 1998). The role of anthocyanins in underground organs (such as tuberous roots) that grow under dark conditions is not clear, but it is conceivable
that it is similar to that in seeds (e.g. protection from
pathogens and predators and improvement of preservation and thus reproductive advantage), because
tubers, like seeds, play a reproductive role.
The reason why most sweet potato cultivars have
white or yellow flesh may be because consumers pre* Corresponding author; e-mail [email protected]; fax 81–29–
839–4829.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Hironori Mano ([email protected]).
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.106.094425
1252
fer pale foods, as is the case with other staples, and
because the anthocyanins in the tuber are an obstacle
to industrial starch production. Nevertheless, attention
is now being focused on the multiple physiological
functions of the anthocyanins derived from purplefleshed sweet potatoes, such as their strong antioxidative activity (Kano et al., 2005), antimutagenicity
(Yoshimoto et al., 1999b), antihyperglycemic effect
(Matsui et al., 2002), and hepatoprotective and antihypertensive effects (Suda et al., 2003). New purplefleshed cultivars have been developed recently. The
content of anthocyanins in indigenous purple-fleshed
cultivars was low, and the development of a new purplefleshed cultivar, Ayamurasaki (Yamakawa et al., 1997),
enabled edible dye production from sweet potato.
Trials have been done on the efficient and stable
production of anthocyanins from tissue or cell culture of various plant species, including sweet potato.
For example, optimum conditions for the culture of
anthocyanin-producing sweet potato cells (Nishimaki
and Nozue, 1985; Nozue et al., 1987) or Ayamurasaki
hairy roots (Nishiyama and Yamakawa, 2004) have
been investigated. However, low anthocyanin content
and the light dependency of anthocyanin production
have been noted as problems. Konczak-Islam et al.
(2000) obtained a cell line that had high anthocyanin
content and was derived from the tuberous roots of
Ayamurasaki. This line showed light-independent anthocyanin production and may prove useful in the
improvement of anthocyanin productivity, although
the regulatory elements of anthocyanin biosynthesis
are still unknown.
The mechanisms regulating flavonoid pigments
have been studied in various plant species. Detailed
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
studies have been done in maize, Arabidopsis (Arabidopsis thaliana), and petunia (Petunia hybrida) by numerous mutation analyses, and many regulatory genes
have been identified that control the transcription of
flavonoid structural genes. Transcriptional factors with
MYB or basic helix-loop-helix (bHLH) domains and a
WD40 protein are commonly identified in different
species (for review, see Broun, 2004; Koes et al., 2005).
Although a model has been proposed that these regulators interact with each other and make transcription complexes with the promoters of the structural
genes, the partnership seems to be complex. For example, MYB domain C1 protein regulating the anthocyanin pathway in maize needs a bHLH partner (B/R)
to activate the flavonoid structural gene dihydroflavonol reductase (DFR) promoter, whereas MYB domain
P protein regulating the phlobaphene pathway can
activate the promoter without a bHLH partner (Sainz
et al., 1997). Moreover, other regulatory genes, such as
TTG2, a WRKY transcription factor (Johnson et al.,
2002), TT1, a zinc finger protein (Sagasser et al., 2002),
TT16, a MADS domain protein (Nesi et al., 2002), and
ANL2, a homeodomain (HD) protein (Kubo et al.,
1999), have been reported. Some of these genes are
involved in various aspects of plant development besides flavonoid biosynthesis, including development
of the trichome and root hair (TTG2, WRKY; TTG1,
WD40; GL3 and EGL3, bHLH; Zhang et al., 2003), root
(ANL2, HD), and seed (TT1, zinc finger; TT16, MADS;
AN1, bHLH; AN11, WD40; Spelt et al., 2002), acidification of vacuoles (AN1 and AN11), or mucilage formation (TTG2, TTG1, GL3, and EGL3).
The genes responsible for the color differences
among cultivars have been revealed. One amino acid
alternation in bHLH protein created the green formae
of perilla from red formae (Gong et al., 1999), and a
retrotransposon-induced mutation on the promoter
of the MYB gene created white-skinned grape (Vitis
vinifera) cultivars from red-skinned ones (Kobayashi
et al., 2004). In the case of rice, red or brown grain was
selected out by ancient breeders. Two loci, Rc and Rd,
that are responsible for red seed color were identified
by classical genetic analysis (Kato and Ishikawa, 1921),
and recently they were revealed to encode the bHLH
and DFR genes, respectively (Sweeney et al., 2006;
Furukawa et al., 2007).
As mentioned above, numerous reports have described the genes regulating flavonoid pigmentation in
flowers, leaves, seeds, and fruits but not in underground organs such as tuberous roots. In sweet potato,
no MYB, bHLH, or WD40 protein has been reported
thus far; instead, a MADS-box gene, IbMADS10, was
recently isolated and suggested to be involved in
anthocyanin pigmentation (Lalusin et al., 2006), although its involvement in the underground organ is
unclear.
We report here the isolation of a new R2R3-type
MYB gene, IbMYB1, from a purple-fleshed sweet potato cDNA library and its predominant expression in
the tuberous roots of purple-fleshed cultivars. Tran-
sient and stable forced expression of the IbMYB1 gene
resulted in intense anthocyanin pigmentation in various tissues. These results suggest that the IbMYB1
gene is responsible for purple pigmentation in the
flesh of tuberous roots of sweet potato.
RESULTS
Isolation of cDNA Clones with Tissue- and
Cultivar-Specific Expression
We constructed cDNA libraries from the tuberous roots of the purple-fleshed sweet potato cultivar
Ayamurasaki and the yellow-fleshed sweet potato cultivar Kokei-14, and we determined the sequences of 3,783
randomly isolated cDNA clones from Ayamurasaki and
2,804 from Kokei-14. The cDNA clones were clustered,
and 25 sequence groups were selected according to
their frequency of appearance in the libraries and/or
specificity to the Ayamurasaki library. The 25 selected
groups were subjected to reverse transcription (RT)PCR analysis, and the results indicated that five
groups, IT1, IT4, IT4785, IT666, and IT4097, were
expressed in a tissue-specific manner (Fig. 1). Whereas
IT948 was identical to the Actin gene and was expressed equally in all tissues tested, IT1 and IT4 were
expressed predominantly in tuberous-root tissue and
IT4097 was expressed in the tuberous roots and the
calli. IT4785 and IT666 were expressed in a cultivarspecific manner; that is, they were expressed only in
Figure 1. Genomic PCR and RT-PCR performed on five clones isolated
from a cDNA library derived from sweet potato tuberous roots revealed
tissue- and/or cultivar-specific expression. Amplified products (30
cycles) were size fractionated on a 1.0% agarose gel. Names of clones
are indicated at left and identities of the clones at right. A, Ayamurasaki;
K, Koukei-14.
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Mano et al.
probes and only a small number of signals in the case
of the EcoRV and HindIII digests, one would expect a
small number of loci to encode several similar-type
MYB genes in the two cultivars of sweet potato. However, although the band patterns were similar, the
presence of small variations indicated that a few of the
MYB-like genes had structures that differed between
Ayamurasaki and Kokei-14.
