J. AMER. SOC. HORT. SCI. 125(4):398–403. 2000.

Expression Pattern of Homologues of Floral Meristem
Identity Genes LFY and AP1 during Flower
Development in Apple
Nobuhiro Kotoda,1 Masato Wada, Sadao Komori,2 Shin-ichiro Kidou,3 Kazuyuki Abe, Tetsuo
Masuda, and Junichi SoejimaApple Research Center, National Institute of Fruit Tree Science, Shimokuriyagawa, Morioka 020-0123, Japan
ADDITIONAL INDEX WORDS. AP1, flower development, LFY, MADS-box, Malus sylvestris var. domestica
ABSTRACT. Two apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] homologous fragments of FLO/LFY andSQUA/AP1 (AFL and MdAP1, respectively) were analyzed to determine the relationship between floral bud formation andfloral gene expression in ‘Jonathan’ apple. The AFL gene was expressed in reproductive and vegetative organs. By contrast,
the MdAP1 gene, identified as MdMADS5, which is classified into the AP1 group, was expressed specifically in sepals
concurrent with sepal formation. Based on these results, AFL may be involved in floral induction to a greater degree than
MdAP1 since AFL transcription increased ≈2 months earlier than MdAP1. Characterization of AFL and MdAP1 should
advance the understanding of the processes of floral initiation and flower development in woody plants, especially in fruit
trees like apple.

In contrast to herbaceous plants, apple trees (Malus sylvestrisvar. domestica) flower and set fruit only after an extended juvenile
phase that may last several years. Thus, it is important to understandthe mechanism of floral bud formation because apple is one of themost commercially important tree fruits in the world. However, thegenetic factors controlling floral initiation in apples have not been
investigated in detail.
In the last decade, several genes related to flower initiation anddevelopment have been isolated and the function of these genes isgradually becoming clearer (Liljegren and Yanofsky, 1996; Ma,
1994; Mandel and Yanofsky, 1995; Parcy et al., 1998; Pineiro and
Coupland, 1998; Ratcliffe et al., 1998). In snapdragon (Antirrhinum
majus L.) and arabidopsis [Arabidopsis thaliana (L.) Heynh.],
FLORICAULA (FLO)/LEAFY (LFY) (Coen et al., 1990; Schultzand Haughn, 1991; Weigel et al., 1992; Weigel and Nilsson, 1995)
and SQUAMOSA (SQUA)/APETALA1 (AP1) (Huijser et al., 1992;
Irish and Sussex, 1990; Mandel et al., 1992) play central roles in thetransition from the inflorescence meristems into floral meristems.
Both FLO and LFY are expressed in the floral meristem prior tofloral organ primordia formation and LFY in Arabidopsis activates
AP1 (Mandel and Yanofsky, 1995). SQUA and AP1 are members of
the MADS-box (MCM1, AGAMOUS, DEFICIENCE, and SRF,
serum response factor) family of a transcription factor, which has aconserved DNA-binding/dimerization region (Coen andMeyerowitz, 1991; Purugganan et al., 1995; Riechmann andMeyerowitz, 1997; Schwartz-Sommer et al., 1990) and they function as both floral meristem and organ identity genes (Bowmann etal., 1993; Guftafson-Brown et al., 1994; Weigel and Meyerowitz,
1994).
In this paper, we describe isolation and characterization of two
apple homologue fragments of FLO/LFY and SQUA/AP1 genes
Received for publication 13 Apr. 1999. Accepted for publication 7 Mar. 2000.
National Institute of Fruit Tree Science contribution 1169. The cost of publishing
this paper was defrayed in part by the payment of page charges. Under postal
regulations, this paper therefore must be hereby marked advertisement solely toindicate this fact.
1
To whom reprint requests should be addressed.
2
Current address: Okinawa Subtropical Station, JIRCAS, Maesato, Ishigaki 9070002, Japan.
3
Associate professor, Cryobiosystem Research Center, Faculty of Agriculture,
Iwate University, Ueda, Morioka 020-8550, Japan.

