The Horticulture Journal 89 (3): 225–236. 2020.
doi: 10.2503/hortj.UTD-146

The Japanese Society for
JSHS

Horticultural Science

http://www.jshs.jp/

Accumulation of Polymethoxyflavones and O-methyltransferase Gene
Expression in Various Citrus Cultivars
Fatema Tuz Zohra1, Shotaro Takematsu2, Yuri Itami3 and Nobuhiro Kotoda1,2,3*
1

The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan
Faculty of Agriculture, Saga University, Saga 840-8502, Japan
3
Graduate School of Agriculture, Saga University, Saga 840-8502, Japan
2

Polymethoxyflavones (PMFs) are naturally occurring O-methylated flavones predominantly accumulated indifferent citrus tissues that show numerous human-health promoting activities. O-methyltransferase (OMT)
family members are involved in the biosynthesis of PMFs in plant species including citrus. Although the
distribution of PMFs has been determined in citrus cultivars, the relation between the accumulation of PMFs
and the related OMT gene expression in leaves and flavedos (exocarps) of citrus cultivars remains to beclarified. In this study, we investigated the accumulation of nobiletin and tangeretin in the leaves and flavedosof various citrus cultivars widely grown in Japan. As expected, the accumulation of nobiletin and tangeretin
varied among the citrus cultivars and the two tissues, and nobiletin and tangeretin accumulated at higher
levels in flavedos than in leaves. In particular, we clearly demonstrated that the nobiletin content showed asignificant and positive correlation (r = 0.824, P = 0.00182) between leaves and flavedos. Based on these results,
the concentration of PMFs in leaves could be utilized as an early selection marker for seedlings in the juvenile
phase that are expected to accumulate a higher amount of PMFs in fruit, resulting in a shortening of thebreeding period. We also identified two novel candidate genes, CreOMT1 and CreOMT4, putatively involved in
PMF biosynthesis in citrus. Notably, the expression of CreOMT1 showed a significant correlation (r = 0.700,
P = 0.0243) with the nobiletin content in the flavedos of 10 citrus cultivars. Our results provide valuable
information for developing new citrus cultivars that could accumulate higher PMFs and insight into
elucidating the PMF biosynthetic pathway in citrus in the future.
Key Words: flavedo, flavone, HPLC, nobiletin, tangeretin.

Introduction
Citrus species are some of the most popular andwidely cultivated fruits in the world. Many citrus
species are considered to be rich in secondary metabo‐lites (present in the peel, pulp, seed, pressed oil, juice orwhole fruit) that play important roles in plant cells andReceived; September 25, 2019. Accepted; November 25, 2019.
First Published Online in J-STAGE on February 15, 2020.
This work was financially supported by the Cabinet Office,
Government of Japan, Cross-ministerial Strategic Innovation
Promotion Program (SIP), “Technologies for creating next-generation
agriculture, forestry and fisheries” (funding agency: Bio-oriented
Technology Research Advancement Institution, NARO), IchimuraFoundation for New Technology, and Yanmar Environmental
Sustainability Support Association (YESSA).
Part of this study was presented at the Autumn Annual Meeting of
The Japanese Society for Horticultural Science, 2018.
* Corresponding author (E-mail: koto@cc.saga-u.ac.jp or nobu_fruits@yahoo.co.jp).

human health due to their functional properties (Patilet al., 2006; Tripoli et al., 2007). Polymethoxyflavones(PMFs) are naturally occurring O-methylated flavonoid
compounds found exclusively in citrus that belong to
the Rutaceae family, as well as in plant species in the
Lamiaceae and Asteraceae families (Fig. 1; Berim andGang, 2016; Kefford and Chandler, 1970; Li et al.,2009; Patnayak et al., 1942). The peels of citrus fruits
contain higher amounts of PMFs than other edible partsof the fruit (Ke et al., 2017; Nogata et al., 2006). Citrus
fruits are used in the juice industry for juice production
as a raw material, as well as in the food, beverage, cos‐
metic, and pharmaceutical industries as food additives,
spices, cosmetic ingredients, and chemoprophylactic
drugs, respectively (Braddock, 1999; Lv et al., 2015).
The peels are often discarded as waste materials fromjuice production and other industries and citrus peel
byproducts are utilized as a source of functional ingre‐
dients (Braddock, 1999; Manthey and Grohmann, 2001;© 2020 The Japanese Society for Horticultural Science (JSHS), All rights reserved.

226

F. T. Zohra, S. Takematsu, Y. Itami and N. Kotoda

3’
8

5’

7
6

3
5

Compound

R1

Sinensetin
Tangeretin
Nobiletin
Heptamethoxyflavone

4’

Total no. of
-OCH3

R2

R3

H

H

OCH3

5

H

OCH3

H

5

H

OCH3

OCH3

6

OCH3

OCH3

OCH3

7

Fig. 1. Chemical structures of polymethoxyflavones found in
citrus.

Rafiq et al., 2018). Nobiletin and tangeretin are two
major PMFs that are particularly abundant in citrus
peels (Choi et al., 2007; Inafuku-Teramoto et al., 2011;
Nogata et al., 2006). Recently, citrus PMFs have attract‐
ed great interest due to their protective effects against
memory impairment in Alzheimer’s disease (Braidy
et al., 2017; Kimura et al., 2018; Onozuka et al., 2008),
as well as their anti-tumor, anti-hyperglycemia, antiobesity, and anti-neuroinflammation and insulin resis‐
tance activities (Ho and Kuo, 2014; Kawaii et al., 1999;
Lee et al., 2010, 2013; Minagawa et al., 2001; Miyata
et al., 2008; Onda et al., 2013; Surichan et al., 2018;
Wang et al., 2014).
In plants, O-methyltransferase (OMT) is considered amajor methyltransferase group. Methylation of the hy‐
droxy group of flavonoids in plants is carried out by
OMT using S-adenosyl-L-methionine (SAM) as amethyl group donor (Ibrahim et al., 1998; Roje, 2006;
Struck et al., 2012). In PMFs, the hydroxy group(s) is
replaced with a methoxy group(s). Citrus PMFs are a
special group of O-methylated flavonoids that have
many methoxy groups in their parent molecules. OMTproteins that are encoded by the OMT gene familytransfer a methyl group from SAM to the hydroxygroup, and they are considered to be involved in thebiosynthesis of PMFs (Berim and Gang, 2016; Itohet al., 2016). The functions of plant OMT genes inflavonoid biosynthesis have been investigated in many
plant species, such as barley, Madagascar periwinkle,
mango, and rice (Byeon et al., 2015; Chidley et al.,2016; Christensen et al., 1998; Schröder et al., 2004).
Recently, five flavonoid-O-methyltransferase (FOMT)
genes were identified from Shiikuwasha (Citrus
depressa Hayata) that is known to accumulate nobiletinin its fruit peel (Itoh et al., 2016). Moreover, 58 OMTgenes were identified from the entire C. sinensis

