Mol Genet Genomics (2007) 278:295–305
DOI 10.1007/s00438-007-0250-0

ORIGINAL PAPER

Constitutive expression of two apple (Malus ⴛ domestica Borkh.)
homolog genes of LIKE HETEROCHROMATIN PROTEIN1aVects Xowering time and whole-plant growth in transgenic
Arabidopsis
Naozumi Mimida · Shin-ichiro Kidou ·Nobuhiro Kotoda

Received: 18 February 2007 / Accepted: 15 May 2007 / Published online: 19 June 2007
© Springer-Verlag 2007

Abstract Fruit trees, such as apple (Malus £ domestica
Borkh.), are woody perennial plants with a long juvenile
phase. The biological analysis for the regulation of Xowering time provides insights into the reduction of juvenile
phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1(LHP1) is involved in epigenetic silencing of the target
genes such as Xowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and
MdLHP1b. These genes may have been generated as aresult of ancient genome duplication. Although the putative
MdLHP1 proteins showed lower similarity to any otherknown plant LHP1 homologs, a chromo domain, a chromo
shadow domain, and the nuclear localization signal motifs
were highly conserved among them. RT-PCR analysis
showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout
the growing season. Constitutive expression of MdLHP1a
or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tX2 mutant, suggesting that apple LHP1 homolog

Communicated by S. Hohmann.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00438-007-0250-0) contains supplementarymaterial, which is available to authorized users.
N. Mimida · N. Kotoda (&)
Apple Breeding and Physiology Research Team,National Institute of Fruit Tree Science,
92-24 Shimo-kuriyagawa, Morioka,
Iwate 020-0123, Japane-mail: koto@aVrc.go.jp
S.-i. Kidou
Cryobiosystem Research Center,
Iwate University, Morioka, Iwate 020-8550, Japangenes are involved in the regulation of Xowering time andwhole-plant growth. Based on these results, LHP1 homolog
genes might have rapidly evolved among plant species, but
the protein functions were conserved, at least between Arabidopsis and apple.
Keywords Apple (Malus £ domestica Borkh.) ·Flowering time · LIKE HETEROCHROMATIN PROTEIN1(LHP1) · TERMINAL FLOWER2 (TFL2) · Ploidy ·Hybridization
Abbreviations
FBS
Fruit-bearing shootLD
Long-daySD
Short-daySS
Succulent shootVS
Vegetative shootIntroduction
The transition from juvenile to adult phase in plant species
requires environmental cues, such as temperature, lightintensity, and day length, and internal cues, such as phytohormones, age, and body size and thus the perennial fruit
trees, such as apple (Malus £ domestica Borkh.), have along juvenile phase lasting about 4-8 years (Hackett 1985;
Dennis Jr 2003). The long juvenile phase is the primaryfactor that limits the eYcient breeding and the genetic andmolecular analyses of fruit trees. Therefore, the studies ofthe molecular basis of Xower diVerentiation would provide
insights into the solution of such issues in the area of horticulture. Because apple is one of the most economicallyimportant fruit tree crops in the world, many researchers
have studied this one genetically and physiologically from

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the view point of both industry and scientiWc interest. A
decade ago, molecular studies on the morphogenesis inapple also started and several genes related to plant development have been isolated and characterized. Recent studies on the genetic control of Xowering time in apple suggestthat MdTFL1, a TERMINAL FLOWER1(TFL1)-like gene ofapple, is a key regulatory gene involved in the maintenanceof the juvenile and vegetative phase in apple (Kotoda et al.
2006). However, little is known about other genes regulating the Xowering time of apple, and much less about thegenetic framework of the life cycle of apple.
On the other hand, studies on Arabidopsis thaliana have
led to the identiWcation of many genes controlling the Xowering time. Previous studies revealed that some mutants
lacking the genes encoding Polycomb group (PcG) proteinsor chromatin-remodeling factors show early Xowering.
CURLY LEAF (CLF) was Wrst identiWed as a PcG gene inplant, which represses AGAMOUS (AG) expression (Goodrich et al. 1997). The EMBRYONIC FLOWER2 (EMF2)
encoding a PcG protein with a zinc Wnger motif is required
for maintaining the vegetative development and regulating
the development of the inXorescence and Xoral meristem(Yoshida et al. 2001). These PcG family proteins form
large multimeric complexes and regulate developmental
processes by preventing inappropriate expression of target
genes (Chanvivattana et al. 2004). For example, CLF/
SWINGER (SWN), EMF2, FERTILISATION-INDEPENDENT ENDOSPERM (FIE), and MULTICOPY SUPPRESSOR OF IRA1 (MSI1) constitute a PcG complex,
which predominantly maintains AG expression in a transcriptionally repressed state (Chanvivattana et al. 2004).
Arabidopsis LIKE HETEROCHROMATIN PROTEIN1(LHP1) [TERMINAL FLOWER2 (TFL2)] is a single-copy
gene encoding a homolog protein of HETEROCHROMATIN PROTEIN 1 (HP1), which is composed of two characteristic domains: an N-terminal chromatin organization
modiWer (chromo) domain (CD) and a C-terminal chromo
shadow domain (CSD) (Gaudin et al. 2001; Kotake et al.
2003). The HP1-family proteins are known to regulate
chromatin function from mammals to yeast, and may act
similarly as PcG proteins by silencing genes (Maison andAlmouzni 2004). Animals have three isoform proteins,HP1, HP1, and HP1, whereas plants harbor a singlecopy gene encoding a homologous protein more related toHP1 according to its euchromatin localization (Minc et al.
2000; Zemach et al. 2006). The loss-of-function lhp1/tX2
allelic mutants showed the pleiotropic phenotypes, such as
early Xowering, terminal Xower, small plant size, andcurled leaf (Larsson et al. 1998; Gaudin et al. 2001; Kotake
et al. 2003). In addition, the double mutants of tX1 and tX2
showed a more severe phenotype than that of a single
mutant (Larsson et al. 1998). DNA microarray analysis in
the tX2 mutant showed that the expression levels of euchro-123