398(AFL and MdAP1) and the relation between expression patterns of
AFL and MdAP1. Furthermore, the morphological changes of apicalmeristems during flower development in apple were studied indetail.
Materials and Methods
RNA EXTRACTION. RNA was isolated from ‘Jonathan’ apple [age:14 to 15 years; rootstock: Maruba kaido (Malus prunifolia var. ringo
Asami), location: Morioka, Japan] in 1997 and 1998 by acetyltrimethylammonium bromide (CTAB)-based method (Changet al., 1993) modified by Yamamoto and Mukai as follows (notpublished). About 0.1 g of plant tissue was frozen in liquid nitrogenand then ground to fine powder. The powder was mixed with 1 mL
2× CTAB (2% CTAB, 0.1 M Tris-HCl pH 9.5, 20 mM EDTA, 1.4 M
NaCl, and 1% 2-mercaptoethanol) then incubated at 65 °C for 10
min. The solution was extracted with 24 chloroform : 1 isoamyl
alcohol (v/v) and RNA in the aqueous phase was precipitated with
0.25 volume of 10 M lithium chloride at –20 °C for 2 h. Nucleic acids
were pelleted by centrifugation at 12,000 gn for 10 min at 4 °C. The
pellet was dissolved in TE (10 mM Tris-HCl pH 8.0, and 1 mM
EDTA) then extracted with TE saturated phenol and phenol–
chloroform. The aqueous layer was extracted sequentially withchloroform–isoamyl alcohol [24:1 (v/v)] then RNA in the aqueous
phase precipitated with 0.1 volume 3 M sodium acetate and 2.5
volumes ethanol. RNA was pelleted by centrifugation at 12,000 gn
for 10 min at 4 °C. The pellet was dissolved in TE (10 mM Tris-HCl
pH 8.0, and 1 mM EDTA).
LIGHT MICROSCOPY. Shoot apices at each developmental stage
were fixed in FAA (1.8% formaldehyde, 5% acetic acid, and 45%
ethanol) for 24 h at 24 °C. The fixed shoot apices were dehydrated
with an ethanol series and embedded in Paraplast plus (Sigma
Chemical, St. Louis, Mo.). Longitudinal sections (10 µm) wereprepared for morphological observation with a rotary microtome(Yamato Kohki, Asaka, Japan). Sections were stained with 0.5%
hematoxylin, 1.0% safranine, and 1.0% fast green FCF. The samples
were dehydrated with ethanol transferred to xylene and mountedwith Entellan Neu (Merk Chemical, Darmstadt, Germany). The
sections were viewed through a Nikon Labophot YF microscope(Nikon, Tokyo, Japan) and photographs were taken using Fujichrome
Sensia II 36 film (Fujifilm, Tokyo, Japan).
J. AMER. SOC. HORT. SCI. 125(4):398–403. 2000.

GCAA/AT/CTIGAT/CACIGC-3', 3' primers 5'-TTT/CTGT/CTCT/
CTGA/G/TATIGCT/CTT-3' and 5'-GCIGCA/GAAA/GCAICCIAA/G-3') were used to amplify homologous sequences of SQUA/
AP1 from apple cDNA for the RT-PCR using the following reaction
conditions: 40 cycles of 1 min at 94 °C, 1 min at 52 °C, and 2 min
at 72 °C. Two overlapping 110- and 567-bp fragments were
amplified and cloned into pBluescript II SK+ vectors. The sequences of two clones of each fragment containing an insert inpBluescript II SK+ were analyzed as described above.
RNA BLOTTING ANALYSIS. RNA was isolated from leaves, leavesof tissue cultures of shoots, stems, roots, whole flower buds atvarious developmental stages, and sepals, petals, stamens, andcarpels of ‘Jonathan’ apple. RNA was separated on a 1.2% agarose
gel containing 5.0% (v/v) formaldehyde. Total RNA (10 µg) was
loaded per lane. The gels were blotted onto Hybond-N nylon
membrane (Amersham). For AFL , hybridization was done with
DIG-labelled RNA probes, which were prepared by in vitro transcription using the DIG RNA labelling kit (Boehringer Mannheim,
Mannheim, Germany) according to the protocol provided by the
manufacturer. The pBluescript II SK+ containing a 447-bp fragment of AFL was linearized and used as a template to make probes.
Hybridization and washing were performed according to a standard
protocol (Boehringer Mannheim). For MdAP1, the 567-bp fragment of the coding region of MdAP1 was used as a template to makeprobes. Hybridization was done with 32P-labelled cDNA probes at
65 °C in 1 M NaCl, 10% dextran sulfate, and 1% SDS for 12 hfollowed by one rinse in 2× SSC and three washes in 2× SSC and
0.1% SDS and one wash in 2× SSC. RNA blot analysis probed with
MdMADS, which contains a 75-bp fragment in MADS-box genes inapple (Sung and An, 1997), was performed as a control to MdAP1
expression. The pBluescript II SK+ vector containing a 75-bp
fragment in MADS-box genes in apple ‘Jonathan’ amplified by RTPCR with two oligonucleotides (5' primer 5'-CGTCAAGTCACTTTTTGCAAACGT-3' and 3' primer 5'-AGCATCACAGAGAACAGAGAGC-3') were used to generate antisense DIG-labeled
RNA probes.
Fig. 1. Growth of current shoot of ‘Jonathan’ apple from the cluster base. (A) Current
shoots from the cluster base were collected for measuring shoot lengths and thetotal RNA was extracted from apical buds. Scale bar = 7 cm. (B) The lengths of
40 to 50 shoots were measured after flowering through October. Population of
apical buds was synchronized at each developmental stage. Vertical bars = SD.