Isolation of MYB Gene Family from Sweet Potato
Sequence analysis of 39 randomly isolated clones of
genomic PCR products revealed the presence of at
least another four MYB members, named IbMYB2-1 to
IbMYB2-4. Although the predicted ORF sequences of
IbMYB1 and IbMYB2 were quite similar, the predicted
lengths of their second introns differed (Fig. 3). The
deduced amino acid sequence encoded by all of these
Figure 2. Schematic representation of IbMYB1 cDNA and Southernblot analysis. A, Schematic representation of IbMYB1 cDNA with
R2R3-MYB domain (shaded box). Positions of primers (arrows) used for
probe preparation are indicated. MYB and Specific probes were
prepared with F8/R13 and F9/gRp primer sets, respectively. B, Southernblot analysis of cultivars Ayamurasaki and Kokei-14 using the MYB
and Specific probes of IbMYB1. Twenty micrograms of genomic DNA
was digested with EcoRI (EI), EcoRV (EV), and HindIII (H), fragmented
on a 0.8% agarose gel, transferred to nylon membrane, and hybridized
with MYB (left) and Specific (right) probes. Asterisks indicate differences between Ayamurasaki and Kokei-14. Aya, Ayamurasaki; Ko,
Koukei-14. Molecular marker sizes are indicated in kilobase pairs at
left.
Ayamurasaki, not in Kokei-14. IT4785 was homologous to the flavonoid 3#-hydroxylase of Ipomoea tricolor, and IT666 showed partial identity to R2R3-type
MYB genes. We therefore acquired a novel MYB-like
gene, IT666, with tissue- and cultivar-specific expression. As there have been no reports of MYB-like genes
in sweet potato, to our knowledge, we named this
IT666 gene IbMYB1. The IbMYB1 cDNA was predicted
to have an open reading frame (ORF) of 750 bp, including 312 bp of the R2R3-type MYB-like region in
the first half; the latter half had no marked similarity
(Fig. 2A).
Southern-blot analysis using IbMYB1 probes (MYB
probe for the putative R2R3 DNA binding domain and
Specific probe for the C-terminal region of IbMYB1)
indicated that a number of MYB genes existed in sweet
potato (Fig. 2B). As there were few differences between the blots achieved using the MYB and Specific
Figure 3. Schematic representation of genomic organization of IbMYB
genes, with exons (white boxes) and introns (lines between exons).
Positions of primers (arrows) used for RT-PCR analysis (Fig. 5) are
indicated, and resulting amplification products (lines) are shown.
Product sizes from amplification of genomic DNA and fully processed
mRNAs (shown in parentheses) are given for each PCR reaction. FA/
RA2 and FB/RB2 annealed specifically to the IbMYB1 and IbMYB2
genes, respectively.
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
Figure 4. IbMYB genes show features of an R2R3-MYB-binding domain protein. A, Amino acid comparison of the R2 and R3
DNA-binding regions of IbMYB1 and IbMYB2-1 with selected set of R2R3-type MYB factors from various plant species shows
high sequence conservation (amino acids identical with those of IbMYB1 are marked in gray). Previously published MYB gene
amino acid sequences were retrieved from the DNA data bank of Japan/EMBL/GenBank databases (grape VvmybA1
[BAD18977], grape VlmybA1-1 [BAC07537], morning glory Ipmyb1 [AAY54243], morning glory InMYB1 [BAE94389],
morning glory InMYB2 [BAE94709], morning glory InMYB3 [BAE94710], petunia AN2 [V26 allele; AAF66727], tomato ANT1
[AAQ55181], Arabidopsis PAP1 [AAG42001], Arabidopsis PAP2 [AAG42002], snapdragon ROSEA1 [ABB83826], gerbera
GMYB10 [CAD87010], maize Pl [AAA19819], maize C1 [AAA33482], Arabidopsis TT2 [CAC40021], and strawberry FaMYB1
[AAK84064]). The amino acid residues shown to be required for interaction of maize C1 with bHLH cofactor R (L77, R80, R83,
and L84) are marked with asterisks. B, Deduced amino acid sequence of IbMYB1. The R2R3-binding domain is shaded in gray.
Underlined sequence is aligned in Figure 4D. Boxed sequence is KPRPR(S/T) F-like motif. Splice-site locations of two introns are
marked with asterisks. C, Phylogenetic analysis of a selected set of R2R3-type MYB factors from various plant species.
Phylogenetic trees were produced by the neighbor-joining method of Saitou and Nei (1987) for the aligned amino acid
sequences of R2R3 DNA-binding domains. For construction of the trees, we used only the R2R3 MYB domain sequence (104
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Mano et al.
members had a length of 249 amino acids, with more
than 99% identity of amino acid sequences among
the protein encoded by IbMYB2 members and around
93% identity between that encoded by IbMYB1
and IbMYB2 members. Clones belonging to IbMYB1,
IbMYB2-1, IbMYB2-2, and IbMYB2-4 were found in
both Ayamurasaki and Kokei-14, and no differences
in the sequences of the areas tested were detected between the two cultivars. In contrast, IbMYB2-3 was found
only in Kokei-14.
Phylogeny of Sweet Potato R2R3-Type MYB Factors
The sequences of the IbMYB1 and IbMYB2 genes
were compared with those encoding other R2R3type MYB factors in various plant species: VvmybA1
(Kobayashi et al., 2004) and VlmybA1-1 (Kobayashi
et al., 2002) from grape; Ipmyb1 (Chang et al., 2005),
InMYB1, InMYB2, and InMYB3 (Morita et al., 2006)
from morning glory (Ipomoea nil); AN2 (Quattrocchio
et al., 1999) from petunia; ANT1 (Mathews et al.,
2003) from tomato (Lycopersicon esculentum); PAP1,
PAP2 (Borevitz et al., 2000), and TT2 (Nesi et al.,
2001) from Arabidopsis; ROSEA1 (Schwinn et al.,
2006) from snapdragon (Antirrhinum majus); GMYB10
(Elomaa et al., 2003) from gerbera (Gerbera hybrida); C1
(Paz-Ares et al., 1987) and Pl (Cone et al., 1993) from
maize; and FaMYB1 (Aharoni et al., 2001) from strawberry (Fragaria spp.). The R2R3-binding domain of the
deduced amino acid sequence encoded by the IbMYB1
and IbMYB2 genes showed high sequence similarity
with those encoded by other MYB genes of other plant
species (Fig. 4A), and splice-site locations and intron
phases were conserved with the majority of the Arabidopsis (77 of 130) and rice (45 of 85) MYB genes (Fig.
