Plant Physiol. Biochem. 40 (2002) 955–962
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Original article

Anthocyanin biosynthetic genes are coordinately expressed during redcoloration in apple skin
Chikako Honda a,*, Nobuhiro Kotoda b, Masato Wada b, Satoru Kondo c, Shozo Kobayashi d,Junichi Soejima b, Zilian Zhang a, Tomomi Tsuda a, Takaya Moriguchi aaNational Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan
Department of Apple Research, National Institute of Fruit Tree Science, Morioka, Iwate 020-0123, Japanc
Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japand
Department of Grape and Persimmon Research, National Institute of Fruit Tree Science, Akitsu, Hiroshima 729-2494, Japanb

Received 12 December 2001; accepted 4 June 2002

Abstract
Five genes of anthocyanin biosynthetic enzymes, chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9),
dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), anthocyanidin synthase (ANS; EC 1.14.11.X), and UDP glucose:flavonoid 3-Oglucosyltransferase (UFGluT; EC 2.4.1.X), were isolated, and their expression was investigated to elucidate the molecular mechanism forred coloration in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] skin. In ‘Orin,’ a yellow apple cultivar, no significant
levels of anthocyanin were detectable, whereas in ‘Jonathan’ and ‘Fuji,’ both red apple cultivars, anthocyanin concentrations increased
during fruit development. At the ripe stage, the level of anthocyanin concentration was about three times higher in ‘Jonathan’ than in ‘Fuji.’
The accumulation of transcripts for the five genes was induced at the later developmental stages in all three cultivars. The levels for the
expression of the five genes basically corresponded to the anthocyanin concentrations; that is, the induction of the genes in ‘Orin’ was lesspronounced, and that in ‘Fuji’ and ‘Jonathan’ was notable, with much higher expression levels in ‘Jonathan’ than in ‘Fuji’. These results
indicate that the five genes are coordinately expressed during fruit development and that their levels of expression are positively related tothe degree of anthocyanin concentration. This is the first report that characterizes the relationship between the expression of anthocyanin
biosynthetic genes and apple fruit coloration. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Anthocyanin; Apple skin; Flavonoid biosynthetic genes; Fruit development; Gene expression; Red coloration

1. Introduction
The red coloration of apple [Malus sylvestris (L.) Mill.
var. domestica (Borkh.) Mansf.] skin is derived fromchemical components called anthocyanins that belong to aclass of flavonoids. The accumulation of anthocyanins isAbbreviations: ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; CTAB, cetyltrimethylammonium bromide;
DAFB, days after full bloom; DFR, dihydroflavonol 4-reductase; EDTA,
ethylenediamine-N,N,N',N'-tetraacetic acid; EST, expressed sequence tag;
F3H, flavanone 3-hydroxylase; HPLC, high performance liquid chromatography; PAL, phenylalanine ammonia-lyase; RT–PCR, reverse transcription–polymerase chain reaction; UFGalT, UDP galactose: flavonoid 3-Ogalactosyltransferase; UFGluT, UDP glucose: flavonoid 3-Oglucosyltransferase* Corresponding author.
E-mail address: hondac@affrc.go.jp (C. Honda).
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 5 4 - 7

influenced by environmental stimuli, such as light, temperature, and nutrition [13,19], as well as by genetic factors. The
regulatory mechanisms of anthocyanin biosynthesis attract
interest because red skin coloration is an important factor inmarket acceptance for many apple cultivars. There are
distinguished varieties of red-skinned apples: (a) red cultivars, such as ‘Jonathan’ and ‘Starking Delicious,’ whichbear well-colored fruits; (b) red cultivars, such as ‘Fuji’ and‘Jonagold,’ which are pale in color under certain conditions;and (c) non-red cultivars that accumulate some anthocyanins after shade-pretreatment by bagging, such as ‘Orin,’‘Golden Delicious,’ and ‘Mutsu.’
During the past decade, cloning of structural genes
encoding enzymes in the anthocyanin biosynthetic pathway
and the identification of genes encoding transcription factors that regulate the expression of structural genes have