Mol Genet Genomics (2007) 278:295–305

matin genes, such as FLOWERING LOCUS T (FT), which
is a Xowering integrator gene, and APETALA3, PISTILLATA, AG, and SEPALLATA3, which are Xoral homeotic
genes, are up-regulated (Kotake et al. 2003; Takada andGoto 2003; Nakahigashi et al. 2005; Germann et al. 2006).
Thus, the pleiotropic phenotypes of the lhp1/tX2 mutants
are likely to be reXected by the fact that LHP1 represses theexpression of multiple genes.
In this study, we report the isolation and characterizationof two LHP1 homolog genes in apple, MdLHP1a and
MdLHP1b. These genes showed lower identity to otherknown LHP1 homologs in plant species, but constitutive
expression of MdLHP1a or MdLHP1b rescued the Arabidopsis lhp1/tX2 mutant. Consequently, LHP1 homolog genes
in the apple of woody perennial may also be involved in theregulation of the Xowering pathway, and conserved functionally in plant species. In addition, these apple LHP1
homolog genes may have been duplicated because the
Maloideae subfamily possesses the complex genome
derived from the origin of polyploidy.

Materials and methods
Plant materials
DiVerent organ tissues and shoot apices of apple(Malus £ domestica Borkh.) cultivars (cv.) ‘Fuji’ and ‘Jonathan’ were collected from the experimental Weld at the
National Institute of Fruit Tree Science in Morioka, Japan.
These cultivars were used for the seasonal and the tissue
speciWc expression analyses because they are popular cultivars and the genetic background is not so diVerent between
them. The seedlings from ‘Fuji’ apple were used for the
expression analysis of MdLHP1a and MdLHP1b in thejuvenile phase. The A. thaliana tX2-2 loss-of-function
mutant was obtained from the Arabidopsis Biological
Resource Center at the Ohio State University and employed
for the mutant rescue experiment.
cDNA library construction and cloning of MdLHP1aand MdLHP1b
Total RNA was isolated from Xower buds of ‘Fuji’ apple
collected on April 26, 2004, in Morioka, Japan, with the
CTAB method as described by Kotoda et al. (2000). Poly(A)+ RNA was isolated using a PolyA tract mRNA puriWcation kit (Promega, Madison, WI, USA). The cDNA library
was constructed using a ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA, USA) and Packagene® Lambda DNA
Packaging System (Promega). The average size of theinserted cDNAs was about 1.3 kbp. PCR ampliWcation of
Arabidopsis LHP1 DNA fragments was carried out with aMol Genet Genomics (2007) 278:295–305

pair of primers (LHP1F: 5⬘-TAG GAA ACG GAA GCG
CAA ATA TGC AG-3⬘; LHP1R: 5⬘-CCA ACA CTT CCT
GGA CAT TGT CTG AT-3⬘). The PCR-ampliWed fragment(gLHP1, 696 bp) was cloned into the pT7Blue vector(Novagen, Darmstadt, Germany). The gLHP1 labeled with
[-32P]dCTP (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) was used as a probe to screen 1.0 £ 105 pfu
of the apple cDNA library. A positive plaque was excised tothe pBluescript SK(¡) phagemid clone (MdLHP1a1), andthis clone was then sequenced. Then, 5.0 £ 105 pfu of thecDNA library was screened with a MdLHP1a1 probelabeled with digoxigenin (DIG, Roche Diagnostics, Mannheim, Germany), which was ampliWed by PCR with a pair ofprimers designed for the 5⬘ region (MdLHP1F-NheI: 5⬘GTA GCT AGC ACA AAT GAG AAC CAA GG-3⬘ with
NheI site in bold and ATG in italic; MdLHP1-600R: 5⬘GAC GAT GCC AAT TTC CAC AT-3⬘). The hybridization
was performed in DIG Easy Hyb (Roche Diagnostics) at
42°C for 16 h followed by two rinses in 2£ SSC containing
1% (w/v) SDS at room temperature for 5 min and two
washes in 0.5£ SSC containing 1% (w/v) SDS at 68°C for
20 min. Chemiluminescent signals were visualized using the
LAS1000 image analyzer (Fuji Photo Film, Aichi, Japan).
PCR ampliWcation of MdLHP1a genomic DNA was carriedout in a mixture of a pair of primers [MdLHP1F-NheI and
MdLHP1a1R-XhoI: 5⬘-TGC CTC GAG ACT AAA ATG
TAG GGT TGT AT-3⬘ with XhoI site in bold and CTA (stopcodon) in italic], EX-Taq DNA polymerase (Takara Biomedical, Tokyo, Japan), and 250–300 ng of genomic DNA
as a template. PCR was programmed for preheating at 96°C
for 3 min followed by 30 cycles of 96°C for 20 s, 58°C for
30 s, and 72°C for 5 min. A 4.6-kb PCR-ampliWed productwas cloned into the pT7Blue vector (Novagen).
Sequence analyses
The nucleotide sequence was determined using a DTCS
Quick Start Kit for Dye Terminator Cycle Sequencing(Beckman Coulter, Fullerton, CA, USA) and an automated
DNA sequencer CEQ 8000 (Beckman Coulter). Nucleotide
and amino acid sequences were analyzed using CLUSTAL
X multiple sequence alignment program Version 1.83
(Jeanmougin et al. 1998) and GeneDoc (Nicholas et al.
1997). The phylogenetic tree was displayed using the
Njplot program (Perrière and Gouy 1996). A homology
search was performed with BLAST2 of the GenomeNet(http://www.genome.ad.jp/).