4B; Jiang et al., 2004). We performed phylogenetic
analysis of a selected set of R2R3-type MYB factors
from various plant species. Using the neighbor-joining
method of Saitou and Nei (1987), phylogenetic trees
were produced of the aligned amino acid sequences of
R2R3 DNA-binding domains (Fig. 4C). The result indicated that the IbMYB1 and IbMYB2 genes were located
in the N09 subgroup comprising Arabidopsis PAP1 and
PAP2 and petunia AN2 (Jiang et al., 2004). The predicted C-terminal regions of the IbMYB1 and IbMYB2
genes showed similarity to those of the InMYB2 and
InMYB3 genes, but there was very limited sequence
identity in the case of the other MYB proteins outside the R2R3 domain, except for the presence of a
KPRPR(S/T) F-like motif (boxed in Fig. 4, B and D),
which was previously reported for the subgroup
of R2R3 MYB genes comprising petunia AN2 and
Arabidopsis AtMYB75 (PAP1), AtMYB90 (PAP2), and
AtMYB113 (Stracke et al., 2001).
Expression Analysis of IbMYB1 and IbMYB2s
The expression patterns of IbMYB1 and IbMYB2
were analyzed by RT-PCR using specific primers: FA/
RA2 for IbMYB1 and FB/RB2 for IbMYB2 members. As
shown in Figure 5, when genomic DNAs were used as
templates for the reaction, the band patterns were
consistent with the predictions from Figure 3: that is, a
single band of about 800 bp was found in IbMYB1 and
multiple bands of about 900 to 1,250 bp in IbMYB2s.
Whereas IbMYB1 mRNA accumulated predominantly
in the tuberous roots of Ayamurasaki and slightly in
the calli, no signals of IbMYB2 members were detected
in these tissues. Two transcripts of different sizes detected in the IbMYB1 mRNA amplification may indicate alternative splicing, as the smaller transcript was
consistent in size with mature mRNA and the larger
transcript with retention of the second intron. Whereas
the first intron of IbMYB1 was under the GT-AG rule,
the second intron had GC-AG at the ends; this may
have been the cause of the alternative splicing. However, it is not clear whether the larger transcript of the
IbMYB1 gene has any functions; many MYB genes of
Arabidopsis and rice undergo alternative splicing and
are suggested to have multiple biological processes, as
some of these splice variants are differentially regulated (Li et al., 2006).
Tissue-Specific Expression of Genes Involved in
Anthocyanin Biosynthesis and Accumulation
In Ayamurasaki, various tissues besides the tuberous roots, such as the nontuberous roots, stems (ST),
young leaves (YL), stressed leaves (RL), flower buds
(FB), and open flowers (FL), accumulate anthocyanins
and exhibit pigmentation. Anthocyanins were extracted from each tissue and quantified (Fig. 6A), and
expression analysis of the genes involved in anthocyanin biosynthesis and accumulation was performed
at the same time (Fig. 6B).
The IbMYB1, IbMYB2, and IbMADS10 genes were
candidates for regulators of anthocyanin biosynthesis; CHS, CHI, F3H, DFR, ANS, and 3GT (encoding
chalcone synthase, chalcone isomerase, flavanone3-hydroxylase, DFR, anthocyanidin synthase, and
flavonoid 3-glucosyl-transferase, respectively) were
genes involved in parts of the anthocyanin biosynthesis pathway; VP24 (encoding vacuolar protein) was
considered to be involved in anthocyanin vacuolar
Figure 4. (Continued.)
amino acid residues; Fig. 4A) of each selected MYB-related protein. The matrix of sequence similarities was calculated by the
Clustal program (ClustalW 1.83; Thompson et al., 1994) and subjected to neighbor-joining analysis to generate branching
patterns. N09, N08, and N14 are the subgroups clustered by Jiang et al. (2004). D, Amino acid comparison of C-terminal region.
KPRPR(S/T) F-like motif is boxed. Genes harboring KPRPR(S/T) F-like motif correspond to the genes of the N09 subgroup in
Figure 4C.
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
seems to be not related to those of other tissues, such as
the tuber skins, flowers, leaves, and stems. For example, the flesh colors were not related to the tuber skin
colors or young leaf colors (Fig. 7A). The family trees
of the cultivars used are shown in Figure 7B. The
Figure 5. Expression analysis of IbMYB1 and IbMYB2s. Genomic PCR
and RT-PCR were performed using the FA/RA2 and FB/RB2 primer sets
indicated in Figure 3. Amplified products (28 cycles) were size fractionated on a 1.0% agarose gel. Amplification of Actin was used as a
control. A, Ayamurasaki; K, Koukei-14. Molecular marker sizes are
indicated in kilobase pairs at left.
transport and/or trapping (Nozue et al., 1997; Xu
et al., 2001).
The structural anthocyanin genes (CHS to 3GT) were
expressed in pigmented tissues, with the exception of
flesh of stored (i.e. stored for few months before use)
tuberous roots (sTF) and FL (Fig. 6B). IbMYB1 gene
expression was detected mainly in flesh of developing
(i.e. used just after harvest) tuberous roots (dTF), less
in peeled outer layer (less than 1 mm thick) including
the skins of developing and stored tuberous roots (dTS
and sTS), and a little in sTF, red and white nontuberous roots (RR and WR), and ST. Slight expression
of the IbMYB2 genes was detected in dTF and dTS.
The IbMADS10 gene was detected a little in dTF, sTF,
RR, WR, and FB. Expression of the VP24 gene was
detected in all tissues tested except for expanded green
leaves (EL).
Anthocyanin Accumulation and Gene Expression
in Seven Sweet Potato Cultivars
The relationship between anthocyanin accumulation and gene expression was examined in seven sweet
potato cultivars: three purple fleshed, one orange fleshed
(containing carotenoids), two yellow fleshed, and one
white fleshed (Fig. 7A). Among various sweet potato
cultivars, the color and/or color intensity of the flesh
Figure 6. Anthocyanin accumulation and gene expression in Ayamurasaki tissues. A, Photometric determination of anthocyanin content in
methanolic extracts of various tissues of Ayamurasaki. N (number of
samples) is indicated below. Error bars represent SD. B, RT-PCR analysis
of genes hypothesized to be involved in anthocyanin biosynthesis and
accumulation. Amplified products (30 cycles) were size fractionated on
a 1.0% agarose gel. Actin was used as a control. See text for abbreviation descriptions.
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Mano et al.
Figure 7. Seven sweet potato cultivars were tested: three purple-fleshed
cultivars, one orange-fleshed cultivar, and three yellow or whitefleshed cultivars. A, Appearance of
flesh or skin of mature tuberous roots
or young developing tuberous roots
or adaxial side of young leaf of the
seven cultivars. AM, Ayamurasaki;
MM, Murasakimasari; PS, Purple
Sweet Lord; AK, Ayakomachi; KK,
Kokei-14; BA, Beniazuma; JW, Joy
White. B, Family trees of the sweet
potato cultivars used in the experiments. Purple- or orange-fleshed cultivars are indicated by purple or
orange text. The cultivars used
are indicated by framed rectangles.