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contributed to our understanding of the molecular basis ofanthocyanin biosynthesis in higher plants, such as maize,
snapdragon, and petunia [6]. In fruit trees, the anthocyanin
biosynthetic genes from grapes (Vitis vinifera L.) have been
investigated carefully [20], and UDP glucose:flavonoid
3-O-glucosyltranseferase (UFGluT) was found to be a key
enzyme controlling the color red in grape skin [1]. Morerecently, Kobayashi et al. [11] found that there were nodifferences in either coding or promoter sequences of the
UFGluT gene between white-skinned grape cultivars (‘Italia’ and ‘Muscat of Alexandria’) and their red-skinned sport
cultivars (‘Ruby Okuyama’ and ‘Flame Muscat’). They
concluded that a mutation of a regulatory gene controlling
UFGluT gene expression caused the change from white to
red in the sports.
In apples, on the other hand, there have been only a few
molecular studies on the anthocyanin biosynthesis pathway.
Dong et al. [3] investigated the changes in the expression of
the anthocyanin biosynthetic genes during apple flower
development and showed that the genes were controlled byboth development and light. Cheng et al. [2] isolated
co-dominant randomly amplified polymorphic DNA(RAPD) markers linked to apple skin color, which showedthat the trait of red skin color was primarily controlled by
one or more dominant genes at a single locus in apple. To
date, however, little is known about the roles of these genes
on the anthocyanin accumulation during red coloration inapple skin.
There are two peaks of anthocyanin biosynthesis in apple
fruits, one in young fruitlets and the other in ripening fruits.
We focused on the later stage, which is economicallyimportant for market value, and studied the relationshipbetween anthocyanin accumulation and the expression ofanthocyanin biosynthetic genes at this stage. In this study,
we describe the isolation of the five genes of anthocyanin
biosynthetic enzymes, chalcone synthase (CHS), flavanone
3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR),
anthocyanidin synthase (ANS), and UFGluT (Fig. 1), from
‘Jonathan.’ We also indicate that the five genes are coordinately expressed during apple fruit development and that thelevels of expression are positively correlated with thedegree of anthocyanin concentrations in ripening fruits of‘Orin,’ ‘Fuji,’ and ‘Jonathan.’

2. Results
2.1. Gene cloning and sequence analysis
The cDNAs containing the complete coding region(MdCHS, MdF3H, and MdANS) or the cDNA fragments(pDFR and pUFGluT) for the anthocyanin biosynthetic
genes were isolated from ‘Jonathan’ in two ways. First, inthe course of expressed sequence tag (EST) analysis from acDNA library of ‘Jonathan’ floral buds, we identified
MdCHS, MdF3H, and MdANS. The CHS partial sequence in

Fig. 1. The putative anthocyanin biosynthetic pathway in apple skin. CHS,
chalcone synthase; CHI, chalcone isomerase; F3H, flavanone
3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; DFR, dihydroflavonol
4-reductase; ANS, anthocyanidin synthase; UFGluT, UDP glucose:flavonoid 3-O-glucosyltransferase; UFGalT, UDP galactose:flavonoid 3-Ogalactosyltransferase; glu, glucose; gal, galactose. The enzymes whosegenes were used for Southern and northern blot analyses are underlined.

Malus sp. (DDBJ accession no. X68977) was previously
reported, and our MdCHS from ‘Jonathan’ was the firstisolated gene with the complete coding region. The coding
region of MdCHS was 1176 bp long, with a 391-deduced
amino acid sequence and a predicted molecular mass of
42.6 kDa. MdCHS was highly homologous with the CHSs
from other plants. The deduced amino acid sequence of
MdCHS showed 89.7% identity with that of Citrus sinensis
CHS (AB009350), 89.7% identity with that of Lycopersiconesculentum CHS (X55195), 89.2% identity with that of
V. vinifera CHS (X75969), 87.7% identity with that of
Antirrhinum majus CHS (X03710), and 83.9% identity withthat of Zea mays CHS (X60204) (data not shown). In
addition to MdCHS, cDNA clones for MdF3H and MdANS
containing the complete coding regions were also isolated
from this library. Both of the corresponding genes havealready been isolated from ‘Fuji’ (AF117270 andC. Honda et al. / Plant Physiol. Biochem. 40 (2002) 955–962

