See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/358522791

Difference Between Coloration and Endogenous Abscisic Acid Accumulation

Patterns in Two Red Grape Cultivars, ‘Aki Queen’ and ‘Ruby Roman’ (Vitis
labruscana Bailey) Berries

Article in The Horticulture Journal · February 2022

DOI: 10.2503/hortj.UTD-342

CITATION READS

1 73

7 authors, including:

Ayako Katayama-Ikegami Yuta Sugiyama

Kyoto University Gunma University
53 PUBLICATIONS 1,342 CITATIONS 21 PUBLICATIONS 450 CITATIONS

SEE PROFILE SEE PROFILE

Takane Katayama
Kyoto University
166 PUBLICATIONS 5,417 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Plant Alkaloid Engineering View project

Alkaloid production View project

All content following this page was uploaded by Ayako Katayama-Ikegami on 11 April 2022.

The user has requested enhancement of the downloaded file.

This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication.

The Horticulture Journal Preview The Japanese Society for

doi: 10.2503/hortj.UTD-342
JSHS Horticultural Science
http://www.jshs.jp/

Difference Between Coloration and Endogenous Abscisic Acid Accumulation

Patterns in Two Red Grape Cultivars, ‘Aki Queen’ and ‘Ruby Roman’ (Vitis
labruscana Bailey) Berries

Ayako Katayama-Ikegami1**, Yuta Sugiyama1***, Takane Katayama1,2, Akiko Sakamoto1,

Ryo Shimada1, Chiho Miyazaki1 and Mei Gao-Takai1*

1
Ishikawa Prefectural University, Nonoichi 921-8836, Japan
2
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

Changes in anthocyanin and endogenous abscisic acid (ABA) contents in the berry skins of Vitis labruscana ×
V. vinifera cultivars, ‘Aki Queen’, and ‘Ruby Roman’ were investigated during the fruit development period.
Color development of ‘Aki Queen’ berries mainly occurred within 20 days post véraison, while that of the
‘Ruby Roman’ was prolonged for about 40 days. In both cultivars, the ABA level in the berry skin started to
increase a few days before véraison; however, ABA accumulation ceased approximately 10 days after véraison
in ‘Aki Queen’, while it continued until the late stage of maturation in ‘Ruby Roman’. In addition, higher
indole-3-acetate aspartate (IAA-Asp) and lower isopentenyladenine (iP) contents were observed in ‘Ruby
Roman’ than in ‘Aki Queen’ at the late development stage. The expression analysis of genes involved in ABA
metabolism revealed that V. vinifera 9-cis-epoxycarotenoid dioxygenase 3 (VviNCED3), which is assumed to play
a major role in ABA biosynthesis, remained relatively higher in ‘Ruby Roman’ than in ‘Aki Queen’ after
véraison. Considering that ABA plays a regulatory role in grape maturation, these results may indicate that
the coloration of ‘Ruby Roman’ at the later stage of maturation is partly attributable to an increased ABA
pool in berry skins.

Key Words: ABA metabolism, anthocyanin, berry maturation, endogenous plant hormone, véraison.

ripening processes, and these hormones may be

Introduction
The quantities and composition of anthocyanins are
determined by the skin color of the grape berry. In
grapes, anthocyanins are accumulated after the onset
of ripening, which is known as véraison. For non‐
climacteric fruits, such as grape, abscisic acid (ABA),
brassinosteroids, and ethylene have been proposed
to promote berry ripening, while auxins, cytokinins,
gibberellins, and jasmonates delay some associated
Received; September 28, 2021. Accepted; December 18, 2021.
First Published Online in J-STAGE on February 11, 2022.
Part of this study was presented at the 2019 Spring Meeting of the
Japanese Society for Horticultural Science.
This study was supported by grants from JSPS, numbers of
JP17K07648 and 26850019 and JP17K07649, and by the Donated
Fund Research from Actree Corporation, Ishikawa Prefecture, Japan.
* Corresponding author (E-mail: mtakai@ishikawa-pu.ac.jp).
** Present address: Graduate School of Agriculture, Kyoto
University, Kyoto 606-8502, Japan.
*** Present address: Center for Food Science and Wellness, Gunma
University, Maebashi 371-8510, Japan.
involved in ripening regulation through complex inter‐
actions (Böttcher et al., 2015; Corso et al., 2016; Fortes
et al., 2015; Gouthu and Deluc, 2015; Jia et al., 2016;
Kuhn et al., 2014; Pilati et al., 2017; Ziliotto et al.,
2012). The degree of anthocyanin accumulation is pri‐
marily determined by genetic variation and is also
affected by environmental factors, such as light, tem‐
perature, and available assimilates, which limit the car‐
bon flow of secondary metabolism.
Based on genetics, Ban et al. (2014) reported quanti‐
tative trait loci (QTLs) for anthocyanin content in link‐
age groups (LGs) 2 (MYB haplotype), 8, and 14 in
interspecific hybrid grapes. Costantini et al. (2015) also
detected 19 QTLs for the total anthocyanin content in a
V. vinifera cross (‘Syrah’ × ‘Pinot Noir’). The number
and type of functional MYB haplotypes at the color
locus (LG2) are the major genetic factors that determine
the variation of anthocyanin content in grape berry skin
(Azuma, 2018). The locus appears to be a tandemly
arrayed MYB gene that spans a region of 200-kb. In this