Numbers in parentheses after
purple-fleshed cultivar names are
relative anthocyanin contents of
their tuberous roots. (Yamakawa
et al., 1995, 1997, 1999; Kumagai
et al., 2002; Tamiya et al., 2003).
literature-based relative anthocyanin contents of mature tuberous roots of the purple-fleshed cultivars are
also presented in Figure 7B, and our measurements are
presented in Figure 8A. Anthocyanins in the flesh of
tuberous roots were detected only in the three purplefleshed cultivars, but in the outer layer of the tuberous
roots, they were also detected in orange- and yellowfleshed cultivars (Fig. 8A).
All tested genes were detected in almost every
cultivar tested by genomic PCR (Fig. 8B), with exceptions being IbMADS10 in Ayakomachi and Joy White.
By RT-PCR, we found that, in the flesh of small dTF,
the IbMYB1 gene and the structural anthocyanin genes
were expressed predominantly in purple-fleshed cultivars (Fig. 8C). Expression of IbMYB2 was scarcely
detected in each cultivar. Expression of IbMADS10 and
VP24 was not associated with anthocyanin content. In
the peeled outer layer containing the skin, the expression patterns of the genes encoding the transcription
factors were similar to those in the flesh. The structural
anthocyanin genes were expressed in the outer layers
of the six red-skinned cultivars.
Transient Expression Analysis of IbMYB1
Using a particle gun, the IbMYB1 gene was transiently expressed in sweet potato leaves, tuberous roots,
and calli under the control of the cauliflower mosaic
virus (CaMV)-derived 35S promoter or sweet potatoderived IT394 promoter (pIT394). The constructs used
for the bombardment are shown in Figure 9. The green
fluorescent protein (GFP) gene was used as the marker
of bombarded cells. Anthocyanin pigmentation was
observed in the bombarded cells of leaves (Fig. 10A),
tuberous roots (Fig. 10C), and calli (data not shown)
when the IbMYB1 gene was introduced with the GFP
gene (Fig. 10, A and C), whereas no pigmentation was
detected when the IbMYB1 antisense fragment was
introduced with GFP (Fig. 10, B and D). The GFP fluorescence in most leaf cells diffused to neighboring cells,
whereas the anthocyanin pigment stayed in the bombarded cells. Detection of anthocyanin pigmentation
was delayed until 1 or 2 d after the detection of GFP
fluorescence. The pigmentation deepened over more
than 1 week and then remained even after the GFP
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
Figure 8. Anthocyanin accumulation
and gene expression in different sweet
potato cultivars. A, Anthocyanin contents of mature tuberous roots (including
skin and flesh, grown in the field, longer
than 15 cm) and flesh or peeled outer
layer (less than 1 mm thick) including the
skin of young developing tuberous roots
(grown in the greenhouse, smaller than
10 cm) of each cultivar were determined. Three samples were tested in all
experiments. Error bars represent SD. B,
Genomic PCR of 10 sweet potato cultivars. About 30 ng of genomic DNA was
subjected to PCR. Amplified products
(40 cycles) were size fractionated on
1.0% agarose gel. Flesh colors of tuberous roots are indicated above the cultivar names. C, RT-PCR of flesh or outer
layer (including the skin) of young developing tuberous roots of seven cultivars. Amplified products (30 cycles for
IbMYB1, IbMYB2s, and IbMADS10; 28
cycles for the rest of the genes) were size
fractionated on 1.0% agarose gel. Actin
was used as a control. Flesh or skin colors
of tuberous roots are indicated above
the cultivar names. AM, Ayamurasaki;
MM, Murasakimasari; PS, Purple Sweet
Lord; TM, Tanegashimamurasaki; AK,
Ayakomachi; HK, Hamakomachi; SR,
Sunny Red; KK, Kokei-14; BA, Beniazuma;
JW, Joy White.
fluorescence had disappeared. The speed of accumulation of GFP and anthocyanin was slower in the tuberous roots than in the leaves. Differences in promoter
(CaMV 35S or pIT394) or the cultivar of bombarded
cells (Ayamurasaki or Kokei-14) did not affect these results (data not shown).
Overexpression of IbMYB1 in Transgenic Plants
Forced expression analysis of the IbMYB1 gene was
also performed in sweet potato calli and heterologous
plants (Arabidopsis and rice) with stable transformation. In Arabidopsis, overexpression of IbMYB1 induced
ectopic pigmentation in seedlings (Fig. 11A), roots (Fig.
11B), flowers, leaves, and stems (Fig. 11C), and even in
seeds (Fig. 11D). However, most of the plants with
severe pigmentation were sickly and could not survive
without the addition of Suc to the culture medium. In
rice, we could not produce a pigmented plant. In sweet
potato, overexpression of IbMYB1 induced strong pigmentation in transgenic calli (Fig. 12A). We obtained
17 independent pigmented transgenic calli by transformation of about 3 g of embryogenic calli of Kokei-14.
Ten of the 17 lines of pigmented transgenic calli grew
well and were subjected to anthocyanin quantification
and gene expression analysis. Sometimes untransformed
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DISCUSSION
Figure 9. Constructs used for transient expression analysis and for
stable transformation. Plasmid vector names of pBluescriptII background are represented. Names of binary vector (pPZP2H-lac) background are represented in parentheses.
calli of Kokei-14 or Ayamurasaki show patchy pigmentation (Fig. 12A), but we could not obtain calli as
severely and uniformly pigmented as the pHM158
(35STIbMYB1) transformants when the calli were not
transformed or were transformed with other constructs, such as pHM150b (35STGFP) or pHM159
(35STanti-IbMYB1; data not shown). The amount of
anthocyanin in the transgenic calli was increased by
nearly 200 times that in the nontransgenic Kokei-14
calli (Fig. 12B) and was comparable to that in the tuberous roots of purple-fleshed cultivars (Fig. 8A). The
growth rates of the transgenic callus lines were inversely related to the levels of anthocyanin accumulation (Fig. 12C) and were much higher than those of
nontransgenic calli (Fig. 12B). We also assessed the influence of light on anthocyanin accumulation and
growth rate. There was a tendency for anthocyanin accumulation to increase and growth rate to decrease in
calli grown in light, and light-exposed nontransgenic
calli of Ayamurasaki showed significantly higher rates
of anthocyanin accumulation than did dark-exposed
calli of this cultivar (Fig. 12D).
The IbMYB1 gene and the structural anthocyanin genes
were predominantly expressed in the pigmented transgenic calli and pigmented untransformed Ayamurasaki
calli (Fig. 13). Scant expression of the IbMYB2 genes
and the IbMADS10 gene was detected in some transgenic or nontransgenic calli. The VP24 transcripts were
decreased when the calli (Ayamurasaki, Kokei-14, and
pHM158-2) were grown in the dark, but the expression
pattern was not correlated with the anthocyanin content. Whereas two transcripts of different sizes were
detected by RT-PCR analysis of IbMYB1 in untransformed Ayamurasaki tissues (Figs. 5, 6, and 13), a single
band (of a size consistent with introduced IbMYB1
cDNA and the same size as the smaller transcript
detected in untransformed tissues) was detected in
pHM158 transgenic calli (Fig. 13).