AF117269, respectively). The coding sequence of‘Jonathan’ MdF3H (1098 bp long) showed 94.5% identitywith that of ‘Fuji’ F3H at the nucleotide level, and the
deduced amino acid sequence of MdF3H showed 96.7%
identity with that of ‘Fuji’ F3H at the amino acid level (datanot shown). The coding sequence of ‘Jonathan’ MdANS(1074 bp long) was substituted by only two nucleotides andone amino acid residue compared with that of ‘Fuji’ ANS(data not shown).
Secondly, the partial fragments for DFR and UFGluT
were amplified by reverse transcription–polymerase chain
reaction (RT–PCR) using specific primers based upon thecorresponding genes from ‘Fuji’ (AF117268 for DFR and
AF117267 for UFGluT). A single band of the respective
expected size (1.0 kbp for DFR and 1.2 kbp for UFGluT)
was amplified from cDNA synthesized from the total RNA
of ‘Jonathan’ fruit skin, then sub-cloned into a pCRII vector,
and finally sequenced to confirm the homology. The partial
fragments for pDFR (943 bp long) and pUFGluT (1175 bplong) of ‘Jonathan’ showed 99.6% and 98.8% identities tothose of ‘Fuji’ at the nucleotide level, respectively.
These clones of MdCHS, MdF3H, MdANS, pDFR, andpUFGluT have been deposited in DDBJ under the accession
numbers AB074485, AB074486, AB074487, AB074488,
and AB074489, respectively.
2.2. Southern blot analysis
To estimate the copy number of these structural genes in
the apple genome, the genomic DNA from ‘Jonathan’ leaves
was digested with EcoRI, BamHI, DraI, KpnI, HindIII, or
XbaI, and Southern blot analysis was performed under highstringency using the five genes listed above as the probes(Fig. 2). Hybridization with MdCHS as the probe gave one
to three major fragments in the DraI, HindIII, and XbaI
digestions. A number of weak bands were observed in all
digestions, indicating that CHS was encoded by a multigene
family in the apple genome. Two major fragments andanother weak fragment hybridized with the MdF3H probe
were detected in the DraI and HindIII digestions, and two
major fragments were found in the XbaI digestion. pDFR
showed two weak fragments in the BamHI digestion, twomajor fragments in the DraI digestion, and three major
fragments in the EcoRI digestion. MdANS showed one
major band in the DraI digestion, two weak bands in the
KpnI digestion, and three major bands in the XbaI digestion.
pUFGluT showed one major band in the BamHI digestion,
three major bands in the DraI digestion, and two bands inthe EcoRI digestion. Collectively, these results suggested
that a few copies for each F3H, DFR, ANS, and UFGluT
might be present in the apple genome.
2.3. Total anthocyanin accumulation
To investigate anthocyanin accumulation, we chose threeapple cultivars, ‘Orin,’ ‘Fuji,’ and ‘Jonathan,’ which differed957

Fig. 2. Southern blot analysis of the five anthocyanin biosynthetic genes inthe ‘Jonathan’ apple. Each lane contained 10 µg of genomic DNA from
‘Jonathan’ leaves digested with each restriction enzyme. B, BamHI; D,
DraI; E, EcoRI; H, HindIII; K, KpnI; X, XbaI. Positions of the molecularmass markers are shown at left.