© 2022 The Japanese Society for Horticultural Science (JSHS), All rights reserved.
 2 A. Katayama-Ikegami, Y. Sugiyama, T. Katayama, A. Sakamoto, R. Shimada, C. Miyazaki and M. Gao-Takai
cluster, the VviMYBA1 locus and the VviMYBA2 locus
are functionally crucial for berry pigmentation in
V. vinifera. Since the two adjacent MYB alleles in the
color locus are inherited together, they are described as
MYB haplotypes (Azuma, 2018; Azuma et al., 2011).
Among these haplotypes, Hap A is nonfunctional
(Kobayashi et al., 2004, 2005; Walker et al., 2007). In
the interspecific hybrid grapes of V. vinifera and
V. labrusca, three functional MYB alleles derived from
V. labrusca, named VlMYBA1-2, VlMYBA1-3, and
VlMYBA2, have been identified (Azuma et al., 2011).
The allele combination of VlMYBA1-2 and VlMYBA1-3
has been named Hap E1, while the VlMYBA2 and
VlMYBA1-3 combination was named Hap E2. These
two haplotypes are identified only in hybrid grapes
(Azuma, 2018).
‘Ruby Roman’ is a red table grape cultivar selective‐
ly bred from seedlings of ‘Fujiminori’, a tetraploid
black table grape obtained from progeny of Ikawa
Selection 682 × ‘Pione’ (Yamada and Sato, 2016),
while ‘Aki Queen’ is a self-bred seedling of ‘Kyoho’.
‘Ruby Roman’ and ‘Aki Queen’ bear red berries and
their LG2 loci have similar components, which consist
of three nonfunctional Hap As and one Hap E1 (Azuma
et al., 2011). The pattern of berry coloration, however,
is slightly different between them. The accumulation of
anthocyanin in ‘Ruby Roman’ berries was slow until 15
days after véraison and accelerated from 15 to 35 days
(Matsuda et al., 2020), while it accumulated within 20
days after véraison in ‘Aki Queen’ (Katayama-Ikegami
et al., 2017; Yamane and Shibayama, 2006).
ABA promotes the biosynthesis of anthocyanins
through upregulation of MYBAs and resultant genes
encoding enzymes together with the transporters
involved (Jeong et al., 2004; Katayama-Ikegami et al.,
2016b; Koyama et al., 2010; Pilati et al., 2017). During
the late stage of maturation, exogenous ABA treatment
promoted the expression of VlMYbA1-2, VlMybA1-3,
and UFGT in ‘Aki Queen’, but did not significantly
promoted UFGT expression for ‘Ruby Roman’
(Katayama-Ikegami et al., 2017). Comparison of the
expressions of these three genes demonstrated that they
tended to be higher in ‘Ruby Roman’ than in ‘Aki
Queen’ at the late stage of maturation, implying the
presence of a trait(s) other than the LG2 locus that con‐
trols the coloration of grape berries.
Therefore, it was assumed that the endogenous ABA
content in ‘Ruby Roman’ may be higher than that in
‘Aki Queen’, resulting in the coloration of ‘Ruby
Roman’ at the later stage. In this study, to clarify the
coloration during the later stage of maturation in ‘Ruby
Roman’, we investigated the differences between these
two cultivars in terms of endogenous ABA metabolites
and associated gene expressions. As mentioned above,
the complex and coordinated regulation of endogenous
plant hormones affects berry development, especially
from the onset of maturation to ripening. The detailed
function and/or combinations of these plant hormones
are not fully understood. Therefore, in order to better
understand the ripening process from the differences
between the two cultivars, in addition to ABA, we also
analyzed the contents of auxin and cytokinins and their
metabolites.
Materials and Methods
Plant materials
A 30-year-old ‘Aki Queen’ (Vitis labruscana Bailey
× V. vinifera L.) and two 8-year-old ‘Ruby Roman’
(Vitis labruscana Bailey × V. vinifera L.) grapevines
grown in the same vinyl house at Ishikawa Prefectural
University were used for two years, 2014 and 2015, in
this study. The ‘Aki Queen’ vine was trained with long
cane pruning, and the ‘Ruby Roman’ vines were trained
with short cane pruning. The flower clusters were tradi‐