genome (Liu et al., 2016). However, little is known
about the distribution of PMF accumulation, the genes
responsible for PMF biosynthesis, or their expression in
the leaves and flavedos (exocarps) of various citrus cul‐
tivars. Quantification of nobiletin and tangeretin in vari‐
ous citrus cultivars and analysis of related OMT genes
are needed because of their broad spectrum of biologi‐
cal activities and the value of citrus as a functionalfood.
In this study, therefore, the accumulation of nobiletin
and tangeretin in the leaves and flavedos, and the ex‐pression of the related OMT genes that are putatively
involved in PMF biosynthesis, was investigated in vari‐
ous citrus cultivars. Notably, the expression of two
OMT genes, CreOMT1 and CreOMT4, was possibly
related to the accumulation of PMFs. Identifying the
OMT genes related to PMF biosynthesis and elucidating
their functions in relation to PMF accumulation inleaves and flavedos could play an important role in elu‐
cidating the detailed mechanism of PMF accumulationin citrus.

Materials and Methods
Plant materials
We preliminarily screened 121 citrus accessions, in‐cluding wild citrus plants, for PMF accumulation inmature leaves (data not shown). According to the PMF
accumulation, we classified them into three categories,that is, high, medium and low PMF-accumulating
plants. From these, we selected 11 citrus cultivars fromthe high, medium and low group for this study. Tissue
samples (leaves and flavedos) of 11 citrus cultivars,‘Amanatsu’ (C. natsudaidai Hayata), ‘Aoshima’(C. unshiu Marcow), ‘Benibae’ (‘HF No. 9’ ×
‘Encore’), ‘Hirakishu’ (C. kinokuni hort. ex Tanaka),‘Kiyomi’ (C. unshiu Marcow × C. sinensis Osbeck),‘Mihaya’(‘Tsunonozomi’ × ‘No. 1408’),‘Ogimikuganii’ (C. depressa Hayata), ‘Ota’ Ponkan(C. reticulata Blanco), ‘Seinannohikari’ (‘EnOw
No. 21’ × ‘Youkou’), ‘Shiranuhi’ [‘Kiyomi’ × ‘Nakano
No. 3’ (C. reticulata)], and ‘Yoshida’ Ponkan(C. reticulata), grafted onto trifoliate orange [Poncirus
trifoliata (L.) Raf.], were collected in January 2018from the citrus germplasm collections at Saga Universi‐
ty, Japan. Of the 11 cultivars, 9, 10, and 11 cultivars
were used for semi-quantitative reverse-transcription(RT)-polymerase chain reaction (PCR), quantitative RTPCR (qRT-PCR), and quantitative analysis for PMFs,
respectively. The mature leaves were collected random‐ly, whereas flavedos were collected from ripened citrus
fruits (flavedos were sliced from the peels), and bothtissues were quickly frozen in liquid nitrogen and storedat −70°C until further analysis.
Sample preparation for high-performance liquid chro‐
matography (HPLC) analysis
Tissue samples were freeze-dried overnight andTable 1.
Identified OMT-related genes from the latest C. clementina
Hort. J. 89 (3): 225–236.
2020.
227 genome database (vecrushed to a fine powder by a multi-beads shocker(Yasui Kikai Corp., Osaka, Japan) using a standard pro‐gram. Fifty milligrams of the powdered sample wereadded to 500 μL of 80% methanol (Wako Pure Chemi‐
cal Industries, Ltd., Osaka, Japan) and mixed by vortex‐
ing, then extracted using an ultrasonic cleaner US-3R(AS ONE Corp., Osaka, Japan) for 10 min. The mixture
was then stored in the dark at room temperature (r.t.).
Subsequently, the sample was centrifuged at 10000 rpm
with a TOMY MX-200 (TOMY Seiko Co., Ltd., Tokyo,
Japan) for 1 min at r.t., and the supernatant was collect‐ed. Then, 500 μL of 80% methanol was added, mixed
by vortexing, and centrifuged at 10000 rpm for 1 min at
r.t. (this step was repeated twice). Finally, the extracted
supernatant was adjusted up to 2.5 mL by adding 80%
methanol, filtered through a 0.45 μm RephiQuik PTFE
syringe filter (RephiLe Bioscience, Ltd., Shanghai,
China) into a 2 mL vial (Agilent Technology, Santa
Clara, CA, USA) and stored at −20°C until analysis.
The experiment was performed in three biological repli‐
cates.

Table 1.

HPLC analysis
Samples were analyzed using an HPLC system(pump: PU-2089 plus, autosampler: AS-2057 Plus, de‐tector: UV-2075; Jasco Corp., Tokyo, Japan; column
oven: CTO-10AC; Shimadzu Corp., Kyoto, Japan)
equipped with an Inertsil® ODS-3 column (3 μm, 4.6 ×
33 mm; GL Sciences Inc., Tokyo, Japan) under the fol‐lowing conditions: solvent A was 10% methanol with
0.1% phosphoric acid, and solvent B was 90%
methanol with 0.1% phosphoric acid. The HPLC sepa‐
ration condition was as follows: 87.5% solvent A from0 to 4 min, 87.5–40.0% A from 4 to 10 min, 40.0% A
from 10 to 20 min, 40–87.5% A from 20 to 21 min, and87.5% A from 21 to 30 min. The flow rate was
1.2 mL·min−1, and the UV was measured at 254 nm.
The column oven temperature was set at 40°C and thesample injection volume was 5.0 μL. The analysis timewas 30 min per sample.

Isolation of OMT genes from ‘Yoshida’ Ponkan
Leaves of ‘Yoshida’ Ponkan were used to isolate thecDNA of three OMT genes corresponding to CcOMT1,CcOMT2, and CcOMT4. The cDNA was amplified
by PCR using KOD FX (TOYOBO Co., Ltd.,
Osaka, Japan) from the RT products. Each amplified
PCR product was cloned into a pGEM®-T Easy
vector (Promega, Madison, WI, USA) after an adenine
nucleotide addition to the products. The isolated
genes were designated as CreOMT1 (accession no.
LC507211), CreOMT2 (acc. no. LC507212), andCreOMT4 (acc. no. LC507213), respectively. To obtain
the genomic DNA of CreOMT1, CreOMT2, andCreOMT4, the same primer sets as those for cDNA iso‐lation were used with the total DNA of ‘Yoshida’
Ponkan as a template. Sequences were confirmed using
an ABI 3130xl DNA sequencer (Life Technologies
Corp., Carlsbad, CA, USA). The primer sets used ingene cloning are listed in Table 2.