in fruit color genotypes according to a report by Cheng et al.
[2]. Apple skin was collected three times during fruit
development, and total anthocyanin concentrations were
spectrophotometrically measured at 530 nm (Fig. 3). Since
the ripe stages differed among the three cultivars, the last
harvest dates were dependent on them. The harvest dates
were described as days after full bloom (DAFB). In ‘Orin,’a significant accumulation of anthocyanin was not detectedin any stage (data not shown). In ‘Fuji,’ no anthocyanin
accumulation occurred at 106 DAFB, but it was notably
induced at 142 DAFB, resulting in a 4.2-fold increase from
142 to 176 DAFB. In ‘Jonathan,’ no significant accumulation of anthocyanin was detectable at 106 DAFB; thereafter,a rapid accumulation from 142 to 154 DAFB occurred, witha 2.2-fold increase during the last 12 d.

Fig. 3. Change of total anthocyanin concentrations during fruit maturation.
Fruits of ‘Fuji’ and ‘Jonathan’ were harvested on the indicated day. Three
to six skin disks were used for each measurement. Vertical lines indicate
standard deviation. DAFB, days after full bloom.

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Fig. 4. Northern blot analysis of the five anthocyanin biosynthetic genes
during the ripening of ‘Orin,’ ‘Fuji,’ and ‘Jonathan’ fruits. Apple fruits were
harvested on the indicated day. Each lane contained 10 µg of total RNA
from apple skin. In the lower panel, the total RNA on the gel was stainedwith ethidium bromide to confirm equivalent loading. DAFB, days after
full bloom. Lanes 1–3, ‘Orin;’ lanes 4–6, ‘Fuji;’ lanes 7–9, ‘Jonathan.’Fig. 5. HPLC analysis of anthocyanins in ‘Fuji’ and ‘Jonathan.’ The
identity and the molar ratio were established by chromatography ofauthentic standards. The concentration of each standard is 8.3 ppm. cy
3-gal, cyanidin 3-galactoside; cy 3-glu, cyanidin 3-glucoside; cy 3-ara,
cyanidin 3-arabinoside.

2.4. Expression patterns of the five anthocyanin
biosynthetic genes during fruit development

fied were 3-glucoside and 3-arabinoside [21,23]. Recently,
Lister et al. [14] showed by reverse-phase high performanceliquid chromatography (HPLC) that the apple peel of‘Splendour’ contained cyanidin 3-galactoside. GómezCordovés et al. [5] also showed by reverse-phase HPLC thatcyanidin 3-galactoside was the major anthocyanin in the‘Starking Delicious’ apple. However, there is no available
information by HPLC on ‘Orin,’ ‘Fuji,’ or ‘Jonathan.’ Thus,
the molecular components of the anthocyanin pigments inthe three cultivars, ‘Orin,’ ‘Fuji,’ and ‘Jonathan,’ wereanalyzed by reverse-phase HPLC. A representative result of
the chromatograms is shown in Fig. 5. None of the
3-glycosides was detected by HPLC, not even in the ripe
fruit of ‘Orin’ at 168 DAFB (data not shown). In ‘Fuji’ at
176 DAFB, 3-glucoside was not detected; instead,
3-galactoside and 3-arabinoside were identified with amolar ratio of 95 to 5. Furthermore, 3-glucoside was also
not detected, and 3-galactoside and 3-arabinoside wereidentified with a molar ratio of 96 to 4 in ‘Jonathan’ at
154 DAFB.