tionally shaped (left 3.5 cm floret from the bottom) and
dipped in a solution of 25 mg·L−1 gibberellic acid (GA3)
and 5 mg·L−1 forchlorfenuron (Kyowa Hakko Bio Co.,
Ltd., Japan) to induce seedlessness at full bloom. Then
the clusters were dipped in GA3 (25 mg·L−1) at 10 days
after full bloom (DAFB) to stimulate berry enlarge‐
ment. The day of full bloom of the ‘Aki Queen’ vine
was May 19, and véraison (beginning of softening) was
June 28 (40 DAFB) in 2014, and May 16 and June 25
(40 DAFB) in 2015. Fruit clusters were thinned for the
‘Aki Queen’ to leave one on each shoot, and were
thinned to leave 30–35 berries on each cluster before
véraison. The day of full bloom of ‘Ruby Roman’ vines
was May 24, véraison was July 5 (42 DAFB) in 2014,
and May 19 and July 3 (45 DAFB) in 2015. The fruit
clusters were thinned for the ‘Ruby Roman’ to keep one
per three shoots, and were thinned to leave 25–30
berries on each cluster before véraison. To investigate
their development at different growth stages, berries
were collected randomly at an interval of seven days
(three days around véraison) from June 4 (16 DAFB) to
August 11 (84 DAFB) for ‘Aki Queen’, and from June
9 (16 DAFB) to August 18 (86 DAFB) for ‘Ruby
Roman’ in 2014, and seven to 10 days (four days
around véraison) from June 1 (16 DAFB) to August 19
(95 DAFB) for ‘Aki Queen’, and from June 5 (17
DAFB) to August 20 (93 DAFB) for ‘Ruby Roman’ in
2015 (see Figures for actual date). Fifteen (2014) and
30 (2015) berries for each sampling were divided into
three or five replicates (n = 3 in 2014, n = 5 in 2015).
Note that the berry samples used in this 2014 study
were the same as previously published for analysis of
anthocyanin accumulation and expression of antho‐
cyanin biosynthetic genes, VlMybA1-2, VlMybA1-3, and
VviUFGT (Katayama-Ikegami et al., 2017).
Investigation of berry quality and skin coloration
After measuring the berry diameter and weight, the
pulp of peeled berries was squeezed, and the content of
total soluble solids (TSS) and titratable acidity (TA) of
 Hort. J. Preview
the juice was determined. TSS (°Brix) was measured
using a digital refractometer (PAL-1; Atago Co., Ltd.,
Japan). TA was measured by titration with 0.1 mol·L−1
NaOH to a phenolphthalein endpoint. TA content was
expressed as the mass (in grams) of tartaric acid equiva‐
lent per 100 mL juice.
Sugars were extracted in 1 mL 80% (v/v) ethanol
from 20 mg lyophilized and ground fine powder of
berry skin for 30 min at 30°C on an orbital shaker. After
centrifuging at 14,000 rpm, the supernatants were fil‐
tered using a 0.22 μm polyvinylidene difluoride filter
(EMD Millipore Co., Germany). Sucrose (Suc), glucose
(Glu), and fructose (Fru) were quantified using a high-
performance liquid chromatograph equipped with an
Asahipak NH2P-50 4E (4.6 mm × 250 mm column;
Showa Denko K.K., Japan). Elution was performed
at 1 mL·min−1 with 75% acetonitrile and monitored
with a charged aerosol detector (Corona Veo; Thermo
Fisher Scientific Inc., USA) as described previously
(Sugiyama et al., 2016). Standard curves were created
using known concentrations of the sugars. Samples
obtained before August were diluted 40 times with
acetonitrile, and samples after August were diluted 80
times before injection.
The total anthocyanin content in the berry skins was
measured as described by Shiraishi et al. (2007), with a
slight modification. One skin disc was collected from
each berry using a cork borer (Φ = 8 mm) from the
berry apex (2014) or equatorial site (2015). Antho‐
cyanins were extracted in 50% (v/v) aqueous acetic acid
for 24 h at 4°C in the dark, followed by filtration
through a 0.45 μm PVDF filter (EMD Millipore). The
absorbance of the extract at 520 nm was measured
using a spectrophotometer (Biospec-1600; Shimadzu
Corp., Japan). The total anthocyanin content was