The pathways of biosynthesis of flavonoid pigments
have been investigated well, perhaps because of the
color that they add to plants. Their regulatory genes in
aerial parts such as flowers, leaves, seeds, and fruits
have been identified in various plant species, whereas
little is known about their regulation in underground
organs such as tuberous roots. Although the role of
anthocyanins in underground organs is not clear, the
fact that many sweet potato cultivars with not only
purple flesh but also orange, yellow, or white flesh
retain anthocyanins in the skin of their tuberous roots
suggests that they play an important role.
Sweet potato accumulates anthocyanins in various
tissues such as the flowers, leaves, stems, and nontuberous roots, as well as the tuberous roots (Fig. 6A).
The IbMYB1 gene was expressed predominantly in
tuberous roots, whereas the expression of IbMYB2
genes was scarcely detected (Fig. 6B). WR was the only
tissue that showed expression of the IbMYB1 gene in
the absence of anthocyanin detection (Fig. 6). The fact
that the expression levels of structural anthocyanin
genes were similar to that in RR indicates that anthocyanin biosynthesis was already active in WR but anthocyanin had not accumulated to levels high enough
to detect. There was no tissue that expressed the IbMYB1
gene without structural anthocyanin gene expression.
We sometimes could not detect IbMYB1 expression in
tuberous roots when the samples had been stored for a
few months (e.g. sTF in Fig. 6B; also in sTS in some
cases) or in seed tubers (data not shown). These tissues
may have been losing their anthocyanin biosynthesis
activity, because they also showed diminished expression of the structural anthocyanin genes. In any case, a
striking correlation of expression in the tuberous roots
was observed between IbMYB1 and the structural anthocyanin genes.
We did not detect IbMYB1 expression in flowers and
leaves despite the anthocyanin accumulation and the
strong expression of structural anthocyanin genes (YL,
RL, and FB in Fig. 6). IbMYB1 expression was also not
detected in the skins of tuberous roots of non-purplefleshed cultivars (Ayakomachi, Kokei-14, and Beniazuma
in Fig. 8C). These results suggest that IbMYB1 acts
specifically in the flesh of tuberous roots and that other
regulatory genes are active in the other tissues. Contamination of the outer layer samples with flesh tissue
may be the reason why IbMYB1 was detected in the
outer layers of the purple-fleshed tuberous roots (dTS
and sTS in Fig. 6; Ayamurasaki, Murasakimasari, and
Purple Sweet Lord in Fig. 8C). The difference in
anthocyanin composition of the outer layer and the inner portion of Ayamurasaki tuberous root (Yoshimoto
et al., 1999a) may reflect a difference in the genes
responsible for the regulation of anthocyanin biosynthesis. The fact that, among various sweet potato
cultivars, the color and/or color intensity of the flesh
were not related to those of other tissues (Fig. 7A) also
supports these ideas.
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
Figure 10. Transient expression analysis of IbMYB1. GFP (pHM160) and
IbMYB1 (pHM196) or IbMYB1 antisense (pHM197) were cobombarded
into sweet potato leaves (A and B) and
sections of tuberous roots (C and D). A
and C, Cobombardment of pHM160
and pHM196. B and D, Cobombardment of pHM160 and pHM197. Each
section contains bright-field images
(top) and fluorescent images (bottom).
Times after bombardment are indicated
above. Exposure times were identical
for each bright-field image (0.2 s) and
fluorescent image (2.0 s). Magnifications were identical for all images.
Scale bar in bottom left corner represents 100 mm.
The results of bombardment of the IbMYB1 gene
into leaves (Fig. 10A) indicated the existence of mechanisms of anthocyanin biosynthesis common to different tissues. However, Ayamurasaki leaves display a
complex change in color; they are red when they are
young, turn green as they develop, and turn red again
with stress (YL, EL, and RL in Fig. 6A). Therefore, we
can assume that multiple regulatory genes and a degradation mechanism are active in these leaves.
This is, to our knowledge, the first report of the
identification of MYB genes in sweet potato, and other
transcription factors related to anthocyanin biosynthesis, such as the bHLH and WD40 genes, are still unknown in sweet potato. However, several members of
the MYB, bHLH, and WD40 gene families have been
isolated from morning glory and their differential
expression pattern reported; InMYB1 is expressed in
the flower and InMYB2 is expressed in the petiole,
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Mano et al.
Figure 11. Overexpression of IbMYB1 in Arabidopsis
under the control of the CaMV 35S promoter. Fiveday-old seedlings (A), a root (B), flowers, leaves, and
stems (C), and seeds (D) of Arabidopsis plants transformed with the construct pHM158 (CaMV35ST
IbMYB1) accumulated ectopic pigmentation.
stem, and root, but expression of InMYB3 has not been
detected (Morita et al., 2006). According to phylogenic
analyses (Fig. 4C), the InMYB2 and InMYB3 genes are
more closely related to IbMYB1 and IbMYB2 than to
InMYB1. These results indicate that the MYB genes
working in flowers and the MYB genes working in
roots were divided before the division of morning
glory and sweet potato.
A number of MADS-box genes have been isolated
from sweet potato, and one of them, IbMADS10, has
been suggested to be involved in anthocyanin biosynthesis (Lalusin et al., 2006). However, the level of correlation of this gene with anthocyanin accumulation or
expression of the structural anthocyanin genes was not
high in our experiments. It is possible that another transcription factor for IbMADS10 activity, such as a bHLH
transcription factor that needs an MYB transcription
factor to activate it, is involved.
Sometimes red-pigmented cells appear in sweet
potato calli grown under light conditions (Fig. 12A).
Nishimaki and Nozue (1985) used the yellow-fleshed
cultivar Kintoki to establish cell lines (ALD and ALND)
that accumulate anthocyanins. The ALD cell line produced anthocyanins in the light but not in darkness,
and the ALND line produced anthocyanins under both
light and dark conditions. However, the content of anthocyanins in the dark was one-fourth that in the light
(Nozue et al., 1997). On the other hand, our transgenic
calli lines, made by forced expression of the IbMYB1
gene in the yellow-fleshed cultivar Kokei-14, accumulated anthocyanins in a light-independent manner
at high levels comparable to those in the tuberous
roots of purple-fleshed cultivars (Figs. 8A and 12B).
These features are similar to those of cell lines selected
and established from Ayamurasaki tuberous roots
(Konczak-Islam et al., 2000). It is possible that accumulation of anthocyanin in calli in a light-dependent
manner is caused by the activation of regulator genes
that act in the aerial parts and light-independent accumulation is caused by the activation of regulator genes
that act in the underground organs.
Light induced anthocyanin accumulation in
Ayamurasaki calli (Fig. 12D); sometimes IbMYB1 expression was detected in this tissue (Fig. 13) and sometimes not. Unlike cell lines, the callus samples may not
have been homogeneous and may have included diverse cells pigmented by the activation of various
regulatory genes.
However, the determinant of growth rate was not
clear; the growth rates of most of the transgenic calli
were much higher than those of the nontransgenic calli
(Fig. 12B). It is conceivable that the accumulated anthocyanins protected the callus from the inhibitory
effects of light and thus promoted their propagation.