Northern blot analysis was done using the same probes asin Southern blot analysis. Each of the five probes generated
one band corresponding to the expected size in the hybridization (Fig. 4). In ‘Orin,’ the expression of the fiveanthocyanin biosynthetic genes was hardly detectable atboth 106 and 142 DAFB (lanes 1 and 2); a very low
expression of MdCHS, MdF3H, pDFR, MdANS, and pUFGluT was then observed at the ripe stage (168 DAFB; lane3). In ‘Fuji,’ the accumulation of the transcripts corresponding to the five genes was hardly detectable at 106 and142 DAFB (lanes 4 and 5), and all five genes were induced
at the ripe stage (176 DAFB; lane 6). In ‘Jonathan,’ little
expression of the five biosynthetic genes was detected at
106 DAFB (lane 7), and a slight expression was observedfor MdF3H, pDFR, MdANS, and pUFGluT at 142 DAFB
(lane 8). At the ripe stage (154 DAFB), all five genes were
induced at high levels (lane 9). These results indicated thatthe five genes are coordinately expressed during fruit
development and that the levels of expression are positively
related to the degree of anthocyanin concentration. However, considering that the anthocyanin accumulation had
partially started at 142 DAFB (Fig. 3), the beginning for thesignificant induction of the five genes was delayed.
2.5. HPLC analysis of anthocyanin in apple skinPrevious works using paper chromatography revealed
that the major pigment in the red skin of apple cultivars wascyanidin 3-galactoside and that the minor pigments identi-

3. Discussion
We demonstrated that anthocyanin accumulation was
positively correlated with the expression of at least the fiveanthocyanin biosynthetic genes (MdCHS, MdF3H, pDFR,
MdANS, and pUFGluT) in apple skin (Figs. 3, 4) and alsosuggested that the levels for the expression of the five genes
basically corresponded to the degree of anthocyanin concentration; that is, the induction of the genes in a yellow
cultivar, ‘Orin,’ was less pronounced, and that in the red

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cultivars, ‘Fuji’ and ‘Jonathan,’ was notable, with muchhigher expression levels in ‘Jonathan’ than in ‘Fuji.’ At
present, the reason for the time lag between the notableinduction of the gene expression and the anthocyanin
accumulation is still unclear. We assume that slight expression of the genes may be sufficient to produce some color,which might act as a trigger for anthocyanin accumulation.
A little expression of the biosynthetic genes did not induce
anthocyanin accumulation in ripening fruits of ‘Orin’ at
168 DAFB, but gave rise to its accumulation in immature
fruits of ‘Jonathan’ at 142 DAFB, suggesting that there areother factors controlling red coloration during fruit ripening
in addition to the regulation of gene expression.
Some enzymes involved in the anthocyanin biosynthetic
pathway in apple skin were studied during fruit development. Lister et al. [16] demonstrated that the activities ofphenylalanine ammonia-lyase (PAL; EC 4.3.1.5), chalcone
isomerase (CHI; EC 5.5.1.6), and UDP galactose:flavonoid
3-O-galactosyltransferase (UFGalT; EC 2.4.1.X) in the‘Splendour’ apple were correlated with anthocyanin accumulation during fruit ripening. Ju et al. [7,8] showed that
PAL activity was not correlated with anthocyanin accumulation in the ‘Delicious’ apple and that CHS activity was notcorrelated with anthocyanin accumulation in the ‘Delicious’and ‘Ralls’ apples exposed to light, whereas UFGalT
activity was positively correlated with its production in both
apple varieties. Moreover, they found that the rapid accumulation of anthocyanin was correlated with an increase in
DFR activity in the ‘Delicious’ apple [9]. In this paper, we
could not find a key gene whose expression was induced
accompanied by anthocyanin accumulation in the present
apple cultivars, such as UFGluT in grape [1,11]. These
results indicate that one single gene was not responsible forthe anthocyanin accumulation but at least five genes wereinvolved in the red coloration of apple skin.
The main anthocyanin identified in apple skin is cyanidin
3-galactoside, while the cyanidin 3-glucoside level is verylow (Fig. 5). This suggests that the key enzyme controlling
red coloration is not UFGluT but UFGalT in apple skin.
Recently, the genes for UFGalT have been isolated fromtwo plants, Vigna mungo [17] and Petunia hybrida [18].
Recombinant UFGalT from P. hybrida transferred the galactosyl moiety only to flavonoids, but not to anthocyanidins[18], whereas recombinant UFGluT from V. vinifera transferred the glucosyl moiety primarily to anthocyanidins [4].
The deduced amino acid sequence of the apple UFGluTfrom ‘Fuji’ possesses sequences in some regions that are
similar to the UFGluTs and also to the UFGalTs from otherplants (Fig. 6). The putative UDP-glucose binding domain[10] in the apple UFGluT region from 358 to 381 wasconserved among the UFGluTs and UFGalTs from otherplants. The amino acid sequences of the UFGluTs fromV. vinifera, Z. mays, and Hordeum vulgare showed 52.6%,
37.5%, and 35.9% identity, respectively, to the UFGluTfrom ‘Fuji’ (Table 1). The amino acid sequences of the
UFGalTs from P. hybrida and V. mungo showed 48.2% and959