expressed as nmol·cm−2 skin of equivalent cyanidin 3-
glucoside chloride standard (TOKIWA Phytochemical
Co., Ltd., Japan).
Determination of endogenous plant hormone contents
A range of endogenous plant hormones in the berry
skins was analyzed as described by Gao-Takai et al.
(2019) according to the method of Chiwocha et al.
(2003) and Gouthu et al. (2013), using liquid
chromatography-triple quadrupole mass spectrometry
(LC: Waters 2695 Separations Module, MS/MS: Waters
micro mass Quattro micro API Mass Spectrometer;
Waters Corp., USA), including ABA and ABA-derived
compounds, such as ABA glucosyl ester (ABA-GE),
dihydrophaseic acid (DPA) and phaseic acid (PA;
two ABA catabolites that are produced by ABA
8'-hydroxylation), neophaseic acid (neoPA; an ABA
catabolite that is made by ABA 9'-hydroxylation), and
7'-hydroxy ABA (7'-OH-ABA); indole-3-acetic acid
(IAA; an auxin) and IAA-amino acid conjugates, such
as IAA-aspartate (IAA-Asp), IAA-alanine (IAA-Ala),
and IAA methyl ester (IAA-ME); and cytokinins,
3
including trans-zeatin (tZ), isopentenyl adenine (iP),
and their sugar conjugates [tZ-riboside (tZR) and iP-
riboside (iPR)].
Expression analysis of ABA metabolism-related genes
Total RNA was extracted from the berry skins using
the hot borate method (Wan and Wilkins, 1994) and was
then treated with DNaseA (Thermo Fisher Scientific)
in the presence of RNasin Ribonuclease Inhibitors
(Promega Corp., USA). Single-strand cDNAs were then
synthesized using SuperScript III Reverse Transcriptase
(Thermo Fisher Scientific). The gene expressions
encoding ABA metabolism-related genes, including
VviNCED3 (VviNCED1 in Gao-Takai et al., 2019) and
VviNCED2, which encode a key ABA biosynthesis
enzyme, VviCYP707A1 and VviCYP707A4, which are
ABA catabolism genes, VviABA-BG1, which encodes
β-glucosidases and is associated with the release of free
(active) ABA from ABA-GE, VviABA-GT, which
encodes glucosyltransferases and is related to the inacti‐
vation of ABA to produce ABA-GE, and VviABF2,
which encodes ABA-responsive element binding fac‐
tors and is associated with ABA signal transduction
were investigated. Most of primer sequences for quanti‐
tative real-time polymerase chain reactions (qRT–PCR)
were used as described in Gao-Takai et al. (2019), and
the primer pairs were (forward: 5'-AAGGAGAAGAGG
TTAGCCGACA-3'; reverse: 5'-GCGATTTGGTCATT
GGTCAG-3') for VviCYP707A4 (XM_002282197).
PCR was performed on 1 μL cDNA from each sample
using a StepOnePlus Real-Time PCR System (Applied
Biosystems, USA) and the SYBR Green system with
SYBR Pre-mix Ex-Taq II (Takara Bio Inc., Japan). The
standard amplification protocol consisted of an initial
denaturing step at 95°C for 30 s, followed by 45 cycles
at 95°C for five seconds, 60°C for 30 s. Data were cal‐
culated from the calibration curve and normalized using
a VviGAPDH expression curve (Katayama-Ikegami
et al., 2016a). PCR was performed at least in duplicate
on each sample of three replicates.
Results
Berry growth, TSS, TA, sugar, and anthocyanin accu‐
mulation
In the two trial years, berry diameter and weight were
approximately 35 mm and 30 g for ‘Ruby Roman’ and
30 mm and 18 g for ‘Aki Queen’ at harvest (Fig. 1).
The changes in TSS and TA during berry development
were not different between the two cultivars. The vérai‐
son occurred generally when the TSS was around 8
°Brix and the TA started to reduce (vertical dashed line
in Fig. 1). The fructose, glucose and sucrose contents
indicated similar changes between the two cultivars,
which decreased from the immature stage to the lowest
levels at about one week before véraison, then
increased. The glucose content around véraison in ‘Aki
Queen’ seemed to be higher than in ‘Ruby Roman’
 4 A. Katayama-Ikegami, Y. Sugiyama, T. Katayama, A. Sakamoto, R. Shimada, C. Miyazaki and M. Gao-Takai