On the other hand, the growth rates of the transgenic
callus lines were inversely related to the levels of
anthocyanin accumulation (Fig. 12C). It is possible that
the growth rates of some transgenic lines were so fast
that anthocyanin accumulation did not reach saturation in these lines. The fact that the growth rates of all
transgenic lines exceeded 2.0 (i.e. the weight of the calli
doubled in 2 weeks; Fig. 12B), whereas the anthocyanin contents of the leaf or tuber cells increased for
more than 1 week (Fig. 10, A and C), may support this
assumption.
The fact that overexpression of the IbMYB1 gene
induces ectopic pigmentation not only in sweet potato
but also in heterologous Arabidopsis (Fig. 11) suggests
a common regulatory mechanism. On the other hand,
the fact that most of the transgenic Arabidopsis plants
with severe pigmentation were sickly and unable to
survive without Suc in the culture medium suggests a
difference among species. Quattrocchio et al. (1998) and
Gong et al. (1999) also found common and different
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
Figure 12. Overexpression of IbMYB1 in sweet potato calli under the control of the CaMV 35S promoter. A, Kokei-14-derived
calli transformed with the construct pHM158 (CaMV35STIbMYB1) turned a deep purple. The calli of 10 independent transgenic
lines and untransformed calli derived from Ayamurasaki (Aya) and Kokei-14 (Ko) were grown under light conditions. One of the
transgenic callus lines (pHM158-2) and the untransformed calli were also grown under dark conditions. B, Growth rate and
anthocyanin content of each callus were determined. The fresh weight (FW) of each callus after culture for 2 weeks was divided
by the initial fresh weight (FWi) to determine the growth rate. 1 to 15, pHM158-1 to 15 transgenic lines; D, grown in dark
conditions; L, grown under light conditions. N (number of samples) is indicated below. Error bars represent SD. C, Relationship
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Mano et al.
actions of anthocyanin regulatory genes in heterogonous
plants; ectopic expression of petunia AN2 and JAF13
induced CHS gene expression in heterologous maize
but not in petunia. Both bHLH alleles of perilla, MYCRP and MYC-GP, induced anthocyanin accumulation
in heterologous tobacco (Nicotiana tabacum) plants,
whereas only MYC-RP acted as a regulator in perilla.
The biosynthesis of anthocyanins occurs in the cytosol, whereas the end products accumulate in the
vacuole. This may be the reason why the anthocyanin
pigment induced by bombardment with IbMYB1 remained in the bombarded cells, whereas the GFP fluorescence diffused to other cells. It is proposed that
glutathione-S-transferase-like proteins such as maize
BZ2 (Marrs et al., 1995), petunia AN9 (Mueller et al.,
2000), and Arabidopsis TT19 (Kitamura et al., 2004)
deliver their flavonoid substrates to the transporter,
such as the maize multidrug resistance-associated protein (MRP)-type transporter ZmMRP3 (Goodman et al.,
2004) or the Arabidopsis multidrug and toxic compound extrusion transporter TT12 (Debeaujon et al.,
2001). In sweet potato, it has been suggested that VP24
precursor protein is involved in vacuolar transport
and/or accumulation of anthocyanin (Nozue et al.,
1997; Xu et al., 2001).
Transient and stable forced expression analysis of
IbMYB1 suggested that IbMYB1 induces not only structural anthocyanin genes but also anthocyanin transporters such as ZmMRP3 that are expressed under the
control of the regulators of anthocyanin biosynthesis,
R and C1 (Goodman et al., 2004). We examined whether
IbMYB1 induced VP24 expression (Fig. 13). The VP24
gene was expressed not only in transgenic calli
but also in unpigmented nontransgenic light-grown
Kokei-14 callus, but it was scarcely detected in darkgrown pHM158-2 callus and light-grown pHM158-12
callus. These results deny the inductivity of VP24 gene
expression by IbMYB1. We also examined the expression pattern of VP24 in sweet potato tissues (Figs. 6B
and 8C). The relatively abundant expression in older
tissues (i.e. more abundant in sTF, sTS, and FL than in
dTF, dTS, and FB in Fig. 6B) suggests that VP24 acts in
the retention of anthocyanin rather than in transport
and/or accumulation. There may be other IbMYB1inducible genes that are involved in anthocyanin transport and accumulation in sweet potato.
However, the IbMYB1 transcript was not detected in
the tissues of Kokei-14, and there were no differences
in base sequences of the IbMYB1 coding region between Ayamurasaki and Kokei-14. The fact that the
overexpression of IbMYB1 in Kokei-14 induced a large
amount of anthocyanin pigmentation suggests that mutation(s) on the promoter region of the IbMYB1 gene
caused alteration in the flesh color of sweet potato,
Figure 13. RT-PCR analysis of the genes hypothesized to be involved in
anthocyanin biosynthesis and accumulation was performed in sweet
potato calli. Amplified products (30 cycles for IbMYB1, IbMYB2s, and
IbMADS10; 28 cycles for the rest of the genes) were size fractionated on
1.0% agarose gel. Actin was used as a control.
as in the case for grape skin color; the insertion of a retrotransposon into the 5#-flanking region of VvmybA1
was associated with loss of pigmentation in whiteskinned cultivars, whereas the coding sequences were
identical with those of red-skinned cultivars (Kobayashi
et al., 2004). Alternatively, mutation(s) on regulator(s)
of the IbMYB1 gene may have caused the alteration of
flesh color, although the regulators of the anthocyanin
regulatory genes are unknown.
All three purple-fleshed cultivars expressed the
IbMYB1 gene in their tuberous roots (Fig. 8C). Presumably, they inherited the active promoter of the IbMYB1
gene from a common ancestor, Yamagawamurasaki
(Fig. 7B). However, their contents of anthocyanin differ
Figure 12. (Continued.)
between growth rates and anthocyanin content of transgenic callus lines. D, Influence of light conditions on growth rates and
anthocyanin content. The anthocyanin content of Ayamurasaki calli was increased significantly by light.