Identification of OMT genes
To identify flavonoid OMT-related genes across thelatest C. clementina genome database (ver. 1.0), a Basic
Local Alignment Search Tool (BLAST) search was
conducted in Phytozome (https://phytozome.jgi.doe.
gov/), the Plant Comparative Genomics portal of the
Department of Energy’s Joint Genome Institute, basedon the amino acid sequence of a flavonoid 7-OMT gene
(CAA54616) in barley. The flavonoid 7-OMT gene inbarley has been found to have a function of Omethylating apigenin and the hydroxy group at the 7thposition of naringenin (Christensen et al., 1998). From
this search, 16 candidate genes for citrus OMT (desig‐
nated as CcOMT) were obtained in descending order ofhomology (Table 1).

Identified OMT-related genes from the latest
C. clementina genome database (ver. 1.0) in Phytozome.
Gene ID

Locus name

CcOMT1CcOMT2CcOMT3CcOMT4CcOMT5CcOMT6CcOMT7CcOMT8CcOMT9CcOMT10CcOMT11CcOMT12CcOMT13
CcOMT14CcOMT15
CcOMT16

Ciclev10015705m
Ciclev10015708m
Ciclev10015723m
Ciclev10015630m
Ciclev10015762m
Ciclev10017559m
Ciclev10018099m
Ciclev10015724m
Ciclev10015993m
Ciclev10017872m
Ciclev10017930m
Ciclev10018226m
Ciclev10015685m
Ciclev10017683m
Ciclev10017585m
Ciclev10017649m

Multiple sequence alignment and construction of aphylogenetic tree
In order to predict the function of identified OMTgenes in citrus, putative amino acid sequences were an‐alyzed using the ClustalX2 multiple sequence align‐
ment program ver. 2.0.5 (Jeanmougin et al., 1998) andthe BioEdit program ver. 7.7.0 (http://www.mbio.ncsu.
edu/Bioedit/bioedit) with the known genes from otherplant species that are related to the O-methylation offlavonoids. The tree was constructed using theneighbor-joining (N-J) method for the deduced aminoacid sequences of the OMT genes from alfalfa[Medicago sativa (L10211)], American golden sax‐
ifrage [Chrysosplenium americanum (U16794)],
Arabidopsis [Arabidopsis thaliana (U70424)], barleyTable 2. Primer sequences used in gene cloning, semi-qRT-PCR and qRT-PCR.
F. T. Zohra, S. Takematsu, Y. Itami and N. Kotoda
228
Table 2. Primer sequences used in gene cloning, semi-qRT-PCR and qRT-PCR.
GeneCloning
CreOMT1
CreOMT2
CreOMT4
Semi-qRT-PCR/qRT-PCR
CreOMT1
CreOMT2
CreOMT4
Reference gene
CuActinPrimerOligonucleotide sequence (5′→3′)

Ccl_7OMT1_F1
Ccl_7OMT1_R1
Ccl_7OMT2_F1
Ccl_7OMT2_R1
Ccl_7OMT4_F1
Ccl_7OMT4_R1

TAACCCTACTTAGCCTAATAACTCTGGTGGCCCTAGGGCCATATCAATTAACCCTACTTAGCC
GCCCTAGGATCGAGAAACAACTCGCTTCTTGTTAATGACAG
TATTGGGAAGACACAGGATC

qPCR_7OMT-1_F
qPCR_7OMT-1_R
qPCR_7OMT-2_F
qPCR_7OMT-2_R
qPCR_7OMT-4_F
qPCR_7OMT-4_R

TGTTGTACTCAAGTGGATATTG
TTCAGTAGACTCATCATTCCC
GGTTCTATTCAAGTGGATATTATTTCAATAGACTCAGAATCCCTTACTCAAGTGGGTTCTGCATA
GGTTGAGTCCTTGTCTATGC

CuActin_F (-86 > -66)
CuActin_R (-1 < 19)

GAGCGATAGAGAGAATCGACATATCCTCAGCATCGGCCATT[Hordeum vulgare (CAA54616)], corn [Zea mays
(DR811764)], Madagascar periwinkle [Catharanthus
roseus (AY127568)], mango [Mangifera indica(KP993176)],
peppermint[Mentha × piperita(AY337457, AY337458, AY337459, AY337460,
AY337461)], Shiikuwasha [C. depressa (LC126059)],
rice [Oryza sativa (DQ288259, DQ530257)], vanilla[Vanilla planifolia (DQ400399, DQ400400)], wheat[Triticum aestivum (DQ223971)] with Clementine[C. clementina hort. ex Tanaka (CcOMT1 to
CcOMT16)] and ‘Yoshida’ Ponkan (CreOMT1,CreOMT2, and CreOMT4). The phylogenetic tree wasdisplayed using the N-J plot with bootstrap values for
100 trials in each branch. In addition, multiple sequencealignment was conducted for CreOMT1, CreOMT2,
and CreOMT4 with three other OMT proteins from bar‐ley (CAA54616), Madagascar periwinkle (AY127568),
and peppermint (AY337459).
RNA isolation
Total RNA was isolated from the leaves of 10 citrus
cultivars(‘Amanatsu’,‘Aoshima’,‘Benibae’,‘Hirakishu’, ‘Kiyomi’, ‘Mihaya’, ‘Ogimi kuganii’,‘Ota’ Ponkan, ‘Seinannohikari’, and ‘Shiranuhi’) using
a CTAB-based method and from flavedos using an SV
Total RNA Isolation System (Promega) according to
the manufacturer’s instructions. Isolated total RNA was
purified with an RNeasy Mini Kit (Qiagen GmbH,
Hilden, Germany). The quality and concentration ofRNAs were determined using a BioSpec-mini(Shimadzu). Prior to the cDNA synthesis, the concen‐
tration of extracted RNA was adjusted to 200 ng·μL−1.
Expression analysis of citrus OMT genes
The first-strand cDNA was synthesized from 1 μg of