297

apple, was digested with EcoRI, PstI, BamHI, or EcoRVand then electrophoresed on a 0.8% agarose gel. The DNA
bands were blotted onto a nylon membrane Hybond-N+(GE Healthcare Bio-Sciences Corp.) and hybridized withthe full-length cDNA of MdLHP1a1 labeled with DIG by
PCR as a probe. The hybridization signals were detected in
the same manner as described in the section of cloning.
Expression analyses by RT-PCR
and Northern hybridization
Total RNAs were extracted from Xower buds (late April),fruit receptacles, and apical buds of fruit-bearing shoots(FBS) and succulent shoots (SS) (from June to October in2004) of adult ‘Jonathan’ apple trees and stems, apical budsof vegetative shoots (VS), and leaves of 1-month-old seedlings and cotyledons and roots of 6-day-old seedlings. For
RT-PCR analysis, Wrst-strand cDNAs were synthesizedfrom 2 g of total RNAs in 30 l of reaction mixture using
the ReverTra Ace kit (Toyobo, Tokyo, Japan). The subsequent PCR reactions were performed with 0.5 l of the
Wrst-strand cDNA reaction mixture as templates. A fragment of MdLHP1a1/-a2 or MdLHP1b1/-b2 was ampliWedwith the following primers: sense primer (5⬘-CAG CAGCGT TCT ACT GAT CCT ATT TAC AAT-3⬘) and antisense primer (5⬘-GAT CCA GAT ACA AAT CTC ATAATA TTT CCT AAT ACA G-3⬘) for MdLHP1a1/-a2 andsense primer (5⬘-CAG AGC AAT CGC AGC ACA CTG
GA-3⬘) and antisense primer (5⬘-CTC ATC CAA ACT
CCG ACT AGT CCC-3⬘) for MdLHP1b1/-b2. As an internal control, a fragment of apple Histone H3 was ampliWedwith the following primers: sense primer (5⬘-TGA AGA
AGC CCC ACA GAT A-3⬘) and antisense primer (5⬘-ACACAA GAA ACT ATA AAC C-3⬘) as described by Kotoda
et al. (2006). Thermal cycle programs were as follows:
96°C for 3 min followed by 40 cycles of 96°C for 20 s,
65°C for 30 s, and 72°C for 3 min for MdLHP1a1/-a2 and
MdLHP1b1/-b2 and 96°C for 3 min followed by 32 cyclesof 96°C for 20 s, 56°C for 30 s, and 72°C for 30 s for Histone H3. For Northern analysis, total RNA from wild-type
and transgenic Arabidopsis plants was electrophoresed on a
denaturing 1.2% agarose gel with formaldehyde and thenblotted onto a Hybond N+ membrane (GE Healthcare BioSciences Corp.). The blotted membrane was hybridized
with a DIG-labeled MdLHP1a1 probe. The hybridization
was performed in DIG Easy Hyb at 50°C for 16 h. The
wash and detection were performed in the same manner asthat used for DNA.

Southern blot analysis
Construction of transformation vector
Southern blot analysis was carried out according to themethod described by Sambrook et al. (1989). Fifteen
micrograms of genomic DNA, isolated from the leaves of

To construct a vector for the constitutive expression of
MdLHP1a, the coding region of MdLHP1a1 was ampliWed

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by PCR with a pair of primers MdLHP1F-NheI and
MdLHP1a1R-XhoI. The PCR products were subsequently
digested with NheI and XhoI and then cloned into the XbaI–SalI sites of the plasmid vector pBI9526 (a derivative of
pBI221) bearing the cauliXower mosaic virus (CaMV) 35S
promoter fused with the  sequence (Gallie and Walbot
1992) and the nopaline synthase terminator (Tnos) (designated as 35S:MdLHP1a1:Tnos/pBI9526). After
35S:MdLHP1a1:Tnos/pBI9526 was digested with
EcoRI, the DNA fragment of 35S:MdLHP1a1:Tnos wascloned into the respective sites of the pSMAK312Blue
binary vector. The binary vector obtained was named35S:MdLHP1a1. To construct a vector for constitutive
expression of MdLHP1b, the coding region of MdLHP1b1
was ampliWed with a pair of primers MdLHP1F-NheI and
MdLHP1b1R-KpnI [5⬘-ATC GGT ACC TTA AAA TGTAGG ATT GTA TCG-3⬘ with KpnI site in bold and TTA(stop codon) in italic]. It was then cloned into the XbaI–
KpnI sites of modiWed pSMAK193E (35S/pSMAK193E)
to be placed between the promoter 35S and the terminatorof the Arabidopsis rubisco gene (TrbcS). The resultant
binary vector was named 35S:MdLHP1b1. The pSMAK
binary vector series, pSMAK312Blue and pSMAK193E,
were made from the pSMAK251 vector. The characterization of pSMAK251, which contains genes encoding -glucuronidase (GUS) and neomycin phosphotransferase II(NPTII) in its T-DNA region, will be published elsewhere(H. Ichikawa, in preparation).
Arabidopsis transformation
Wild-type (ecotype Columbia) and tX2-2 Arabidopsis
plants were transformed with 35S:MdLHP1a1 and
35S:MdLHP1b1 using an Agrobacterium tumefaciens
strain, EHA101 (Hood et al. 1986), by the inWltration
method as described by Bechtold et al. (1993). Bleach-sterilized seeds were place on MS plates (Murashige andSkoog 1962) containing carbenicillin (250 mg/l) and kanamycin (35 mg/l). Arabidopsis seeds of the T3 plant (primary transformants were deWned as T1 plants) were sownon agar plates followed by stratiWcation at 4°C for 3 days
and grown in a growth chamber controlled at 22°C underlong-day (LD) conditions (16 h light/8 h darkness) or shortday (SD) conditions (8 h light/16 h darkness).
Table 1 The features of fourapple LHP1-like cDNAs