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IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis
Table I. Primers used in RT-PCR experiments
Primer Name
Forward Primer
Reverse Primer
IT1-F/R
IT4-F/R
IT4785-F/R
IT666-F/R
IT4097-F/R
IT948-F/R
IbMYB1-FA/RA2
IbMYB2s-FB/RB2
Act-F/R
CHS-F/R
CHI-F/R
F3H-F/R
DFR-F/R
ANS-F/R
3GT-F/R
IbMADS10-F/R
VP24-F/R
5#-CGGAACGTTCTTAGAAAAGCTG-3#
5#-CCATACCAGCTCGGATTTGT-3#
5#-GAAACCGCACCAGTCGATAG-3#
5#-GCGAATTTAGTCCCGATGAA-3#
5#-CATGCTCTCCAAACAATGGA-3#
5#-TGTGGAATTCGAAAGCTGAG-3#
5#-TATGGTCGGGATCGTCTTCG-3#
5#-TATGGTCGGAATCGTCTTCC-3#
5#-GACTACCATGTTCCCCGGTA-3#
5#-GGACTACCAGCTCACCAAGC-3#
5#-GTTAAGTGGAACGGGAAAAG-3#
5#-CGAGATTCCGGTGATATCGT-3#
5#-TCCTGGGAACACAAAGAAGG-3#
5#-ATTTTCGCGGAGGAAAAGAT-3#
5#-AAGTATCGATCGGCGAAATG-3#
5#-CGAACTGAAGCGGATTGAGAAC-3#
5#-CTTGACACTGCCCTCCAGTATG-3#
5#-GGCCAGAAGAAGGTGTACGA-3#
5#-TGGATGCCAACCTTAACTCC-3#
5#-GCGAAGACGAGGTCTTGATAG-3#
5#-CGGTGTTTTCCGTGATTTCT-3#
5#-GTCTGCAGCAAGAAGGTTCC-3#
5#-CCAAACAACACAACAATCCAA-3#
5#-TTCTGAAGATGGGTGTTCCAT-3#
5#-TTCTGAAGATGGGTGTTCCAG-3#
5#-TTGTATGCCACGAGCATCTT-3#
5#-GTCCTCCACTTGGTCCAGAA-3#
5#-GAGACGACCGTTTGTGGAAT-3#
5#-GGGGCATTTTGGGTAGAAAT-3#
5#-GAGCTTCGCAGAGATCATCC-3#
5#-CTTCCTTCTCCAGCCTTCCT-3#
5#-CACGATATGGCCTCCAGAGT-3#
5#-CCAGCTGTTGCTCTAAACTCTG-3#
5#-ACGAGCAAGCTCCAACATAACA-3#
considerably (Fig. 7B). Interaction of IbMYB1 with
other transcriptional factors may affect anthocyanin
biosynthesis activity.
Columns (Amersham Biosciences), and introduced into vector pBluescript II
SK1 (Stratagene) digested with XhoI and EcoRI. From the randomly isolated
cDNA clones, we determined the sequences of 3,783 cDNA clones from
Ayamurasaki and 2,804 cDNA clones from Kokei-14.
RT-PCR and Genomic PCR
MATERIALS AND METHODS
Plant Materials
Sweet potato (Ipomoea batatas) L. Lam. (cultivars Ayamurasaki, Kokei-14,
Murasakimasari, Purple Sweet Lord, Ayakomachi, Beniazuma, and Joy
White) plants were grown in a greenhouse at 26°C under natural light. Sweet
potato calli were induced from the meristems of apical and axillary buds on
media (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L21 myoinositol, 0.4 mg L21 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7,
1 mg L21 4-fluorophenoxyacetic acid, 0.8% [w/v] agarose) in growth chambers at 26°C in the dark, and transformed calli were cultured on selection
media (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L21 myoinositol, 0.4 mg L21 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7,
1 mg L21 4-fluorophenoxyacetic acid, 25 mg L21 hygromycin B, 500 mg L21
carbenicillin, 0.8% [w/v] agarose) in growth chambers at 26°C under 16 h of
light (around 40 mmol m22 s21) and 8 h of dark. Arabidopsis (Arabidopsis
thaliana) ecotype Columbia plants were grown in growth chambers at 22°C
under continuous light.
RNA and DNA Preparation
RNA was isolated from various tissues and calli of sweet potato. After the
samples had been ground into powder in mortars with liquid nitrogen, they
were washed with buffer solution (0.1 M HEPES, 0.05 M L-ascorbic acid, 0.4%
[v/v] polyvinyl pyrrolidone [Mr 40,000], and 2% [v/v] b-mercaptoethanol)
four times. RNA was isolated according to the protocol of the National Science
Foundation’s Potato Genome Project (RNA isolation using phenol protocol;
http://www.tigr.org/tdb/potato/microarray_SOPs.shtml). DNA was isolated from about 0.5 g of young leaves of sweet potato. After the samples had
been ground into powder in mortars with liquid nitrogen, they were washed
with buffer solution as for the RNA preparation, and then a PhytoPure plant
DNA extraction kit (Amersham Biosciences) was used for DNA isolation in
accordance with the manufacturer’s instructions.
Construction of cDNA Libraries from Sweet Potato
cDNA libraries were constructed from tuberous roots of sweet potato
using cDNA Synthesis kit (Stratagene), size fractioned with Size Sep400 Spun
For RT-PCR, first-strand cDNA was synthesized from 500 ng of total RNA
with Superscript III (Invitrogen) in accordance with the manufacturer’s
instructions. Oligo(dT) 20 was used as the primer. Twenty microliters of the
RT product was acquired from each reaction. One microliter of RT product
was subjected to each PCR amplification. PCR was performed using Platinum
Pfx DNA Polymerase (Invitrogen) and primers designed for each gene. The
PCR conditions were 5 min at 95°C, followed by 25, 28, 30, or 35 cycles of 30 s
at 94°C, 30 s at 56°C, and 2 min at 68°C, followed by 7 min at 68°C. At least two
independent repeats of the RT-PCR experiments were performed, and typical
images of electrophoresis are presented. We could not detect any signals when
the reactions were performed under the same conditions without RT. For the
amplification of genomic DNA, the PCR conditions were 5 min at 95°C,
followed by 30 or 40 cycles of 30 s at 94°C, 30 s at 56°C, and 5 min at 68°C,
followed by 7 min at 68°C, using PCR SuperMix High Fidelity (Invitrogen).
The primers used for the PCR are listed in Table I.
Southern Hybridization
Southern-blot analysis of IbMYB1 was performed using the MYB and
Specific probes. Twenty micrograms of genomic DNA of Ayamurasaki and
Kokei-14 was digested with EcoRI, EcoRV, and HindIII, fragmented on a 0.8%
agarose gel, transferred to nylon membrane (Boehringer Mannheim), and
hybridized with the MYB and Specific probes of IbMYB1 (Fig. 2A). The MYB
and Specific probes were prepared by PCR using primers 666-F8 (5#-GTGAGAAAAGGTTCATGGTCC-3#) and 666-R13 (5#-CTTCTTCTGAAGATGGGTGTTC-3#), and 666-F9 (5#-GTGTCTGCCATGGCTTCTTCAA-3#) and
666-gRp (5#-TGGCTGCAGATTACATTCTCAAATTTAATCGTACA-3#), respectively. The probes were labeled and detected using AlkPhos Direct Labeling
and Detection system (Amersham Biosciences) and Hyperfilm ECL (Amersham
Biosciences) in accordance with the manufacturer’s instructions. Membranes
were hybridized at 55°C overnight and washed twice for 10 min at 60°C.
Isolation of Genome Fragments of IbMYB1
The IbMYB1 genome fragments of Ayamurasaki and Kokei-14 were
amplified using the primers 666-Fb2 (5#-ATGGATCCTAAGAATTTCCGACACCCTTC-3#) and 666-R (5#-CGGTGTTTTCCGTGATTTCT-3#) or 666-F3
(5#-CTCATTCTGCGCCTCCATAG-3#) and 666-R14 (5#-GGTGGCAATAGAA-
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Mano et al.