total RNA in 20 μL of a reaction mixture using a
QuantiTect Reverse Transcription Kit (Qiagen) accord‐
ing to the manufacturer’s instructions. Semiquantitative RT-PCR for CreOMT1, CreOMT2, andCreOMT4 was performed with 1 μL of the first-strand
cDNA as a template in a total volume of 20 μL using
PrimeTaq DNA Polymerase (GENETBIO Inc.,
Daejeon, Korea) with gene-specific primer sets (Table
2). CuActin (C. unshiu actin) was used as an internal
control. The RT-PCR products were separated by 2%
agarose gel.
Further expression analysis of CreOMT1 andCreOMT4 was carried out with qRT-PCR. One micro‐
liter of the first-strand cDNA was used as a template ina total volume of 12.5 μL using SYBR® Green RealTime PCR Master Mix (TOYOBO). The qRT-PCR was
performed using a Thermo ScientificTM PikoRealTM
Real-Time PCR System (Thermo Fisher Scientific Inc.,
Waltham, MA, USA) as follows: 95°C for 1 min, 40 cy‐
cles of 95°C for 15 s, and 60°C for 1 min for CreOMT1,CreOMT4, and CuActin. CuActin was used as a refer‐ence gene for normalization of the transcript levels of
CreOMT1 and CreOMT4, as described in Kotoda et al.
(2016). Three technical replicates were performed foreach reaction. The primer sets used in the qRT-PCR are
listed in Table 2.
Statistical analysis
The relationships between PMF contents in leaves
and flavedos and between PMF content and gene ex‐pression were investigated using Pearson’s correlation
analysis and non-correlation test, respectively. All sta‐tistical analyses were performed at a significance levelof P < 0.05 using R-3.2.0 (R Core Team, 2015).

229

Hort. J. 89 (3): 225–236. 2020.

Hirakishu
Hirakishu

Benibae
BenibaeponkanOtaOtaPonkanponkanOtaOtaPonkanOgimikuganiiOgimi
kuganiSeinannohikari
Seinannohikari

Kiyomi
Kiyomi

Hirakishu
Hirakishu

Hirakishu
Hirakishu

OtaOtaPonkanponkanOgimikuganiiOgimi
kugani

Benibae
Benibae

YoshidaPonkanYoshidaponkanKiyomi
Kiyomi

2

YoshidaPonkan
ponkanYoshida4

YoshidaPonkan
ponkanYoshida

6

12
10
8
6
4
2
0

Ogimi
kuganiOgimi
kuganii

8

Shiranuhi
Shiranuhi

Tangeretin in flavedos

14

Kiyomi
Kiyomi10

Seinannohikari
Seinannohikari

18

Benibae
Benibae

Tangeretin (mg·g-1 DW)

12Aoshima
Aoshima

0

16

14

0

1

Amanatsu
Amanatsu

Benibae
Benibae

Hirakishu
Hirakishu

OgimikuganiOgimi
kuganii

OtaOtaPonkanponkanSeinannohikari
Seinannohikari

Shiranuhi
Shiranuhi

Kiyomi
KiyomiMihayaMihayaYoshidaPonkanYoshidaponkanD

Nobiletin in flavedos

16

2

Aoshima
Aoshima18

Aoshima
Aoshima0

Amanatsu
Amanatsu

1

3

Shiranuhi
Shiranuhi

2

4

Aoshima
Aoshima3

5

Shiranuhi
Shiranuhi

4Tangeretin in leaves

6

Amanatsu
Amanatsu

5

7

MihayaMihaya6

Tangeretin (mg·g-1 DW)

Nobiletin in leaves

Amanatsu
Amanatsu

Nobiletin (mg·g-1 DW)

C

B

7

MihayaMihayaNobiletin (mg·g-1 DW)

ASeinannohikari
Seinannohikari

Accumulation of nobiletin and tangeretin in the leaves
and flavedos of citrus cultivars
We investigated the accumulation of PMFs, nobiletin
and tangeretin, in the leaves and flavedos of 11 citrus
cultivars as shown in Figure 2. We found that nobiletin
and tangeretin were widely distributed among the citrus
cultivars and varied in the degree of accumulation in
the both tissues. Nobiletin and tangeretin were accumu‐
lated at higher levels in flavedos (Fig. 2C, D) than inleaves (Fig. 2A, B).
In the leaves, a higher level of nobiletin was found in‘Benibae’ (6.19 mg·g−1 DW), followed by ‘Hirakishu’,‘Ogimi kuganii’, ‘Yoshida’ Ponkan, and ‘Ota’ Ponkan,
whereas nobiletin was present at a lower level in‘Seinannohikari’ (1.64 mg·g−1 DW), followed by‘Shiranuhi’, ‘Kiyomi’, ‘Aoshima’, ‘Mihaya’, and‘Amanatsu’ (Fig. 2A). Similarly, tangeretin was highly
accumulated in the leaves of ‘Benibae’ (5.90 mg·g−1
DW), followed by ‘Hirakishu’, ‘Yoshida’ Ponkan,‘Ogimi kuganii’, and ‘Ota’ Ponkan, while a low tan‐geretin content was observed in ‘Seinannohikari’(0.43 mg·g−1 DW), followed by ‘Kiyomi’, ‘Shiranuhi’,‘Aoshima’, ‘Amanatsu’, and ‘Mihaya’ (Fig. 2B).

In the flavedos, nobiletin was highly accumulated in‘Hirakishu’ (14.81 mg·g−1 DW), followed by ‘Ogimi
kuganii’, ‘Ota’ Ponkan, ‘Benibae’, and ‘Yoshida’
Ponkan, while a lower amount of nobiletin was accu‐
mulated in ‘Kiyomi’ (4.38 mg·g−1 DW), followed by‘Shiranuhi’, ‘Seinannohikari’, ‘Aoshima’, ‘Amanatsu’,and ‘Mihaya’ (Fig. 2C). Similarly, tangeretin was accu‐
mulated at a higher level in the flavedos of ‘Ota’
Ponkan (12.01 mg·g−1 DW), followed by ‘Hirakishu’,‘Yoshida’ Ponkan, ‘Ogimi kuganii’, and ‘Benibae’,
whereas a lower accumulation of tangeretin was ob‐
served in ‘Kiyomi’ (0.74 mg·g−1 DW), followed by‘Amanatsu’, ‘Shiranuhi’, ‘Seinannohikari’, ‘Aoshima’,and ‘Mihaya’ (Fig. 2D).
The nobiletin and tangeretin contents were highest
in the leaves of ‘Benibae’ (6.19 mg·g−1 DW and5.90 mg·g−1 DW, respectively), and the lowest contents
were found in ‘Amanatsu’ (0.17 mg·g−1 DW) and‘Mihaya’ (0.09 mg·g−1 DW), respectively. The highest
amounts of nobiletin and tangeretin were accumulated
in the flavedos of ‘Hirakishu’ (14.81 mg·g−1 DW) and‘Ota’ Ponkan (12.01 mg·g−1 DW), respectively. The
lowest nobiletin and tangeretin contents were found in‘Mihaya’ (0.17 mg·g−1 DW and 0.09 mg·g−1 DW, re‐spectively).