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Results
Isolation of four cDNAs of LHP1 homolog genes
from apple
To isolate the apple LHP1 homolog genes, the PCR-ampliWed DNA fragment (696 bp) of Arabidopsis LHP1 wasused as a probe to screen a cDNA library prepared fromXower buds of the apple cultivar (cv.) ‘Fuji’. One positiveplaque was obtained after screening »100,000 plaques.
The cDNA derived from the plaque was designated as
MdLHP1a1 (DDBJ accession number, AB290726) after
Malus £ domestica LHP1. Moreover, we screened about
500,000 plaques from the cDNA library with an
MdLHP1a1 probe. A total of seven positive plaques wereobtained and then subjected to sequence analysis, revealing that they could be classiWed into four diVerent cDNAs
including MdLHP1a1, three of which were designated as
MdLHP1a2, MdLHP1b1, and MdLHP1b2 (DDBJ accession numbers, AB290727, AB290728, and AB290729,
respectively). The features of four apple LHP1-like
cDNAs are collectively shown in Table 1. The alignment
in nucleotide sequence showed that MdLHP1a1 was 99.1
and 90.4% identical to MdLHP1a2 and MdLHP1b1,
respectively, and that MdLHP1b1 was 99.1% identical to
MdLHP1b2. In comparison with the deduced amino acidsequences of the apple LHP1-like genes, four substitutions
were found between MdLHP1a1 and MdLHP1a2 andbetween MdLHP1b1 and MdLHP1b2. On the other hand,
35 substitutions and Wve deletions/insertions were foundbetween MdLHP1a1 and MdLHP1b1 (Fig. 1a). These
results suggested that a pair of MdLHP1a1 and
MdLHP1a2 and a pair of MdLHP1b1 and MdLHP1b2
would be allelic genes. The predicted amino acidsequences of MdLHP1a1 shared lower similarity to thoseof the HP1 homolog proteins from other plant and animal
species [45.1% similarity to SlLHP1 (tomato), 41.6% to
AtLHP1 (Arabidopsis), 34.4% to ZmLHP1 (maize), 34.2%
to OsLHP1 (rice), 31.3% to DcLHP1 (carrot), 16.7% to
DmHP1 (fruit Xy), and 14.6% to HsHP1 (human)],whereas the CD (79.6, 98.0, 89.8, 87.8, 87.8, 67.3, and63.3% similarities, respectively), CSD (64.8, 62.0, 66.2,
69.0, 71.0, 22.5, and 32.4% similarities, respectively), andthree nuclear localization signal motifs among them were

cDNA inlength (bp)5⬘-UTR in
length (bp)3⬘-UTR in
length (bp)Amino acidsin length

Predicted molecularmass (kDa)

MdLHP1a1

1,845

79

404

453

50.1

MdLHP1a2

1,804

75

367

453

50.2

MdLHP1b1

1,795

152

272

456

50.4

MdLHP1b2

1,793

147

275

456

50.4

Mol Genet Genomics (2007) 278:295–305

299

Fig. 1 Comparison of the predicted amino acid sequences of
MdLHP1s and HP1 homologs from plant and animal species. a Schematic alignment of the predicted amino acid sequences of HP1-likeproteins from apple (MdLHP1a1, MdLHP1a2, MdLHP1b2, and
MdLHP1b2, in this paper), tomato (SlLHP1/LeHP1, AAL25116), Arabidopsis (AtLHP1/TFL2, BAB70689), maize (ZmLHP1, AAM93210),
rice (OsLHP1, NP_920664), carrot (DcLHP1, BAA25905), fruit Xy(DmHP1, P05205), and homo sapiens (HsHP1, NP_036249). Identicalresidues are outlined in white against black. Highly conserved regionsof the chromo domain (CD), chromo shadow domain (CSD), and putative nuclear localization signal (NLS) are indicated by boxes for CD
and CSD and underlines for NLS (Gaudin et al. 2001; Kotake et al.
2003; Zemach et al. 2006). b Phylogenetic tree of the HP1-like proteins. The branch lengths are proportional to the sequence divergence.
Numbers along branches are bootstrap values (1,000 replicates). The
scale bar represents 0.05 substitutions/site

well conserved, except those of DmHP1 and HsHP1(Fig. 1a). The phylogenetic analysis indicated that apple
LHP1 homologs constitute a small clade and are most similar to tomato SlLHP1 in the HP1 family (Fig. 1b).

addition, PCR products (4.6 kb) of apple genomic DNA
ampliWed with primers for MdLHP1a1/-a2 were digested
with each restriction enzyme used in Southern blot analysis
to conWrm the internal restriction enzyme sites in the probe
region (S1). Furthermore, the 4.6-kb PCR products werecloned to characterize the gene structures of MdLHP1a1/-a2.
Eight individual clones were selected randomly, and theirsequences were analyzed. Among them, two clones had thesame coding sequence as MdLHP1a1, which was designated gMdLHP1a1 (DDBJ accession number, AB292418).
The remaining six clones contained the same coding
sequence as MdLHP1a2, designated gMdLHP1a2 (DDBJ
accession number, AB292419). The gMdLHP1a1 sequence
contained PstI and EcoRV sites, whereas gMdLHP1a2 has