(Fig. 2). Anthocyanin accumulation in ‘Ruby Roman’ Endogenous levels of ABA, auxin, and cytokinin
skin started at 3–5 days after véraison and continued metabolites
around the harvest, while that in ‘Aki Queen’ started Endogenous ABA contents in both cultivars (Fig. 3)
soon after véraison and gradually proceeded up to decreased during the immature berry stage until they
approximately 20 days after véraison. reached their lowest levels at approximately one week
before véraison, and then increased. Interestingly, in

2014 2015

‘Aki Queen’ ‘Ruby Roman’ ‘Aki Queen’ ‘Ruby Roman’

40 40 40 40
30 30 30 30
Diameter
(mm)

20 20 20 20
10 10 10 10
0 0 0 0
10 30 50 70 90 10 30 50 70 90 10 30 50 70 90 10 30 50 70 90
40 40 40 40
30 30 30 30
Weight

20 20 20 20
(g)

10 10 10 10
0 0 0 0
10 30 50 70 90 10 30 50 70 90 10 30 50 70 90 10 30 50 70 90
20 20 20 20
15 15 15 15
oBrix

10 10 10 10
5 5 5 5
0 0 0 0
10 30 50 70 90 10 30 50 70 90 10 30 50 70 90 10 30 50 70 90
4 4 4 4
3 3 3 3
Acidity
(%)

2 2 2 2
1 1 1 1
0 0 0 0
10 30 50 70 90 10 30 50 70 90 10 30 50 70 90 10 30 50 70 90
50 50 50 50
Anthocyanin
(nmol∙cm−2)

40 40 40 40
30 30 30 30
20 20 20 20
10 10 10 10
0 0 0 0
10 30 50 70 90 10 30 50 70 90 10 30 50 70 90 10 30 50 70 90
DAFB (Days after full-bloom)

Fig. 1. Changes in berry diameter and weight, total soluble solids (Brix) and titratable acidity in berry pulp, and anthocyanin content in the berry
skins of ‘Aki Queen’ and ‘Ruby Roman’. Error bars represent SE of the mean in 2014 (n = 3) and 2015 (n = 5) (five berries per replication in
both years). Dashed lines indicate the date of véraison. Note that the sample for 2014 is the same as that previously published for antho‐
cyanin accumulation (Katayama-Ikegami et al., 2017).

‘Aki Queen’ ‘Ruby Roman’

1400
1.4 1500
1.4
Fru 2014
1200
1.2 1.2
Glc
1000
1.0 Suc 1.0
1000
0.8
800 0.8
0.6
600 0.6
500
0.4
0.4
400
Sugar (mmol∙g−1 DW)

0.2
200 0.2
00 00
6/4
16 6/14 6/24
37 427/4
45 7/14 7/2470 8/3
74 84 6/9
16 6/19
23 6/29 7/9 517/19 7/29
37 44 65 8/8
76 8/18
86

1.4
1400 1.4
1400
2015
1.2
1200 1.2
1200
1.0
1000 1.0
1000
0.8
800 0.8
800
0.6
600 0.6
600
0.4
400 0.4
400
0.2
200 0.2
200
00 00
16 25 34
6/1 6/11 41 46
6/21 55 7/21
7/1 7/11 66 7/31
76 8/10 95 17 27 36 40 45 52 63 73 93
6/12
6/19
6/26

7/10
7/17
7/24
7/31

8/14
6/5

7/3

8/7

DAFB (Days after full-bloom)

Fig. 2. Changes in sugar content in the berry skins of ‘Aki Queen’ and ‘Ruby Roman’. Error bars represent the SE of the mean in 2014 (n = 3)
and 2015 (n = 5). Arrows indicate the date of véraison.
 Hort. J. Preview 5

‘Aki Queen’ ‘Ruby Roman’

18
18000 18
18000
16
16000 16
16000 2014
14
14000 neoPA 14000
14
PA
12
12000 12000
12
7'-OH-ABA 10000
10
10000
DPA 10
80008 80008
ABA-GE
60006 60006
ABA
40004 40004
20002 20002

(µg∙g−1 DW)
00 00

6/9

7/3
7/7

8/4
8/8
6/16
6/23
6/30

7/10
7/14
7/21
7/28

8/13
8/18
6/4

7/3
7/7

8/1
8/6
6/11
6/18
6/25
6/27
6/30

7/14
7/21
7/28

8/11
16 23 30 37 39 42 45 49 56 63 70 74 79 84 16 23 30 37 40 44 47 51 58 65 72 76 81 86
60006 60006
50005 ABA 50005
40004 DPA 40004
ABA-GE
30003 30003
20002 20002
10001 10001
00 00
16
6/4 23 30 37 39 4245
6/116/186/25 7/2 49
7/9 56 63 70 74 8/6
7/167/237/30 79 84 17 6/166/236/30
6/9 23 30 37 407/7
447/147/217/28
47 51 58 65 8/4
72 76 81 86
8/118/18