44.1% identity, respectively, to the UFGluT from ‘Fuji’(Table 1). Since it is not possible to discriminate thesubstrate specificities (glucosyl moiety or galactosyl moiety,and anthocyanidins or flavonoids) based on their deduced
amino acid sequences, we cannot predict whether or not theapple UFGluT functions as UFGalT transferring the galactosyl moiety to cyanidin. Lister et al. [15] reported that acrude extract of flavonoid glycosyltransferase from apple
skin specially transferred the glycosyl moiety from sugar
nucleotide donors to the 3-position of the flavonoid nucleus
and that UDP-galactose showed the highest activity compared with other sugar donors. Further characterization ofsubstrate and sugar specificity of the apple UFGluT will benecessary to investigate its role in fruit coloration.

4. Methods
4.1. Plant materials
All apple [Malus sylvestris (L.) Mill. var. domestica(Borkh.) Mansf.] samples were obtained from the orchard of
the National Institute of Fruit Tree Science at Morioka,
Japan. Whole ‘Jonathan’ flower buds were collected in the
middle of the growing season (from June to September) forthe construction of a cDNA library. Young ‘Jonathan’ leaves
were collected for genomic DNA isolation. Fruits from the‘Orin,’ ‘Fuji,’ and ‘Jonathan’ varieties were periodicallyharvested at a later developmental stage, and the entire skin
was removed with 1 mm of the cortical tissue for anthocyanin measurement and RNA isolation. Samples were frozen
in liquid nitrogen and stored at –80 °C until analyzed.
4.2. Isolation of MdCHS, MdF3H, and MdANS clones
from ‘Jonathan’Total RNA was extracted from ‘Jonathan’ whole flower
buds in the middle of the growing season (June to September) according to a cetyltrimethylammonium bromide(CTAB)-based method by Kotoda et al. [12]. First- andsecond-strand cDNAs were synthesized from total RNA byusing an oligo(dT) primer (a cDNA Synthesis System Pluskit, Amersham, Little Chalfont, UK). Following ligation
with an EcoRI/BamHI adaptor, double-stranded cDNAs thatwere more than 300 bp long were fractionated by electrophoresis on an agarose gel and ligated into the EcoRI site ofpBluescript II SK+ vectors (Stratagene, La Jolla, CA, USA).
After transformation with DH5α, bacterial white colonies
were randomly selected, and the plasmid DNAs wereextracted for nucleotide sequencing. Sequence analysis was
conducted by the fluorescence detection method on a
Hitachi SQ5500 automated sequencer (Hitachi, Tokyo,
Japan). BLAST searches were done against the sequencedata, and three clones, MdCHS, MdF3H, and MdANS,
showed high homology to the anthocyanin biosynthetic
genes, CHS, F3H, and ANS, respectively, from other plants.

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Fig. 6. Alignment of the deduced amino acid sequence of UDP glucose:flavonoid 3-O-glucosyltransferase (UFGluT) from Malus domestica (AF117267) with
UFGluT from Vitis vinifera (AF000372), UDP galactose:flavonoid 3-O-galactosyltransferase (UFGalT) from Vigna mungo (AB009370), UFGalT fromPetunia hybrida (AF165148), UFGluT from Zea mays (X13502), and UFGluT from Hordeum vulgare (X15694). Identical amino acids are indicated by black
shading; the similarities are shown by gray shading. The putative UDP-glucose binding domain is underlined.