Genomic organization
Southern hybridization was performed to assess the copy
number of LHP1 homolog genes in the apple genome(Fig. 2). The genomic DNA of the apple cv. ‘Fuji’ wasdigested with EcoRI, PstI, BamHI, or EcoRV, and the blotted membrane was then subjected to hybridization withfull-length MdLHP1a1 cDNA. Two to three major hybridizing bands were detected in each digested sample. In

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Mol Genet Genomics (2007) 278:295–305

MdLHP1a was about 28 times longer than that in LHP1
(Fig. 3).
Expression patterns of MdLHP1a and MdLHP1b in various
tissues and in apical buds throughout the growing season
in apple

Fig. 2 Southern hybridization analysis of LHP1 homolog genes inapple. Genomic DNA (10 g) of ‘Fuji’ apple digested with EcoRI, PstI, BamHI, or EcoRV was electrophoresed in a 0.8% agarose gel, blotted onto nylon membranes, and hybridized with an MdLHP1a1 probe.
The molecular size marker is shown in kbp on the left

We performed RT-PCR analysis to clarify the expression
patterns of MdLHP1a and MdLHP1b in apple. Total RNAs
were prepared from various tissues, such as Xoral buds
before bud break in late April; receptacles; apical buds of
FBS in August; leaves of the apple cv. ‘Fuji’ in the adultphase; stems, apical buds of VS, and leaves of 1-month-old
apple seedlings; and cotyledons and roots of 6-day-old
apple seedlings in the juvenile phase. Transcripts of
MdLHP1a were detected mainly in apical buds in both theadult and juvenile phases (Fig. 4a). On the other hand, transcripts of MdLHP1b were detected in all tissues used, withrelatively higher expression in the apical buds of FBS in theadult phase (Fig. 4a). To investigate the change of theexpression of MdLHP1a and MdLHP1b in the apical budsof FBS, which have Xowers and fruit in the following year,
and SS, which are called water sprouts and have no Xowers
or fruit, RT-PCR analyses for MdLHP1a and MdLHP1b
were carried out using total RNAs extracted from the apicalbuds of FBS and SS shoots of the apple cv. ‘Jonathan’ from
June to October 2004. ‘Jonathan’ apple was used in the seasonal expression analysis, because Xower development of
this cultivar had been studied well. It is noteworthy that the
Xowering stage of ‘Jonathan’ apple was early May, and thetime of Xoral bud diVerentiation was early July in Morioka,
Japan (Kotoda et al. 2000). MdLHP1a and MdLHP1b wereexpressed constantly in apical buds of both kinds of shoots
throughout the growing season (Fig. 4b).

only an EcoRV site (Fig. 3). The band pattern of the PCR
products digested with each restriction enzyme (S1) was
consistent with the result of the sequence analysis of them.
These Wndings, together with the results described in thesection above, showed that ‘Fuji’ apple had two loci of
LHP1 homologs, MdLHP1a and MdLHP1b, each having
two alleles, MdLHP1a1/-a2 and MdLHP1b1/-b2, respectively. The MdLHP1a1/-a2 (MdLHP1a) gene comprised
six exons and Wve introns, such as the LHP1 gene (Fig. 3).
Moreover, the length of exons was well conserved between
LHP1 and MdLHP1a, although the fourth intron in

In order to analyze the functions of MdLHP1a and
MdLHP1b using transgenic Arabidopsis, we generated two

Fig. 3 Schematic representation of the apple MdLHP1a gene in comparison with the Arabidopsis LHP1 gene. The PCR products covering
the MdLHP1a1 and MdLHP1a2 alleles are shown as a solid line, andthe arrowheads indicate the region for gene-speciWc primers. Introns

are shown as solid lines, while exons are shown as black boxes andnumbered with Roman numerals above the black boxes. The lengths(bp) of introns and exons are indicated below (introns) and above(exons) the map. The scale bar represents 500 bp

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Rescue of the Arabidopsis tX2 (lhp1) mutant by constitutive
expression of apple MdLHP1a1 or MdLHP1b1

Mol Genet Genomics (2007) 278:295–305

301

Fig. 4 Expression analysis of MdLHP1a and MdLHP1b. a RT-PCR
products of MdLHP1a and MdLHP1b in various tissues. Total RNAs
were isolated from Xower buds, fruit receptacles, and apical buds offruit-bearing shoots (FBS) on August 7, from mature leaves of ‘Fuji’
apple in the adult phase, and from stems, apical buds of vegetative
shoots (VS), and leaves of 1-month-old seedlings and cotyledons androots of 6-day-old seedlings in the juvenile phase. b RT-PCR productsof MdLHP1a and MdLHP1b in apical buds of FBS or in apical buds of
succulent shoots (SS) of ‘Jonathan’ apple from June to October in
2004. The date of sampling the apical buds of both shoots is indicatedabove the lanes. Apple Histone H3 was used as an internal control ofgene expression