25
25000 25000
25 2015
20
20000 20000
20
15
15000 15000
15
10
10000 10000
10
50005 50005

month/day
00 00
(µg∙g−1 DW)

6/1

7/1

6/5

7/1
7/3
7/6
6/10
6/19
6/23
6/26

7/10
7/21
7/31

6/15
6/24
6/28

7/10
7/16
7/21
7/31
16 25 34 38 41 46 55 66 76 17 27 36 40 43 45 48 52 58 63 73
60006 60006

50005 50005

40004 40004

30003 30003

20002 20002

10001 10001

00 00
16 6/8256/156/226/29
6/1 34 38 41 46 55 66
7/6 7/137/207/27 76 17 6/12276/1936
6/5 40 43454852
6/26 58 7/24
7/3 7/10 7/17 63 7/31
73
DAFB (Days after full-bloom)

Fig. 3. Changes in ABA and its metabolites contents in the berry skins of ‘Aki Queen’ and ‘Ruby Roman’. Error bars represent SE of the mean
in 2014 (n = 3) and 2015 (n = 5). Arrows indicate the date of véraison.

‘Aki Queen’, ABA content stopped increasing on day at the lowest level around véraison in both cultivars.
five (2014) or six (2015) after véraison, and then The content of IAA-Asp, an inactive form of IAA,
decreased, whereas in ‘Ruby Roman’, it increased con‐ increased from véraison in both cultivars, and the
tinuously up to 23 days (65 DAFB in 2014) or 13 days increase was more significant in ‘Ruby Roman’ than
(58 DAFB in 2015) after véraison. The ABA content in in ‘Aki Queen’. In both cultivars, the total cytokinin
‘Ruby Roman’ was four times higher than that of ‘Aki content (Fig. 5) decreased from the immature stage,
Queen’ at 60–70 DAFB. The DPA content, a catabolite reached the lowest level around one week to 10 days
of ABA, was high in immature berries, decreased until before véraison, and then increased. The iP and tZ
véraison and could not be detected after véraison. The contents, two active cytokinins, were all observed to
ABA-GE content, an inactive form of ABA, was slight‐ increase during the mature stage, especially for iP, for
ly reduced from immature berries and increased after which the content in ‘Aki Queen’ was twice that of
véraison, similar to ABA. ABA-GE content was incon‐ ‘Ruby Roman’.
sistent in the two-year trials in ‘Ruby Roman’; it was
higher than ABA after 58 DAFB in 2014, but lower Gene expression analysis involved in ABA metabolism
than that from véraison in 2015. The total content of The expressions of VviNCED3 and VviNCED2 were
ABA metabolites in both cultivars was at the lowest high in the immature berries (23 DAFB) in both culti‐
level just before véraison. It continuously increased vars in 2014, then decreased with berry development
after véraison in ‘Ruby Roman’, but did not do so in before véraison. However, in ‘Ruby Roman’, the
‘Aki Queen’ (Fig. 3). As for IAA and its metabolites expression of VviNCED3 increased again after véraison
(Fig. 4), the total contents of IAA metabolites were also and was maintained at a relatively high level until
 6 A. Katayama-Ikegami, Y. Sugiyama, T. Katayama, A. Sakamoto, R. Shimada, C. Miyazaki and M. Gao-Takai

‘Aki Queen’ ‘Ruby Roman’

700 700 2014

600 600 IAA-ME
500 500 IAA-Asp
400 400 IAA
300 300
200 200
100 100
(ng∙g−1 DW) 0 0

6/4

7/7

8/1
8/6

6/9

7/3
7/7

8/8
6/11
6/18
6/25
6/27
6/30

7/14
7/21

8/11

6/16
6/23
6/30

7/10
7/14
7/21
7/28

8/13
8/18
16 23 30 37 39 42 49 56 63 74 79 84 16 23 30 37 40 44 47 51 58 65 76 81 86

700 700 2015

IAA-ME
600 600 IAA-Ala
500 500 IAA-Asp
400 400 IAA
300 300
200 200
100 100

month/day
0 0
6/10
6/19
6/23
6/26

7/10
7/21
7/31
6/1

7/1

6/5

7/1
7/3
7/6
6/15
6/24
6/28

7/10
7/16
7/21
7/31
16 25 34 38 41 46 55 66 76 17 27 36 40 43 45 48 52 58 63 73
DAFB (Days after full-bloom)

Fig. 4. Changes in IAA and its metabolites contents in the berry skins of ‘Aki Queen’ and ‘Ruby Roman’ in 2014 (n = 3) and 2015 (n = 5).
Arrows indicate the date of véraison.