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Table 1Comparison of the deduced amino acid sequence of flavonoid glycosyltransferases from higher plants. Accession numbers are given in Fig. 6
Organism

M. domestica

V. vinifera

P. hybrida

V. mungo

Z. maysH. vulgare

M. domestica
V. vinifera
P. hybrida
V. mungo
Z. maysH. vulgare

100.0
52.6
48.2
44.1
37.5
35.9

100.0
52.6
47.7
40.3
40.3

100.0
48.9
37.4
35.6

100.0
39.1
37.8

100.0
73.6

100.0

4.3. Isolation of pDFR and pUFGluT fragments from
‘Jonathan’Total RNA for RT–PCR was isolated from ‘Jonathan’fruit skin according to the CTAB method. First-strand
cDNA was synthesized from total RNA of ‘Jonathan’ skinusing a first-strand cDNA synthesis kit (Amersham Pharmacia). pDFR was amplified by using the primers 5'gagtccgaatccgtttgtgtca-3' and 5'-atgtttgtgggggctgtcgatg-3',
and pUFGluT was amplified by the primers 5'tccctttcactagccatgcaag-3' and 5'- gtggaggatggagtttttacc -3'
based on the sequences of the genes corresponding to thoseof ‘Fuji’ (AF117268 for DFR and AF117267 for UFGluT).
PCR for isolating pDFR was performed on the first-strand
cDNA under the following reaction condition: three cyclesof 30 s at 94 °C, 30 s at 55 °C, and 90 s at 72 °C; then, 27
cycles of 30 s at 94 °C, 30 s at 55 °C, and 90 s at 72 °C.
PCR for isolating pUFGluT was performed under thefollowing reaction condition: three cycles of 30 s at 94 °C,
30 s at 45 °C, and 90 s at 72 °C; then, 27 cycles of 30 s at
94 °C, 30 s at 50 °C, and 90 s at 72 °C. Amplified fragments
were cloned into the pCRII vector using a TA cloning
system (Invitrogen, San Diego, CA, USA) and sequenced
by the fluorescence detection method using a model 377A
sequencer (Perkin-Elmer Applied Biosystems, Foster City,
CA, USA).
4.4. Genomic DNA extraction and Southern blot
hybridization
Genomic DNA was isolated from mature leaves of a
‘Jonathan’ tree according to a CTAB-based method modified by Yamamoto and Mukai (unpublished). Approximately
1 g of apple leaves was ground to a fine powder in liquid
nitrogen and mixed with 10 ml of an isolation buffer (10%polyethylene glycol 6,000, 0.35 M sorbitol, 0.1 M Tris–HCl(pH 8.0), and 0.5% 2-mercaptoethanol). The mixture wascentrifuged at 7150 × g at 4 °C for 15 min, and the pellet
was suspended with 5 ml of a lysis buffer (0.35 M sorbitol,
0.1 M Tris–HCl (pH 8.0), and 0.5% 2-mercaptoethanol).
After the addition of 0.5 ml of 10% lauroylsarcosine, themixture was kept at room temperature for 10 min, mixed
with an equal volume of 2× CTAB (2% CTAB, 0.1 M
Tris–HCl (pH 9.5), 20 mM ethylenediamine-N,N,N',N'tetraacetic acid (EDTA), 1.4 M NaCl, and 1%
2-mercaptoethanol), and incubated at 65 °C for 20 min. The
mixture was then extracted with an equal volume ofchloroform/isoamylalcohol (24:1, v/v), and nucleic acids inthe aqueous phase were precipitated with an equal volumeof isopropanol and pelleted by centrifugation at 7150 × g at
4 °C for 10 min. Collected nucleic acids were dissolved in
0.75 ml of a TE solution (10 mM Tris–HCl (pH 8.0) and1 mM EDTA) containing 2 µg ml–1 RNase A and kept at
4 °C overnight. DNA was extracted once with an equalvolume of phenol and once with an equal volume ofchloroform/isoamylalcohol (24:1, v/v), and precipitatedwith an equal volume of isopropanol. After being collectedby centrifugation at 7150 × g at 4 °C for 10 min, DNA waswashed once with ethanol/water (7:3, v/v), dried, anddissolved in approximately 0.2 ml of the TE solution. The
quantity of the extracted genomic DNA was determined byelectrophoresis on a 0.7% agarose gel. After being digested
with BamHI, DraI, EcoRI, HindIII, KpnI, or XbaI, thegenomic DNA (10 µg) was electrophoresed on 0.8% agarose gels and blotted on nylon membranes (Hybond-N,
Amersham Pharmacia). Plasmid cDNA fragments of
MdCHS, MdF3H, pDFR, MdANS, and pUFGluT from
‘Jonathan’ were used to generate the probes using a DIG
DNA labeling kit (Roche Diagnostics, Mannheim, Germany). Prehybridization (2 h) and hybridization (overnight)
were done in a hybridization buffer containing 5× SSC, 1%
blocking reagent (Roche Diagnostics), 0.1% lauroylsarcosine, and 0.02% SDS at 68 °C. After hybridization, themembranes were washed twice at 68 °C for 15 min with
2× SSC and 0.1% SDS and then twice at 68 °C for 15 minwith 0.1× SSC and 0.1% SDS. The detection was performed
according to the protocol recommended by the supplierusing a DIG-CSPD system (Roche Diagnostics), and themembranes were exposed to X-ray film (Fuji Photo Film,
Tokyo, Japan).
4.5. Total RNA extraction and northern blot hybridization
Total RNA of ‘Orin,’ ‘Fuji,’ and ‘Jonathan’ was isolated
from the fruit skin. Approximately 200 µg of total RNA
were extracted from 10 g for each sample. Total RNA(10 µg) was electrophoresed on 1.1% agarose gels containing 5.0% (v/v) formaldehyde and then blotted on nylon
membrane (Hybond-N). The loading and transferring
equivalent of RNA was confirmed by staining the gels withethidium bromide and the nylon membrane with methylene
blue. Hybridization was done in a hybridization buffer
containing 50% formamide, 5× SSC, 2% blocking, 0.1%
lauroylsarcosine, and 0.02% SDS at 68 °C using the same