GENE CLONING. Homologue fragments of FLO/LFY and SQUA/
AP1 were amplified by reverse transcription (RT)-polymerase
chain reaction (PCR) from the apices of young floral buds and sepalsof mature floral buds in ‘Jonathan’ apple, respectively.
Two oligonucleotides (5' primer 5'-CAGAGGGAGCATCCGTTTATCGTAAC-3' and 3' primer 5'-GACGC/AAGCTTT/
GGTT/GGGA/GACATACCA-3') corresponding to the conserved
domains in the coding sequence of FLO in Antirrhinum and LFY in
Arabidopsis, were used to amplify a homologous sequence fromapples for the RT-PCR under the following reaction conditions: 40
cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C. A 447
base pair (bp) long fragment was amplified and cloned into thepBluescript II SK+ (Stratagene, La Jolla, Calif.) vector. The sequences of one clone of each fragment containing an insert in the
pBluescript II SK+ were analyzed by dideoxy methods using a
Thermo Sequenase premixed cycle sequence kit according to the
manufacturer’s instructions (Amersham, Buckinghamshire, U.K.)
and universal primers in the Bluescript vectors on a Hitachi SQ5500
automated sequencer (Hitachi, Tokyo, Japan).
Four degenerate oligonucleotides (5' primers 5'-AAA/
GGGIAAA/GT/CTITTT/CGAA/GTA-3' and 5'-GAA/GCAA/
J. AMER. SOC. HORT. SCI. 125(4):398–403. 2000.

Results and Discussion
MORPHOLOGICAL OBSERVATION OF FLOWER DEVELOPMENT IN APPLE.
In apple, initiation of flower primordia for the following year occurs
3 to 6 weeks after bloom or after cessation of shoot growth (Buban
and Faust, 1982). We collected current shoots from cluster bases
after bloom and measured the length every 2 weeks from June to
October to determine the time of transition from the vegetative to thereproductive phase in ‘Jonathan’ apple (Fig. 1A). To determine themorphological changes of apical meristems during flower development, sections of apical buds were prepared (Fig. 2).
In ‘Jonathan’ apple, growth of most current shoots from thecluster base ceased in late June in Morioka, Japan (Fig. 1B). From
this observation, it appears the transition from the vegetative phaseto the reproductive phase occurred in late June.
Microscopic examination showed that the apical meristem wasa narrow, pointed dome on 3 July (Fig. 2A), slightly domed on 17
July (Fig. 2B), and was clearly broadened by 31 July (Fig. 2C). It
appears that initiation of floral bud formation occurs in the middleof July in Morioka. Similar results have been reported by Osanai etal. (1990) and Suzuki et al. (1989).
In the middle of September, the apical meristem domed considerably (Fig. 2D), and sepal primordia were formed in early October(Figs. 2E and H). The initiation of stamen primordia occurred
subsequently (Fig. 2H). In the middle of December, several flower399