chimeric constructs, 35S:MdLHP1a1 and 35S:MdLHP1b1(Fig. 5a). The inWltration method (Bechtold et al. 1993) was
employed to introduce those constructs into the tX2-2(lhp1) null mutant (Kotake et al. 2003). Consequently, seventeen and seven independent transgenic lines were obtained
for the constructs 35S:MdLHP1a1 and 35S:MdLHP1b1,
respectively (described as 35S: MdLHP1a1/tX2-2 and35S:MdLHP1b1/tX2-2 for each transgenic plant, respectively). T3 transgenic plants were grown under LD conditions (16 h light/8 h darkness) for the examination of theirphenotypes, such as plant size, inXorescence morphology,
and Xowering time (the number of rosette leaves produced
at Xowering). Five lines of 35S:MdLHP1a1/tX2-2 andfour of 35S:MdLHP1b1/tX2-2 were selected to examine
the phenotypes. In the representative transgenic lines of35S:MdLHP1a1/tX2-2 (#1) and 35S:MdLHP1b1/tX2-2(#2), the phenotypes, such as the small plant size and curled
leaves that are characteristic of the tX2-2 mutant, were rescued (Fig. 5b). InXorescence shoots, including secondaryones, kept producing Xowers in the transgenic lines of35S:MdLHP1a1/tX2-2 and 35S:MdLHP1b1/tX2-2(Fig. 5e, f), showing that both MdLHP1a1 and MdLHP1b1
rescued the determinate inXorescences of the tX2-2 mutant.
The Xowering time of the transgenic plants was slightly
earlier than that of the wild-type plants but later than that of
the tX2-2 mutants (Table 2). 35S:MdLHP1a1/tX2-2 (#1)
plants Xowered with 8.0 rosette leaves, and 35S:MdLHP1b1/tX2-2 (#2) plants Xowered with 8.8 rosetteleaves. In contrast, wild-type plants Xowered with 8.9
rosette leaves, and tX2 plants Xowered with 5.3 rosetteleaves. The cell size in leaves of the lhp1 (tX2) mutant isdiVerent from that of the wild type under LD conditions(Gaudin et al. 2001). The plant size of wild-type Arabidopsis plants increases more when they are grown under SD
conditions (8 h light/16 h darkness) than under LD conditions. Because the tX2 mutant is photoperiod-insensitive,
the cell size of the tX2 mutant was apparently smaller thanthat of the wild type under SD conditions (Fig. 5g, h). We
observed the upper epidermis of a third rosette leaf in the
35S:MdLHP1a1/tX2-2 (#1) plant under SD conditions,
showing partial, if not complete, restoration in the cell sizeof the rosette leaves (Fig. 5i).
To examine whether transgenic Arabidopsis with
35S:MdLHP1a1 or 35S:MdLHP1b1 shows additional
novel phenotypes, these constructs were also introduced
into the wild-type Arabidopsis (Columbia). More than
thirty kanamycin-resistant lines were obtained for eachconstruct (described as 35S:MdLHP1a1/wt or 35S:MdLHP1b1/wt, respectively). Most of the T1 transgenic
plants showed slightly delayed Xowering (70–80% of alllines), but some of them showed pleiotropic phenotypes,
such as the tX2-2 mutants with early Xowering, terminal
Xowers, and curled leaves (10%) (data not shown).
These phenotypes were inherited by subsequent generations. The phenotypes of Wve representative T3 transgeniclines of 35S:MdLHP1a1/wt and four of 35S:MdLHP1b1/

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302

Mol Genet Genomics (2007) 278:295–305

Fig. 5 Constitutive expressionof MdLHP1a1 and MdLHP1b1
in the Arabidopsis tX2-2 mutant
and wild-type plants: eVects on
Xowering time and growth.
a Schematic representation of
the transformation vectors,
35S:MdLHP1a1 and
35S:MdLHP1b1 used in thisstudy. b Seventeen-day-old
plants after sowing. From left toright, tX2-2, wild-type,35S:MdLHP1a1/tX2-2 (#1),
and 35S:MdLHP1b1/tX2-2(#2). c–f InXorescences of theplants shown in b, which were
photographed 23 days aftersowing. g–i Micrographs of theepidermal cells on a third rosetteleaf from tX2-2 mutant plants(g), wild-type plants (h), and35S:MdLHP1a1/tX2-2 (#1)
transgenic plants (i), which weregrown on MS medium plates
under short-day (SD) conditions(8 h light/16 h dark). j Twentythree-day-old wild-type plants
and 35S:MdLHP1a1/wt (#3)
transgenic plants. k Hybridization pattern with an MdLHP1a1
probe for total RNA from transgenic and control Arabidopsis
plants. A photograph of the ethidium bromide-stained gel before
blotting is shown below. An
arrowhead indicates the expected size of the full-length mRNAof MdLHP1a1 and MdLHP1b1.
The molecular size marker isshown in kbp on the right. Scalebars are 2 cm in b and j, 5 cm inc–f, and 20 m in g–i. The plants
shown in b, c–f, and j weregrown in potted soil under longday (LD) conditions (16 h light/
8 h dark)

wt showing delayed Xowering were examined under LD
conditions. 35S:MdLHP1a1/wt (#3) plants Xoweredwith 10.4 rosette leaves, whereas wild-type plants Xowered with 8.9 rosette leaves (Fig. 5j; Table 2). Delayed
growth of transgenic plants with MdLHP1a or MdLHP1b
constructs appeared to develop a few additional rosetteleaves, although the appearance of the transgenic plants
was similar to that of the wild-type plants. Thus, constitutive expression of MdLHP1a1 or MdLHP1b1 could

123

aVect Xowering time in the wild-type Arabidopsis
plants.
Northern hybridization analysis in transgenic Arabidopsis
with MdLHP1a1 or MdLHP1b1
To determine whether or not the expression level of thetransgene is correlated with the degree of the phenotype intransgenic plants, Northern hybridization analysis wasMol Genet Genomics (2007) 278:295–305

303

Table 2 Flowering time of tX2-2 (lhp1) and wt plants transformed
with 35S:MdLHP1a1 and 35S:MdLHP1b1