MihayaMihayaResults

Fig. 2. Accumulation of nobiletin and tangeretin in the leaves and flavedos of 11 citrus cultivars: ‘Amanatsu’, ‘Aoshima’, ‘Benibae’,‘Hirakishu’, ‘Kiyomi’, ‘Mihaya’, ‘Ogimi kuganii’, ‘Ota’ Ponkan, ‘Seinannohikari’, ‘Shiranuhi’, and ‘Yoshida’ Ponkan. (A) The nobiletincontent in leaves. (B) The tangeretin content in leaves. (C) The nobiletin content in flavedos. (D) The tangeretin content in flavedos. The
values are means ± SD of the results from three biological replicates per cultivar.

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F. T. Zohra, S. Takematsu, Y. Itami and N. Kotoda

Correlation of nobiletin content in the leaves and flave‐
dos of citrus cultivars
To evaluate the distribution of nobiletin content inboth the leaves and flavedos of 11 citrus cultivars, we
performed correlation analysis using the R program(R-3.2.0) (R Core Team, 2015). The correlation analy‐
sis revealed that nobiletin content was positively andsignificantly correlated (r = 0.824, P = 0.00182) be‐
tween the leaves and flavedos of 11 citrus cultivars(Fig. 3). In addition, the nobiletin content was sig‐
nificantly correlated with the tangeretin content inboth leaves (r = 0.978, P < 0.000001) and flavedos(r = 0.913, P < 0.0001) (Fig. S1).
Isolation of OMT genes from ‘Yoshida’ Ponkan
We found 16 OMT-related genes in the C. clementina
genome database (ver. 1.0) by conducting a BLAST
search (Table 1). To infer the function of OMT genes in
citrus, we isolated the cDNA of these corresponding
genes from the leaves of ‘Yoshida’ Ponkan. To evaluate
the evolutionary relationships of 16 putative OMTgenes from the C. clementina genome (assigned as
CcOMT), using three isolated genes from ‘Yoshida’
Ponkan [assigned as CreOMT: CreOMT1 (LC507211),
CreOMT2 (LC507212), and CreOMT4 (LC507213)]
and 18 previously reported OMT genes from other plant
species, including Hv7-OMT of barley, we constructeda phylogenetic tree based on the deduced amino acidsequences (Fig. 4). The phylogenetic tree revealed that
CreOMT1, CreOMT2, and CreOMT4 were closely
clustered with Mp8-OMT (AY337459) of peppermint,
Cat3'5'-OMT (CrOMT2, AY127568) of Madagascar

periwinkle, and Hv7-OMT (CAA54616) of barley.
Hv7-OMT is involved in the O-methylation of thehydroxy group at the 7th position of the flavonoid(Christensen et al., 1998). Moreover, Mp8-OMT ofpeppermint and Cat3'5'-OMT of Madagascar periwin‐kle, which were more closely related to CreOMT1,CreOMT2, and CreOMT4 than Hv7-OMT, are involvedin the oxidation and O-methylation of the hydroxygroup at the 8- and 3',5'-positions of flavonoid, respec‐tively (Cacace et al., 2003; Willits et al., 2004). The
identity levels (similarity) between CreOMT1 andCreOMT2, CreOMT1 and CreOMT4, and CreOMT2
and CreOMT4 were 94.5% (96.4%), 54.1% (70.8%),
and 54.4% (71.4%), respectively. CreOMT1 andCreOMT2 was closely related as shown in the phyloge‐
netic tree. In addition, multiple sequence alignment of
CreOMT1, CreOMT2, and CreOMT4 with Hv7-OMT,
Mp8-OMT, and Cat3'5'-OMT showed five consensus
sequences (Fig. 5). These motifs (motif I to V) are theresidues found in SAM-dependent O-methyltransferase.

97
100
90
48
99

56
45

83

97

100

100

100

53
100
100
55

100

100

Ota Ponkan

100
98
57

Yoshida Ponkan

KiyomiMihayaShiranuhi
Seinannohikarir = 0.824
P = 0.00182

Conc. of nobiletin in leaves (mg·g-1 DW)

Fig. 3. Correlation of nobiletin content between the leaves andflavedos of 11 citrus cultivars: ‘Amanatsu’, ‘Aoshima’,‘Benibae’, ‘Hirakishu’, ‘Kiyomi’, ‘Mihaya’, ‘Ogimi kuganii’,‘Ota’ Ponkan, ‘Seinannohikari’, ‘Shiranuhi’, and ‘Yoshida’
Ponkan. Statistical analysis was performed using R-3.2.0 (RCore Team, 2015).

100

85

BenibaeAmanatsu
AoshimaConc. of nobiletin in flavedos (mg·g-1 DW)

Ogimi kuganii

Hirakishu

77

0.05

100

CcOMT7 (Ciclev10018099m)
CdFOMT5 (LC126059)
CcOMT16 (Ciclev10017649m)
CcOMT12 (Ciclev10018226m)
62 CcOMT14 (Ciclev10017683m)
100 CcOMT13 (Ciclev10015685m)
VanOMT3 (DQ400400)
VanOMT2 (DQ400399)
Ta3'4'5'-OMT (DQ223971)
Zm3'5'-OMT (AY323286)
Os3'-OMT1 (DQ288259)
89
Os3'-OMT2 (DQ530257)
100
Mp3'-OMT (AY337460)
At3'-OMT (U70424)
Ca3'5'-OMT (U16794)
Med2'-OMT (L10211)
CcOMT15 (Ciclev10017585m)
Mi4-OMT (KP993176)
CcOMT11 (Ciclev10017930m)
CcOMT10 (Ciclev10017872m)
CcOMT9 (Ciclev10015993m)
Mp4'-OMT (AY337461)
Mp7-OMT1 (AY337458)
Mp7-OMT (AY337457)
Hv7-OMT (CAA54616)
CcOMT3 (Ciclev10015723m)
CcOMT8 (Ciclev10015724m)
CcOMT6 (Ciclev10017559m)
Cat3'5'-OMT (AY127568)
Mp8-OMT (AY337459)
CcOMT5 (Ciclev10015762m)
CreOMT4 (LC507213)
100 CcOMT4 (Ciclev10015630m)
100 CreOMT2 (LC507212)
CcOMT2 (Ciclev10015708m)
CreOMT1 (LC507211)
100 CcOMT1 (Ciclev10015705m)

Fig. 4. Phylogenetic analysis of OMT genes of citrus cultivars to‐gether with other plant species. The tree was constructed by theneighbor-joining (N-J) method and 100 bootstrap trials for the
deduced amino acid sequences of the OMT genes from alfalfa[Med2'-OMT (L10211)], Arabidopsis [At3'-OMT (U70424)],barley [Hv7-OMT (CAA54616)], corn [Zm3'5'-OMT(DR811764)], Madagascar periwinkle [Cat3'4'-OMT (AY12756),
Ca3'5'-OMT (U16794)], mango [Mi4-OMT (KP993176)], pep‐
permint [Mp3'-OMT (AY337457), Mp4'-OMT (AY337461),
Mp7-OMT (AY337458), Mp7-OMT1 (AY337460), Mp8-OMT(AY337459)], rice [Os3'-OMT1 (DQ288259), Os3'-OMT2(DQ530257)], Shiikuwasha [CdFOMT5 (LC126059)], vanilla[VanOMT2 (DQ400399), VanOMT3 (DQ400400)], and wheat[Ta3'4'5'-OMT (DQ223971)], together with 16 CcOMT candi‐date genes from Clementin and three CreOMT genes[CreOMT1 (LC507211), CreOMT2 (LC507212), CreOMT4(LC507213)] isolated from ‘Yoshida’ Ponkan.