‘Aki Queen’ ‘Ruby Roman’

50 50
iPR 2014
40 40
iP
30 30 tZR
20 20 tZ

10 10
0 0
(ng∙g−1 DW)

6/4

7/3
7/7

6/9

7/3
7/7

8/4
6/11
6/18
6/25
6/27
6/30

7/14
7/21
7/28

6/16
6/23
6/30

7/10
7/14
7/21

16 23 30 37 39 42 45 49 56 63 70 16 23 30 37 40 44 47 51 58 72

50 50
2015
40 40
30 30
20 20
10 10
month/day

0 0
6/5

7/1
7/3
7/6
6/15
6/24
6/28

7/10
7/16
7/21
7/31
6/1

7/1
6/10
6/19
6/23
6/26

7/10
7/21
7/31

16 25 34 38 41 46 55 66 76 17 27 36 40 43 45 48 52 58 63 73
DAFB (Days after full-bloom)

Fig. 5. Changes in cytokinins and their metabolites content in the berry skins of ‘Aki Queen’ and ‘Ruby Roman’ in 2014 (n = 3) and 2015
(n = 5). Arrows indicate the date of véraison.

around 51 DAFB, while that in ‘Aki Queen’ was main‐ véraison). However, in ‘Ruby Roman’, it tended to
tained at a low level after 45 DAFB (Fig. 6). In 2015, increase after véraison (48DAFB) and was maintained
the expressions of VviNCEDs at 25 and 27 DAFB in at a relatively high level until 63 DAFB (18 days after
both cultivars were lower than those in 2014. In ‘Aki véraison). The expression of VviABABG1, which is
Queen’, the expression of VviNCED3 tended to increase associated with the release of free (active form) ABA
just before véraison (38 DAFB) and then decreased, from ABA-GE, gradually increased from véraison to
and was hard to detect after 46 DAFB (six days after the harvest stage in both cultivars. The expression level
 Hort. J. Preview 7

2014 2015

‘Aki Queen’ ‘Ruby Roman’ ‘Aki Queen’ ‘Ruby Roman’

0.040 0.040 0.040 0.040
VviNCED3
0.030 0.030 0.030 0.030

0.020 0.020 0.020 0.020

0.010 0.010 0.010 0.010

0.000 0.000 0.000 0.000

23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.025 0.025
0.020 0.020 0.020 0.020 VviNCED2
0.015 0.015
0.010 0.010 0.010 0.010
0.005 0.005
0.000 0.000 0.000 0.000
23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.004 0.004 0.004 0.004
VviABABG1
0.003 0.003 0.003 0.003
Relative expression (gene/GAPDH)

0.002 0.002 0.002 0.002

0.001 0.001 0.001 0.001
0.000 0.000 0.000 0.000
23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.012 0.015
0.010 0.010
0.008 0.010 VviABAGT
0.005 0.005
0.004 0.005

0.000 0.000 0.000 0.000

23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.05
0.04 VviCYP707A1
0.04 0.04 0.04
0.03
0.02 0.02 0.02 0.02
0.01
0.00 0.00 0.00 0.00
23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.012 0.015
0.010 0.010
VviCYP707A4
0.008 0.010
0.005 0.005
0.004 0.005

0.000 0.000 0.000 0.000

23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93
0.025 0.025 0.025 0.025
0.020 0.020 0.020 0.020 VviABF2
0.015 0.015 0.015 0.015
0.010 0.010 0.010 0.010
0.005 0.005 0.005 0.005
0.000 0.000 0.000 0.000
23 30 37 39 42 45 49 56 63 70 74 79 84 23 30 37 40 44 47 51 58 65 72 76 81 86 25 34 38 41 46 55 66 76 95 27 36 40 43 45 48 52 58 63 73 93

DAFB (Days after full-bloom)

Fig. 6. Changes in gene expression involved in ABA metabolism in the berry skins of ‘Aki Queen’ and ‘Ruby Roman’. Error bars represent SE
of the mean in 2014 (n = 3) and 2015 (n = 3). Dashed lines indicate the date of véraison.