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C. Honda et al. / Plant Physiol. Biochem. 40 (2002) 955–962

probes as in Southern blot analysis. Washing and detection
were performed by the same procedure as used in Southernblot hybridization.
4.6. Measurement of total anthocyanin concentration
Three to six peel disks were randomly chosen for eachsample. Apple peel disks were placed in 25 volumes ofhydrochloric acid/methanol (1:99, v/v) at 4 °C at least for
6 h. Absorbance of each extract was measured at 530 nmwith a spectrophotometer (UV-1600, Shimadzu, Kyoto,
Japan).

[7]

[8]

[9]

[10]

[11]

4.7. HPLC analysis
HPLC was carried out with a Gulliver system (JASCO
Corporation, Tokyo, Japan) according to a method by Tada
et al. [22]. Anthocyanins of apple skin were extracted in 30
volumes of hydrochloric acid/methanol (0.1:99.9, v/v) andanalyzed on a reversed-phase column (4.6 mm × 250 mm,
Mightsyl RP-18 GP Aqua, Kanto Chemicals, Tokyo, Japan).
The solvent system used was a linear gradient of 25% to35% solvent B (phosphoric acid/acetic acid/acetonitrile/water, 1.5:20:25:53.5, v/v/v/v) in solvent A (phosphoric acid/water, 1.5:98.5, v/v) over a period of 30 min.
Samples were eluted at 40 °C and a flow rate of 1 ml min–1.
The HPLC elutes were monitored by absorbance at 530 nm,
and the peaks were identified by comparing their retention
time with authentic standards.

[12]

[13]
[14]

[15]

[16]

[17]

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