231

Hort. J. 89 (3): 225–236. 2020.

mo�f Imo�f IVmo�f IImo�f IIImo�f V

Fig. 5. Alignment of OMT proteins derived from ‘Yoshida’ Ponkan (CreOMT1, CreOMT2, and CreOMT4) and other plant species, Madagascar
periwinkle [Cat3'5'-OMT (AY127568)], barley [Hv7-OMT (CAA54616)], and peppermint [Mp8-OMT (AY337458)]. Putative amino acidsequences were aligned using the ClustalX2 multiple sequence alignment program ver.2.0.5 (Jeanmougin et al., 1998).

Expression analysis of CreOMT1, CreOMT2, andCreOMT4
To clarify whether or not CreOMT1, CreOMT2, andCreOMT4 were expressed in the leaves of nine citrus
cultivars in which PMFs accumulate, we analyzed theexpression of these genes by semi-qRT-PCR (Fig. S2).
CreOMT1 was highly expressed in ‘Hirakishu’, ‘Ota’
Ponkan, and ‘Ogimi kuganii’, with a lower expressionin ‘Benibae’ and ‘Amanatsu’, whereas no expression
was found in ‘Seinannohikari’, ‘Shiranuhi’, ‘Kiyomi’,and ‘Aoshima’. On the other hand, no CreOMT2
expression was detected in any of the investigatedcultivars. CreOMT4 was expressed in ‘Benibae’,‘Hirakishu’, ‘Ota’ Ponkan, ‘Ogimi kuganii’, and‘Amanatsu’, but was not expressed in ‘Seinannohikari’,‘Shiranuhi’, ‘Kiyomi’, or ‘Aoshima’.
To further confirm the transcripts of CreOMT1 andCreOMT4 in the leaves and flavedos of 10 citrus culti‐
vars, we performed qRT-PCR. The transcripts of
CreOMT1 and CreOMT4 were detectable in the twotissues of all citrus cultivars (Fig. 6). The transcript ofCreOMT1 was highly accumulated in the leaves of‘Hirakishu’, ‘Ota’ Ponkan, ‘Amanatsu’, and ‘Ogimi
kuganii’, but lower accumulation was observed in‘Benibae’, ‘Mihaya’, ‘Kiyomi’, ‘Shiranuhi’, ‘Aoshima’,and ‘Seinannohikari’ (Fig. 6A). In contrast, the tran‐
scripts of CreOMT1 were relatively abundant in theflavedos of ‘Ogimi kuganii’, ‘Ota’ Ponkan, ‘Shiranuhi’,and ‘Hirakishu’, with only slight accumulation oftranscripts in ‘Benibae’, ‘Mihaya’, ‘Aoshima’,‘Seinannohikari’, ‘Amanatsu’, and ‘Kiyomi’ (Fig. 6B).
The transcript of CreOMT4 accumulated primarily inthe leaves of ‘Ota’ Ponkan, ‘Amanatsu’, ‘Benibae’, and‘Hirakishu’ and remained constant at lower levels in‘Mihaya’, ‘Kiyomi’, ‘Ogimi kuganii’, ‘Shiranuhi’,‘Seinannohikari’, and ‘Aoshima’ (Fig. 6C). In flavedos,
CreOMT4 was expressed at higher levels in ‘Ota’
Ponkan, ‘Ogimi kuganii’, and ‘Shiranuhi’, whereas
lower levels of transcripts were detected in ‘Kiyomi’,‘Mihaya’, and ‘Seinannohikari’ (Fig. 6D).
To investigate the correlation between nobiletin con‐tent and CreOMT gene expression, we conducted corre‐
lation analysis as shown in Figure 7. The result showeda significant correlation of the nobiletin content and
CreOMT1 expression in the flavedos (r = 0.700, P =0.0243; Fig. 7B), with an apparent correlation between
the nobiletin content and CreOMT4 in the flavedos(r = 0.588, P = 0.0740; Fig. 7D), the nobiletin content
and CreOMT1 in the leaves (r = 0.420, P = 0.2278;
Fig. 7A), and the nobiletin content and CreOMT4 in theleaves (r = 0.306, P = 0.3906; Fig. 7C).

Discussion
In 11 citrus cultivars tested, the nobiletin and tan‐geretin contents were generally higher in flavedos thanin leaves, except for nobiletin in ‘Seinannohikari’ andtangeretin in ‘Mihaya’ (Fig. 2). Similarly, a higher ac‐
cumulation of PMFs in flavedos/peels has been ob‐
served in various citrus cultivars as compared to othertissues such as the fruit, juice vesicle, albedo, and seg‐