Discussion

StrainNumberof plantsa

Number ofrosette leavesb

Number ofcauline leavesb

Wt (Col)
tX2-2

15

8.9 § 0.2

2.7 § 0.2

13

5.9 § 0.1

2.6 § 0.1

In this study, we isolated and characterized four cDNAs ofapple LHP1 homologs, MdLHP1a1, MdLHP1a2,
MdLHP1b1, and MdLHP1b2. The genomic Southernhybridization and sequence analyses revealed that the apple
cv. ‘Fuji’ had two loci of LHP1 homologs, MdLHP1a and
MdLHP1b, each having two alleles, MdLHP1a1/-a2 andMdFL2b1/-b2, respectively, and that the genomic structureof MdLHP1a was similar to that of Arabidopsis LHP1
(Fig. 3). A comparison of the amino acid sequences
revealed that the plant LHP1 homologs exhibited lower
similarity with each other, suggesting that a rapid divergence of these genes had occurred among plant species
through evolution (Fig. 1a, b). In accordance with the
dynamic modiWcation of the chromatin structures inthe evolutionary timescale, the LHP1 family regulating thechromatin structure might also have evolved rapidly andbeen widely distributed among plant species. However,
CD, CSD, and nuclear localization signal motifs characteristic of the LHP1 family were highly conserved in apple
and other plant LHP1 homologs (Fig. 1a). In animals, theCD of a human PcG is mainly involved in recognition ofhistone modiWcations and essential for protein-protein
interaction within a multimeric protein complex; the CSD
of HP1 protein is involved in self-association and interaction with the chromatin assembly factor 1 (CAF1) and thetranscriptional intermediary factor 1 (TIF1) proteins(Brasher et al. 2000; Eissenberg 2001; Cammas et al.
2004). In plants, the CD of SlLHP1 (LeHP1, tomato LHP1)
is required for the interaction of SlLHP1 with K9-methylated Histone H3, which was revealed by Glutathione-Stransferase (GST) pull-down assays in vitro (Zemach et al.
2006). Furthermore, the CSD of both AtLHP1 and SlLHP1
is responsible for homo-dimerization (Gaudin et al. 2001;
Zemach et al. 2006) and SlLHP1 CSD could participate ininteraction with chromocenters in Arabidopsis cells(Zemach et al. 2006). In this study, we demonstrated thatthe constitutive expression of MdLHP1a1 or MdLHP1b1
could restore the Xowering time and cell size of rosetteleaves in the tX2-2 mutant (Fig. 5; Table 2). Observationsof transgenic tX2-2 plants with MdLHP1a1/-b1 have shownthat these genes function equivalently to Arabidopsis
LHP1. Based on these results, LHP1 homolog genes inplants are essential for its growth, as they are in animals;
CD and CSD, which are highly conserved in HP1 family,
would especially be important for the biological function of
HP1 homolog proteins.
MdLHP1a and MdLHP1b were constitutively and abundantly expressed in apical buds of proliferating fruit-bearing
and succulent (vegetative) shoots of apple trees throughout
the growing season (Fig. 4). In transgenic Arabidopsis
expressing MdLHP1a1 or MdLHP1b1 both in tX2-2 and35S:MdLHP1a1/tX2-2#1

14

8.0 § 0.3

2.1 § 0.1

#513

7.3 § 0.2

2.4 § 0.2

#617

9.3 § 0.3

3.1 § 0.1#812

7.8 § 0.2

2.4 § 0.2

#910

7.8 § 0.3

2.6 § 0.2

35S:MdLHP1b1/tX2-2#2

12

8.8 § 0.4

2.8 § 0.1

#3

12

7.3 § 0.2

2.6 § 0.2

#4

13

7.2 § 0.4

2.4 § 0.2

#20

12

7.7 § 0.3

2.8 § 0.1

35S:MdLHP1a1/wt#2

12

10.4 § 0.6

2.9 § 0.2

#3

12

10.4 § 0.6

2.4 § 0.2

#5

12

10.3 § 0.4

2.7 § 0.1

#1612

9.2 § 0.3

2.3 § 0.1

35S:MdLHP1b1/wt#47

9.7 § 0.5

2.9 § 0.1

#811

8.9 § 0.4

2.4 § 0.2

#912

7.8 § 0.4

2.1 § 0.1

#1912

9.5 § 0.3

2.7 § 0.2aPlants were grown on the potted soil at 22°C under the day length
conditions at 16 h of light/8 h of darkness (long-day conditions)b
The number of rosette and cauline leaves were counted at Xowering.
Values are mean § standard error (SE)performed using a DIG-labeled MdLHP1a1 probe. As aresult, the expression of the transgenes was conWrmed in alltransgenic lines with the tX2-2 mutant background(Fig. 5k). In contrast, several lines showed little expressionof the transgenes with the wild-type background (Fig. 5k).
The degree of delayed Xowering was not exactly due to theexpression level of MdLHP1a1 or MdLHP1b1 in transgenic
plants, although the delayed Xowering in these transgenic
plants appeared to depend on the expression of MdLHP1a1
or MdLHP1b1 (Fig. 5k). For example, lines 35S:MdLHP1a1/tX2-2 #2 and #9 were considerably diVerent in
the expression level of MdLHP1a1, but there was little
diVerence between them in the total numbers of rosetteleaves produced at Xowering (Fig. 5k; Table 2). A similar
result was obtained in lines 35S:MdLHP1a1/wt #2 and #5(Fig. 5k; Table 2). In addition, smaller than expected sizesof the MdLHP1a1/MdLHP1b1 transcripts were detected,
predominantly in the transgenic plants with the tX2-2 background, as compared with those with the wild-type background (Fig. 5k).