TTTAAATCAAG-3#) and then cloned into vector pCR2.1-TOPO using a
TOPO TA Cloning kit (Invitrogen). The clones were randomly isolated and
sequenced, and the sequences of 39 clones were determined.
Plasmid Construction
The CaMV 35S promoter was isolated from pBI221 (BD Biosciences
CLONTECH) by PCR using primers that added the XbaI site at the 5# end
and the BamHI site at the 3# end; i.e., 35S-F340x (5#-TGGTCTAGAGACTTTTCAACAAAGGGTAAT-3#) and 35S-Rb (5#-CGGGATCCTCTCCAAATGAAATGAACTTCC-3#). pHM144 was constructed by insertion of the CaMV 35S
promoter into the XbaI and BamHI sites of the plasmid pblue-sGFP(S65T)NOS SK, kindly provided by Dr. Y. Niwa of the University of Shizuoka. The
IbMYB1 ORF region was isolated from the cDNA clone IT666 by PCR using
primers that added the BamHI site at the 5# end and the NotI site at the 3# end;
i.e. 666-Fb2 (5#-ATGGATCCTAAGAATTTCCGACACCCTTC-3#) and 666-Rn
(5#-ATGCGGCCGCTTAGCTTAACAGTTCTGAC-3#). The IbMYB1 antisense
fragment was isolated from cDNA clone IT666 by PCR using primers that
added the BamHI site at the 5# end and the NotI site at the 3# end; i.e. 666-Rb
(5#-TAGGATCCTAACGACGGTGTTTTCCG-3#) and 666-Fn2 (5#-ATGCGGCCGCTAAGAATTTCCGACACCCTT-3#). pHM156 and pHM157 were made
by ligation of the IbMYB1 ORF and the IbMYB1 antisense fragment, respectively, with the ScaI-BamHI fragment of 1.4 kb and the ScaI-NotI fragment of
2.2 kb from pHM144.
The IT394 promoter (Mano et al., 2006) was isolated from the sweet potato
genome by PCR using primers that added the SacII site and removed the
SacI site at the 5# end and that added the BamHI site at the 3# end: 394F07s
(5#-TGGCCGCGGATGTTGAcCTCTTTCATTTTGAACCAA-3#) and 394Rb
(5#-CGGGATCCTTTTCACACAGAAGAGAGAAAGACAAGA-3#). pHM160,
pHM196, and pHM197 were constructed by insertion of the IT394 promoter
into the SacII and BamHI sites of the plasmid pblue-sGFP(S65T)-NOS SK,
pHM156, and pHM157, respectively.
The CaMV 35S promoter, IbMYB1 ORF, and Nos terminator sequence and
the CaMV 35S promoter, IbMYB1 antisense fragment, and Nos terminator
sequence were excised from pHM156 and pHM157, respectively, using XbaI
and KpnI and then cloned to the XbaI and KpnI site of the binary vector
pPZP2H-lac, kindly provided by Dr. M. Yano of the National Institute of
Agrobiological Sciences, Tsukuba, Japan (Fuse et al., 2001). The resulting
binary vectors were named pHM158 and pHM159, respectively.
Transient Expression
Transient expression analysis was carried out as described (Higo et al.,
2005). The same amount of plasmids pHM144 (35S: GFP) and pHM156
(35STIbMYB1) or pHM157 (35STanti IbMYB1), and pHM160 (pIT394TGFP)
and pHM196 (pIT394TIbMYB1) or pHM197 (pIT394Tanti IbMYB1) were
bombarded into sweet potato leaves (Kokei-14 and Ayamurasaki), calli
(Kokei-14 and Ayamurasaki), and sections of tuberous root (Kokei-14). The
bombarded tissues were cultured on medium (Murashige and Skoog Plant
Salt mixture [Wako], 100 mg L21 myo-inositol, 0.4 mg L21 thiamine-HCl, 0.05%
[w/v] MES, pH 5.7, 3% [w/v] Suc, 0.1% [v/v] Plant Preservative mixture
[Plant Cell Technology], 0.6% [w/v] agarose) in growth chambers at 26°C
under 16 h of light (photon flux density around 40 mmol m22 s21) and 8 h of
dark for leaves and continuous dark for calli and tuberous roots. Bombarded
tissues were observed under a microscope (IX70, Olympus), and the fluorescence of GFP was observed through a filter set (excitation wavelength, 460–
490 nm; emission, 515–550 nm; dichroic, 505 nm).
Stable Transformation
The binary vector pHM158 was transformed with Agrobacterium tumefaciens strain EHA101 by the freeze-thaw method (Holsters et al., 1978). Using
this Agrobacterium, transformation of the sweet potato calli was performed in
accordance with the method of Otani et al. (1998). Arabidopsis was transformed by the floral dip method (Clough and Bent, 1998).
Anthocyanin Quantification
Extraction and quantification of anthocyanins was performed in accordance with the protocols of Mehrtens et al. (2005), with minor modifications.
One milliliter of acidic methanol (1% [w/v] HCl) was added to 0.3 g of fresh
plant tissue. Samples were incubated for 18 h at 21°C under moderate shaking
(95 rpm). After centrifugation (21,500g, room temperature, 3 min), 0.4 mL of
the supernatant was added to 0.6 mL of acidic methanol. Absorption of the
extracts at wavelengths of 530 and 657 nm was determined photometrically
(DU 640 Spectrophotometer, Beckman Instruments). When the absorption
value exceeded 2.5, extracts diluted with acidic methanol were used for the
measurements. Quantitation of anthocyanins was performed using the following equation: Q (anthocyanins) 5 (A530 2 0.25 A657) 3 M21, where Q
(anthocyanins) is the concentration of anthocyanins, A530 and A657 are the
absorptions at the wavelengths indicated, and M is the fresh weight (in grams)
of the plant tissue used for extraction. The numbers of samples used for the
measurements are indicated in each figure. Error bars indicate the SDs of the
average anthocyanin contents.
Accession Numbers
Accession numbers for the genes isolated in this article are as follows:
AB258984 (IbMYB1 cDNA), AB258985 (IbMYB1 genome), AB258986 (IbMYB2-1
genome), AB258987 (IbMYB2-2 genome), AB258988 (IbMYB2-3 genome), and
AB258989 (IbMYB2-4 genome).
ACKNOWLEDGMENTS
The authors are grateful to Dr. Yasuo Niwa of the University of Shizuoka
for providing the vector pblue-sGFP(S65T)-NOS SK; Dr. Masahiro Yano of the
National Institute of Agrobiological Sciences for the binary vector pPZP2Hlac; the National Institute of Crop Science for the sweet potato; Mr. Yuichi
Minesaki of Hitachi Software Engineering for his help with the cDNA
clustering analysis; and Mrs. N. Kawaguchi, Mrs. K. Kawajiri, and Mrs.
K. Takahashi for their assistance.
Received December 8, 2006; accepted December 20, 2006; published January 5,
2007.
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