232

F. T. Zohra, S. Takematsu, Y. Itami and N. Kotoda

Shiranuhi
Shiranuhi

Ota
OtaPonkan
ponkanOgimikuganiOgimi
kuganii

Ogimi
Ogimikuganii
KuganiOta
PonkanOtaponkan

Hirakishu
Hirakishu

Shiranuhi
Shiranuhi

Benibae
BenibaeMihayaMihayaAoshima
AoshimaSeinannohikari
Seinannohikari

Kiyomi
Kiyomi

0.020
0.015

0.010
0.005
0.000

Amanatsu
Amanatsu

OtaPonkan
ponkanOta

Amanatsu
Amanatsu

Benibae
BenibaeMihayaMihayaHirakishu
Hirakishu

Kiyomi
Kiyomi

0.02

0.025

Hirakishu
Hirakishu

0.04

0.030

Aoshima
Aoshima

0.06

CreOMT4 in flavedos

0.035

Benibae
Benibae

0.08

Ogimi
Ogimikuganii
kugani0.001

Kiyomi
Kiyomi0.10

Shiranuhi
Shiranuhi

0.002

0.040

Relative expression

0.12

Seinannohikari
Seinannohikari

0.003

Hirakishu
Hirakishu

Ota
PonkanOtaponkanAmanatsu
Amanatsu

OgimikuganiiOgimi
kugani

Benibae
BenibaeKiyomi
KiyomiMihayaMihayaCreOMT4 in leaves

0.14

0.004

Seinannohikari
Seinannohikari

0.16

0.005

0

D

0.18

Aoshima
AoshimaRelative expression

Aoshima
Aoshima

0.01

CreOMT1 in flavedos

0.006

Amanatsu
Amanatsu

0.02

0.007

MihayaMihaya

0.03

0.00

0.008

Relative expression

0.04

0

C

B

CreOMT1 in leaves

0.05

Seinannohikari
Seinannohikari

Relative expression

0.06

Shiranuhi
Shiranuhi

A

Fig. 6. Expression profiles of CreOMT1 and CreOMT4 in the leaves and flavedos of 10 citrus cultivars: ‘Amanatsu’, ‘Aoshima’, ‘Benibae’,‘Hirakishu’, ‘Kiyomi’, ‘Mihaya’, ‘Ogimi kuganii’, ‘Ota’ Ponkan, ‘Seinannohikari’, and ‘Shiranuhi’. (A) The expression of CreOMT1 inleaves. (B) The expression of CreOMT1 in flavedos. (C) The expression of CreOMT4 in leaves. (D) The expression of CreOMT4 in flave‐dos. The citrus actin gene (CuActin) was used as a reference gene. The values are means ± SD of the results from three technical replicates
per cultivar.

1

2

3

4

5

0

6

D

10

15

r = 0.588, P = 0.0740

0.015

0.020

0.025

0.030

0.04

0.06

0.08

CreOMT4
expression
data$Cr7.OMT_4_Flavedos

0.10

5

Conc. of nobiletinin flavedos (mg·g-1 DW)
data$Nobiletin_Flavedos

r = 0.306, P = 0.3906

C

0.005

0.02

data$Cr7.OMT_4_Leaves
CreOMT4
expression

Conc. of nobiletin
in leaves (mg·g-1 DW)
data$Nobiletin_Leaves

r = 0.700, P = 0.0243

0.010

0

B

0.000 0.001 0.002 0.003 0.004 0.005 0.006

CreOMT1
expression
data$Cr7.OMT_1_Flavedos

0.05
0.04
0.03
0.02
0.01
0.00

data$Cr7.OMT_1_Leaves
CreOMT1
expressionr = 0.420, P = 0.2278

A

0

1

2

3

4

5

6

data$Nobiletin_Leaves
Conc. of nobiletin
in leaves (mg·g-1 DW)

0

5

10

15

Conc. of nobiletinin flavedos (mg·g-1 DW)
data$Nobiletin_Flavedos

Fig. 7. Correlation of the nobiletin content and the CreOMT gene expression. The citrus cultivars used in this analysis were the same as thoseused in the gene expression experiment. Scatter plot of (A) nobiletin content and CreOMT1 expression in leaves, (B) nobiletin content and
CreOMT1 expression in flavedos, (C) nobiletin content and CreOMT4 expression in leaves, (D) nobiletin content and CreOMT4 expressionin flavedos. Displaying scatter plot and statistical analysis were performed using R-3.2.0 (R Core Team, 2015).

Hort. J. 89 (3): 225–236. 2020.

ment epidermis (Nogata et al., 2006). Of the 11 culti‐
vars, five (‘Benibae’, ‘Hirakishu’, ‘Ogimi kuganii’,‘Ota’ Ponkan, and ‘Yodhida’ Ponkan) accumulated ahigher level of PMFs. Because the parents of ‘Benibae’are HF No. 9 (‘Hayashi’ Satsuma mandarin ×‘Fukuhara’ orange) and ‘Encore’, the high content of
PMFs in ‘Benibae’ may be derived from ‘Encore’,
which accumulated 3.73 mg·g−1 DW of nobiletin in its
leaves (unpublished result). Regarding three mandarins,
Kishu (‘Hirakishu’), Shiikuwasha (‘Ogimi kuganii’),and Ponkan (‘Ota’ and ‘Yoshida’), their genetic back‐ground and genealogy are unclear at present. Funda‐
mentally, our results were consistent with those of
Nogata et al. (2006) and Yamamoto et al. (2019). The
nobiletin content was higher than the tangeretin content
in the leaves of the same cultivar (Fig. 2A, B). The
flavedos also had more nobiletin than tangeretin, except
for ‘Amanatsu’ (Fig. 2C, D). These results indicatedthat nobiletin tended to accumulate at higher levels thantangeretin and that there was a significant correlation
between the accumulation of nobiletin and tangeretin inleaves (r = 0.978, P < 0.000001; Fig. S1A) and flavedos(r = 0.913, P < 0.0001; Fig. S1B) in the citrus cultivars
tested. Nogata et al. (2006) comprehensively investigat‐
ed flavonoids, including PMFs, in the flavedos of 45
citrus cultivars, and similar results were obtained inabout 76% of 21 cultivars with some nobiletin content(> 0.2 mg·g−1 FW). These results may be due to the factthat nobiletin has six methoxy groups and tangeretin
has five, and so the structural difference is only one
methoxy group (Fig. 1), suggesting that their biosyn‐
thetic pathways are similar. In addition, the nobiletincontent in the leaves was found to have a positive corre‐
lation (r = 0.824, P = 0.00182) with the nobiletin con‐tent in the flavedos (Fig. 3). Similarly, tangeretin
content was shown to be positively correlated in theleaves and flavedos (data not shown). These results
suggest that a set of genes that synthesize PMFs ispresent in both leaves and flavedos. In a citrus breeding
program, this could be utilized for early selection of
seedlings in the juvenile phase, which are expected toaccumulate a higher amount of PMFs in fruit, resulting
in a shortening of the breeding period.
The OMT multigene family has been identified inmany plant species, including alfalfa (Maxwell et al.,1993), American golden saxifrage (Gauthier et al.,1995), Arabidopsis (Zhang et al., 1997), barley(Christensen et al., 1998; Zhou et al., 2008), corn (Zhouet al., 2008), Madagascar periwinkle (Cacace et al.,2003), mango (Chidley et al., 2016), peppermint(Willits et al., 2004), rice (Kim et al., 2006), vanilla (Liet al., 2006), and wheat (Wang et al., 2018; Zhou et al.,2006). Recently, five OMT genes for methylation offlavones were isolated from Shiikuwasha (Itoh et al.,2016). In this study, we identified 16 putative OMTgenes in the C. clementina genome database (ver. 1.0)
and isolated three putative OMT genes, CreOMT1,233