123

304

wild-type backgrounds, the amount of the transcripts of
MdLHP1a1 and MdLHP1b1 might not be correlated withthe extent of the phenotypes, such as Xowering time
(Fig. 5k; Table 2). In Arabidopsis, LHP1 and EMF2, which
maintain the gene activity states epigenetically, and are
expressed predominantly in proliferating cells. In addition,overexpression of these genes does not aVect the growthphases. These observations suggest that HP1-family and
PcG genes are post-transcriptionally regulated (Gaudin
et al. 2001; Yoshida et al. 2001; Hsieh et al. 2003; Kotake
et al. 2003). It was consistent with these observations thatthe phenotype of transgenic plants with MdLHP1a1/-b1 was
not necessarily correlated with the extent of transcription
level. In Northern blot analysis, the aberrant or incomplete
transcripts of MdLHP1a1/-b1 were found in transgenic Arabidopsis plants with MdLHP1a1/-b1, predominantly in the
tX2-2 mutant background. On the other hand, few aberrant
transcripts were found in transgenic plants with
MdLHP1a1/-b1 in the wild-type background, although theexpression of MdLHP1a1/-b1 in the wild-type backgroundwas no less than that in the tX2-2 background. The lower
level of aberrant transcripts in the wild-type background
may be due to a mechanism that represses the abnormal
accumulations of the functional transcripts of LHP1-likegenes. In some (10%) of the transgenic Arabidopsis with
35S:MdLHP1a1/-b1 in the wild-type background, downregulation or silencing of endogenous LHP1 might cause
the expression of tX2-2 mutant-like phenotypes by a mechanism, such as co-suppression or RNA interference (RNAi).
In addition to MdLHP1a and MdLHP1b, several pairs of
two duplicated genes regulating Xoral identity or Xowering
time have been identiWed in the apple genome; for example,
pairs of APETALA1 (AP1)-like genes from the apple cv.
‘Granny Smith’, ‘Jonathan’, ‘Fuji’, and ‘Pinova’ (Yao et al.
1999; Kotoda et al. 2000, 2002; our unpublished results),
LEAFY (LFY)-like genes from ‘Jonathan’ and ‘Fuji’ (Wadaet al. 2002; Esumi et al. 2005), and TFL1-like genes from
‘Jonathan’ and ‘Fuji’ (Esumi et al. 2005; Kotoda and Wada
2005; Kotoda et al. 2006; our unpublished results) havebeen isolated and characterized. The pair of paralogous
genes related to apple Xowering seems to have similar regulatory functions, considering the results from the analysisof transgenic Arabidopsis ectopically expressing thosegenes, although the expression levels and patterns in apple
are not identical between the twin paralogs. The fact that
there are pairs of two copy genes in the apple genome supports the hypothesis that the Maloideae subfamily (a basal
haploid number, n = 17), which includes the genera Malus(apple) and Pyrus (pear), had evolved from the polyploidancestor. It was hypothesized that Maloideae subfamily had
been created by hybridization between ancestors of twoother Rosaceae subfamilies, Spiraeoideae (n = 9) and Amygdaloideae (n = 8) (Sax 1933; Luby 2003) or that the origin

123

Mol Genet Genomics (2007) 278:295–305

of Maloideae (n = 18) had occurred from members of alineage containing the ancestors of Gillenia (n = 9), andthen its progeny had lost one chromosome (n = 17) (Evans
and Campbell 2002). Because polyploidy (genome doubling) can easily produce functional divergence among
homolog genes, this is one of the evolutional strategies inplants that enhances environmental adaptation (Adams andWendel 2005). As an example of studies on such an evolutionary polyploid complex genome, the chromosomal rearrangement of amphidiploid Brassica napus (AACC)
resynthesized by hybridization between allogenic B. rapa(AA) and B. oleracea (CC) aVects the gene expression of
FLOWERING LOCUS C (FLC) homologs, leading to adivergence in Xowering time (Pires et al. 2004; Adams andWendel 2005). In consideration for Brassica genome study,
the polyploid complex genome may produce a variety ofphenotypes in apple as well. Sung et al. (2006) reported
that Arabidopsis LHP1 also plays a crucial role in vernalization by regulating FLC. Interestingly, the vernalization
requirement in some Arabidopsis ecotypes for its development resembles the chilling requirement in deciduous trees,such as the apple, which are adapted to survive at cooler
temperatures in winter, although the apple is a perennial
plant and its life cycle is diVerent from that of annual
plants, such as Arabidopsis.
Taken together, LHP1 homolog genes of chromatin regulator might have rapidly evolved and be diverged widely
among plant species following the dynamic modiWcation ofchromatin structures with chromosomal rearrangement during evolution, but the protein functions were conserved, atleast between Arabidopsis and apple. Recently, we produced
transgenic apples with an RNAi construct for MdLHP1a/
MdLHP1b. These transgenic approaches may provide not
only the insight into the practical aspects, such as the manipulation of Xowering and the acceleration of breeding, butalso a novel notion of the mechanism controlling Xoweringor dormancy via epigenetic system in fruit trees.
Acknowledgments We thank Dr. H. Ichikawa, National Institute ofAgrobiological Sciences (Tsukuba, Japan), for kindly providing thebinary vectors, pSMAK312Blue and pSMAK193E. We also thankDr. E.E. Hood, Texas A&M University College Station, for kindly
providing Agrobacterium tumefaciens EHA101. A. thaliana tX2-2 seeds
were kindly provided by the Arabidopsis Biological Resource Center at
Ohio State University. This work was supported by the “PROBRAIN(Program for Promotion of Basic Research Activities for Innovative
Biosciences)” from the Bio-Oriented Technology Research Advancement Institution (BRAIN). This publication constitutes the contribution
number 1445 of the National Institute of Fruit Tree Science.

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