Fig. 2. Morphological changes of apical buds of ‘Jonathan’ apple. Apical buds fromcurrent shoots were fixed in FAA and embedded in Paraplast plus. Longitudinal
sections (10 µm) of apical buds were prepared and stained with 0.5% hematoxylin,
1% safranine, and 1% fast green (hematoxylin stains cell walls, cytoplasmic
matrices, and plastids purple blue to brownish red, safranine stains lignified cell
walls, choromosomes, and nucleoli red, and fast green stains cell walls andcytoplasm green). (A) 3 July; (B) 17 July; (C) 31 July; (D) 12 Sept.; (E) 10 Oct.;(F) 22 Dec. (tf = terminal flower and lf = lateral flower); (G) 26 Apr. (p = petal,
st = stamen, c = carpel, s = sepal, rt = receptacle, and ov = ovule); (H) flower
primordia on 10 Oct. (sp = sepal primordia, stp = stamen primordia, lf = lateral
flower, and tf = terminal flower). Scale bars = 200 µm (A, B, C, D, H), 400 µm(E, F), and 1000 µm (G).

primordia were visible (Fig. 2F). The floral organs expanded rapidly
after the removal of dormancy and were fully differentiated in late
April before bloom (Fig. 2G). In general, the inflorescence of apple
consists of a terminal flower and four lateral flowers (Fig. 2H). The
terminal flower usually grows and opens earlier than lateral flowers.
PCR CLONING AND SEQUENCE ANALYSIS OF FLO/LFY AND SQUA/AP1
HOMOLOGOUS FRAGMENTS IN APPLE. LFY and AP1 are required forfloral differentiation in Arabidopsis (Mandel et al., 1992; Weigel etal., 1992). Homologous fragments of FLO/LFY and SQUA/AP1
were amplified by RT-PCR from the apices of young floral buds andsepals of mature floral buds in apples, respectively, to analyze
regulation of these two genes during flower development of‘Jonathan’ apple.
Two oligonucleotides corresponding to the conserved domains
in the coding sequence of FLO in Antirrhinum and LFY in Arabidopsis
were used to amplify a homologous sequence from apples (Fig. 3A).
400

A 447-bp fragment was amplified
and cloned into the pBluescript II
SK+ (Stratagene) vector. In addition, four degenerate oligonucleotides were used to amplify homologous sequences of SQUA/AP1 fromapple cDNA by RT-PCR (Fig. 3B).
Two overlapping 110- and 567-bp
fragments were amplified and cloned
into the pBluescript II SK+ vectors.
The identity of AFL (Apple
Floricaula/Leafy) at the nucleotide
level was 82% with FLO (Coen etal., 1990) and 78% with LFY (Weigel
et al., 1992). At the amino acid level,
the identity was 91% and 89% with
FLO and LFY, respectively (Fig.
3A). The identity of MdAP1 (Malus
sylvestris var. domestica AP1) at thenuculeotide level was 68% withSQUA (Huijser et al., 1992) andAP1 (Mandel et al., 1992). At theamino acid level, the identity was
63% and 60% with SQUA and AP1,
respectively (Fig. 3B). The deduced
protein sequence of MdAP1 showed
higher homology to SQUA/AP1 thanany other MADS-box proteins in I
(a region between MADS-box andK-box) and K-box regions (Riechmann and Meyerowitz, 1997). The
homology with the corresponding
Antirrhinum and Arabidopsis sequences (Fig. 3) suggest that the
fragments are derived from the FLO/LFY and SQUA/AP1 homologues in apples. Therefore, they werenamed AFL and MdAP1. In addition, MdAP1 was identical to thecorresponding sequence of MADS-box gene, MdMADS5, which
was isolated recently from ‘Granny Smith’ apple (Yao et al., 1999).
Difference between the MdAP1 and MdMADS5 gene was thesubstitution of only a few nucleotides in the 3' noncoding region.
EXPRESSION PATTERN OF AFL AND MdAP1 DURING FLOWER DEVELOPMENT. The 447- and 567-bp fragments were used to analyze theexpression of both AFL and MdAP1 in the apices of apple at different
developmental stages by generating antisense digoxigenin (DIG)labeled RNA and 32P-labeled cDNA probes, respectively. RNA blot
analyses were performed on the total RNA isolated from shootapices of ‘Jonathan’ apple during flower development. The AFLmRNA was detected in all stages of flower development and weakly
detected in vegetative shoots in June (Fig. 4A, 6.19V). It increased
from mid-August to mid-November, with a slight decrease in
December. The MdAP1 mRNA in the floral meristem, on the other
hand, was first detected in mid-October, when expression of LFY
was high, and increased drastically until the following April with theformation of floral organs (Fig. 4B). By contrast, MdMADS mRNAwas detected at a very early stage as well as a late stage (Fig. 4C).
These results agree with the model that LFY is expressed earlier thanAP1 and regulates AP1 in Arabidopsis. However, AFL is expressed
weakly in the apical buds of water sprouts which are constantly ina vegetative phase and never flower (Fig. 4A, 6.19V).
EXPRESSION OF AFL AND MdAP1 IN DIFFERENT TISSUES OF APPLE.
Expression patterns of AFL and MdAP1 were analyzed in various
J. AMER. SOC. HORT. SCI. 125(4):398–403. 2000.