CreOMT2, and CreOMT4, from ‘Yoshida’ Ponkan.
Phylogenetic analysis showed that they were closely re‐lated to OMT genes such as Mp8-OMT (AY337458),
Cat3'5'-OMT(AY127568),
andHv7-OMT(CAA54616). Barley Hv7-OMT is involved in the Omethylation of the hydroxy group at the 7th position offlavones (Christensen et al., 1998). Similarly, Madagas‐
car periwinkle Cat3'5'-OMT (CrOMT2) is involved in
the two sequential O-methylations of the hydroxy groupat the 3'- and 5'-positions of flavonols and in theirbiosynthesis (Cacace et al., 2003). In peppermint, Mp8OMT (MpOMT2) was found to be involved in the Omethylation of the hydroxy group at the C8-position offlavones (Willits et al., 2004). Because Hv7-OMT and
Mp8-OMT methylate hydroxyflavones at C7 and C8,
respectively, these citrus OMT-like genes may be in‐volved in the biosynthesis of nobiletin and tangeretin,
which are methylated at C7 and C8, respectively(Fig. 1). Especially, CreOMT1 and CreOMT4 may cat‐alyze the hydroxy group at the 8th position of flavones,
considering that they were more related to Mp8-OMT
than to Cat3'5'-OMT or Hv7-OMT in the phylogenetictree.
Multiple sequence alignment of CreOMT1,CreOMT2, and CreOMT4 with Hv7-OMT, Cat3'5'OMT, and Mp8-OMT showed several consensus se‐quence motifs (motif I to V; Fig. 5). Basically,
consensus sequence motifs I to IV are characteristic of
SAM-dependent O-methyltransferase, which transfers amethyl group from SAM to a hydroxy group; SAMdependent methylations are important in the biosynthe‐
sis of flavonoids (Christensen et al., 1998; Gana et al.,2013; Ibrahim et al., 1998; Vidgren et al., 1994; Willits
et al., 2004). Thus, CreOMT1, CreOMT2, andCreOMT4 could be expected to be involved in thebiosynthesis of PMFs in citrus.
We investigated the expression of CreOMT1,CreOMT2, and CreOMT4 in the leaves of nine citrus
cultivars by semi-qRT-PCR. The results demonstrated
that CreOMT1 and CreOMT4 were expressed in‘Benibae’, ‘Hirakishu’, ‘Ota’ Ponkan, ‘Ogimi kuganii’,and ‘Amanatsu’ (Fig. S2). This result reflected the ac‐
cumulations of nobiletin and tangeretin in the leaves of
those cultivars, except for ‘Amanatsu’. In contrast, no
CreOMT2 expression was detected in any of the inves‐tigated cultivars, suggesting that CreOMT2 may not be
involved in the biosynthesis of PMFs in the leaves of
citrus cultivars. Therefore, we selected two OMT genes,
CreOMT1 and CreOMT4, for further expression analy‐
sis of both the leaves and flavedos of 10 citrus cultivars.
Transcripts of CreOMT1 and CreOMT4 were detected
by qRT-PCR in both leaves and flavedos. However,there was a tendency for the transcripts of CreOMT1and CreOMT4 in leaves to be much higher than those inflavedos in the same cultivars (Fig. 6). These results
suggest differences in the state of the RNA of evergreen
leaves and senescent flavedos (exocarps) in the matura‐

234

F. T. Zohra, S. Takematsu, Y. Itami and N. Kotoda

tion season. Flavedos almost cease growing in January,
and RNA synthesis is not thought to be very active. Be‐
cause we confirmed seasonal changes in the accumula‐tion of PMFs in the leaves and flavedos of Ponkan andShiikuwasha (Kotoda et al., 2017; Yamaguchi et al.,2015), the expression of the genes and enzymes respon‐sible for the biosynthesis of PMFs may vary with devel‐
opmental stage, although sufficient PMFs had
accumulated in the tissues.
The transcripts of CreOMT1 and CreOMT4 were as‐sociated with the accumulation of PMFs in leaves andflavedos of the cultivars studied (Figs. 2 and 6). The ex‐pression of CreOMT1 and CreOMT4 tended to be more
related to the accumulation of nobiletin in flavedos thanin leaves (Fig. 7). Notably, there was a significant cor‐
relation (r = 0.700, P = 0.0243) between the CreOMT1
expression and the nobiletin content in flavedos(Fig. 7B). In leaves, however, CreOMT1 was expressed
relatively highly in ‘Amanatsu’, which accumulated alower level of PMFs, to the same degree as in ‘Ogimi
kuganii’, which had a higher level of PMFs. In contrast,
there was not as much CreOMT1 expression in‘Benibae’ as expected. In flavedos, on the other hand,
CreOMT1 was expressed relatively highly in‘Shiranuhi’, which was a low PMF-accumulating culti‐
var. In ‘Amanatsu’, which accumulated fewer PMFs inboth leaves and flavedos, CreOMT1 and CreOMT4
were expressed relatively highly, except for the lower
expression of CreOMT1 in flavedos. Based on these re‐sults, ‘Amanatsu’ may have inherited a part of the genesets related to the biosynthesis of PMFs from a PMFaccumulating cultivar, Kishu-mikan (C. kinokuni hort. ex
Tanaka), which could be a pollen parent of ‘Amanatsu’(Shimizu et al., 2016). In ‘Shiranuhi’, on the other
hand, the higher expression of those OMT genes may bederived from the Ponkan genome because the pollen par‐ent of ‘Shiranuhi’ is ‘Nakano No. 3’ Ponkan (Matsumoto,2001). Further study will be needed to clarify the bio‐chemical function of CreOMT1 and CreOMT4.
In this study, we clarified the accumulation of two
PMFs, nobiletin and tangeretin, in the leaves and flave‐
dos of various citrus cultivars grown in Japan and founda positive correlation in the accumulation of PMFs be‐
tween leaves and flavedos. Based on these results, theconcentration of PMFs in leaves could be utilized as an
early selection marker for seedlings in the juvenile
phase as these are expected to accumulate a higher
amount of PMFs in fruit, resulting in a shortening of thebreeding period. In addition, two candidate genes re‐sponsible for O-methylation in the biosynthesis of
PMFs were identified. Notably, the expression of
CreOMT1 showed a significant correlation with the ac‐
cumulation of nobiletin in the citrus cultivars used inthis study. Our results will be useful in breeding new
cultivars that accumulate higher levels of PMFs andalso for elucidating the PMF biosynthetic pathway incitrus in the future.

Acknowledgements
We are grateful to the staff of Analytical ResearchCenter for Experimental Sciences, Saga University, fortechnical assistance.

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