Fig. 3. Comparison of the AFL and MdAP1 sequences with their homologues in
Arabidopsis (Mandel et al., 1992; Weigel et al., 1992) and Antirrhinum (Coen etal., 1990; Huijser et al., 1992). Amino acids at positions of identity between twoor three of these proteins are blocked in black. Black lines above AFL and MdAP1
indicate primer sites for RT-PCR. (A) Alignment of the partial AFL protein
sequence with the Arabidopsis LFY and the Antirrhinum FLO protein sequences.
The partial AFL obtained corresponded to 447 bp of the FLO/LFY coding
sequence. (B) Alignment of the partial MdAP1 protein sequence with the
Arabidopsis AP1 and the Antirrhinum SQUA protein sequences. The partial
MdAP1 obtained corresponded to the entire coding sequence downstream of the
SQUA/AP1 MADS-box. A broken line below SQUA indicates MADS-box and a
black line indicates K-box.

living tissues by RNA blot hybridization. AFL was expressedstrongly in the sepals, leaves, and leaves of tissue cultures of shoots
but very weakly in the petals, carpels, cotyledons, and stems. There
was no expression in roots (Fig. 5A). In Arabidopsis, LFY isexpressed strongly in young flower primordia and cauline leaves,
but very weakly in flower primordia at the late stage (Weigel et al.,1992). In Antirrhinum, FLO shows a similar expression pattern to
LFY (Coen et al., 1990). In contrast to LFY and FLO, AFL RNA wasexpressed in mature leaves and mature floral organs including
sepals, petals, and carpels.
Expression of MdAP1 was detected specifically in the sepals(Fig. 5B). In comparison, AP1 is expressed in the sepals, petals, andpedicels, and SQUA in the sepals, petals and bracts. Both eucalyptus(Eucalyptus globulus Labill.) EAP1 and EAP2 were expressed in
the operculum which is a unique organ with sepals and petals fused,
and in the receptacle (Kyozuka et al., 1997).
We isolated apple homologue fragments of FLO/LFY and SQUA/
AP1 (AFL and MdAP1, respectively) and analyzed the expression
pattern of these genes. Results showed that the AFL gene wasexpressed in reproductive and vegetative organs such as apical budsof water sprouts and mature leaves. In addition, in situ hybridization
probed with AFL revealed that the AFL transcript was present at highlevels in apical meristems and leaf primordia at the beginning offloral bud initiation, whereas it was not detected in apical meristemsof vegetative shoots (data not presented). For example, FLO and

J. AMER. SOC. HORT. SCI. 125(4):398–403. 2000.

Fig. 4. Northern-hybridization expression pattern of (A) AFL, (B) MdAP1, and (C)
MdMADS genes during flower development. The numbers above the lanes
indicate dates of harvest (month.day). Equal amounts (10 µg) of total RNA
isolated from apical buds were blotted onto Hybond N filters. RNA blot analysis
probed with MdMADS, which contains a 75-bp fragment in MADS-box genes inapple (Sung and An, 1997), was performed as a control to MdAP1 expression (Fig.
4C). V = vegetative shoot (water sprout).

401

Fig. 5. Northern-hybridization expression pattern of AFL and MdAP1 genes indifferent organs. (A) Equal amounts (10 µg) of total RNA isolated from flowers(sepals, petals, stamens, and carpels), seedlings (cotyledons, leaves, stems, androots) and leaves of cultured shoots (CS leaves) were subjected to northern
analysis. Hybridization was done with DIG-labeled RNA probes, which wastranscribed from the recombinant Bluescript plasmids containing a 447-bp
fragment of AFL with DIG-UTP as described by the manufacturer. (B) Equal
amounts (10 µg) of total RNA isolated from flowers (sepals, petals, stamens, andcarpels) and seedlings (leaves, stems, and roots) were subjected to northern
analysis. Hybridization was done with 32P-labeled cDNA probes. A MdAP1
cDNA of the 567-bp fragment obtained was used as a probe. Values on the rightindicate the size of transcripts (kb: kilobase).

LFY are expressed in bract and cauline leaf primordia, respectively.
Blazquez et al. (1997) reported that LFY is expressed extensively
during the vegetative phase. The pea (Pisum sativum L.) FLO/LFY
homologue, PEAFLO is expressed strongly in leaves, inflorescences, and lateral shoot primordia and in floral organ primordia(Hofer et al., 1997). In impatiens (Impatiens balsamina L.), Imp-flo
homologous to FLO/LFY is transcribed in all floral organs including
bracts, petals, stamens, and carpels (Pouteau et al., 1997). Comparedwith the examples described above, the expression pattern of AFL
in apple resembles that of FLO, LFY, Imp-flo, and PEAFLO. AFL
mRNA, however, was also detected in fully differentiated tissues
such as mature leaves and mature floral organs including sepals,
petals, and carpels. This result suggests that AFL functions duringleaf and flower morphogenesis at later developmental stages as well
as at the early developmental stage of floral bud formation.
RNA blot analysis probed with MdMADS, which contains a 75bp fragment in MADS-box genes in apple (Sung and An, 1997), wasperformed as a control to MdAP1 expression (Fig. 4C). This probe
is considered to detect all MADS-box genes in apple. The result
showed that MdMADS mRNA was detected at early and late stages
in apical buds. On the other hand, MdAP1 was expressed specifically in sepals and showed a similar expression pattern to SQUA,
AP1, EAP1, and EAP2 in that their expression was also detected in
sepals or sepal-like organs. While this manuscript was in preparation, Yao et al. (1999) reported that MdMADS5, identical to MdAP1402

in corresponding sequences, was expressed strongly in the receptacle, cortex, and skin of apple fruit. MdAP1 expression was firstdetected in mid-October when the sepal primordia began to developand LFY expression was high in apical buds. It has been proposedthat FLO/LFY interacts with and activates SQUA/AP1, so AFL may
also activate MdAP1. Because there was a delay of ≈3 months in
MdAP1 gene expression after floral initiation, MdAP1 may not
trigger floral initiation directly, but it is certain that the gene isinvolved in sepal and fruit formation. Based on the experimental
results, AFL may be involved in floral induction to a greater extent
than MdAP1 since AFL showed increased transcription ≈2 months
earlier than MdAP1.
Characterization of AFL and MdAP1 should advance the understanding of the processes of floral initiation and flower development
in woody plants, especially in tree fruits like apple. A recent study
has shown that an aspen hybrid of two poplar species (Populus
tremula L. x P. tremuloides Michx.), may be induced to generate
flowers after only 5 months growth following insertion of the LFYgene into cells regenerated from aspen stem segments (Weigel and
Nilsson, 1995). Kyozuka et al. (1997) showed that Eucalyptus
EAP1 and EAP2 have homology to AP1 and transgenic Arabidopsis
plants expressing either EAP1 or EAP2 are early-flowering andhave an increased number of flowering stems. One outcome of plantbiotechnology could be reduction of generation time by introducing
floral genes like LFY, AP1, and their homologues through transgenic approaches, which could be useful for hastening breeding
strategies.
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