was lower in ‘Aki Queen’ at the late mature stage and VviCYP707A4, which are thought to be ABA
in 2014, compared to the other cultivars. As for catabolism genes, was high at the immature stage
VviABAGT, which is thought to encode glucosyltrans‐ and decreased before véraison. The expression of
ferases and produce the inactive form of ABA, ABA- VviCYP707A1 increased again around véraison in
GE, the expression level was different between the two- ‘Ruby Roman’, but not in ‘Aki Queen’, and was detect‐
year’s trials; the level in 2014 was higher than that in ed in ‘Ruby Roman’ at a relatively higher level than
2015 in both cultivars. The expression of VviCYP707A1 that in ‘Aki Queen’. There was no noticeable difference
 8 A. Katayama-Ikegami, Y. Sugiyama, T. Katayama, A. Sakamoto, R. Shimada, C. Miyazaki and M. Gao-Takai
in the expression of VviABF2, a responsive ABA gene,
between the two cultivars, although it appeared to
remain relatively higher in ‘Ruby Roman’ than in ‘Aki
Queen’ after véraison especially in 2014.
Discussion
In cultivation practice, the problem of poor coloring
in ‘Aki Queen’ and its parent ‘Kyoho’ is usually sig‐
nificant, especially under high temperatures, while it
is not very serious in ‘Ruby Roman’, a seedling of
‘Fujiminori’. ‘Aki Queen’ and ‘Ruby Roman’ have the
same haplotypes at the coloring locus, and their berries
express red skin. However, the difference in color
development between the two cultivars may indicate
another genetic basis in addition to the coloring locus.
In this study, the three endogenous plant hormones
(ABA, auxin [IAA], and cytokinin), and their metabo‐
lites were compared with the two cultivars to character‐
ize their functions in coloration at the late mature stage.
Some differences in the number of metabolites, such as
ABA, IAA-Asp, and iP were detected between the two
cultivars (Figs. 3, 4, and 5).
The endogenous ABA content at the late mature
stage in ‘Ruby Roman’ was higher than that in ‘Aki
Queen’ in the two-year’s trials (Fig. 3) and this may be
associated with the good coloration in ‘Ruby Roman’.
In previous studies, the accumulation pattern of endoge‐
nous ABA in the berry skin was not affected by differ‐
ent cultivation methods, i.e., each shoot bears one
cluster, or three shoots bear one cluster in ‘Ruby
Roman’, and long cane pruning or short cane pruning in
‘Aki Queen’ (unpublished). Also, we reported that the
ABA content in ‘Ruby Roman’ was almost the same in
2016 and 2017 (Gao-Takai et al., 2019). Therefore, the
difference in the endogenous ABA level is not likely
dependent on the cultivation method or environmental
factors, but rather on the cultivar’s features. The high
level of DPA in immature berries observed in both
cultivars in this study (Fig. 3) was previously reported
in ‘Shiraz’, ‘Merlot’, and ‘Muscat Hamburg’ berries
(Böttcher et al., 2013; Owen et al., 2009; Sun et al.,
2010). The high expression of VviNCEDs and
VviCYP707As, and DPA content in the immature stage
may indicate that active biosynthesis and catabolization
of ABA simultaneously occurred during the early stage
of berry development. Castellarin et al. (2016) men‐
tioned that the initial increases in ABA at véraison may
largely result from decreased catabolism in 8' hydroxy‐
lation. However, in this study, the expression of
VviCYP707As in ‘Ruby Roman’ was higher than those
in ‘Aki Queen’ after véraison, although the ABA con‐
tent was higher in ‘Ruby Roman’ than in ‘Aki Queen’.
Alternatively, the higher expression of VviNCED3 in
‘Ruby Roman’ until around 60 DAF (Fig. 6) may partly
account for the higher ABA in this cultivar at the late
mature stage. Possible reasons for the high level of
ABA metabolites in ‘Ruby Roman’ after 60 DAFB are;
i) the enzyme for ABA biosynthesis remaining active in
berry skins during the late mature stage, and ABA accu‐
mulation from immature stage and that is not easily
degraded ii) the translocation of ABA from other
organs, and iii) other VviNCEDs, such as VviNCED5 or
VviNCED6 being involved in ABA biosynthesis in
grape berries as reported by Pilati et al. (2017) and
Cramer et al. (2020). Additionally, VviABAGT and
VviABABG1 may contribute to maintaining the amount
of ABA at a constant level each year during the mature
stage. The difference in the expression of VviABAGT
between the two years (Fig. 6) may have been affected
by the environment. The ambient temperature during
the berry development period in 2014 was higher than
that in 2015, resulting in elevated expression of
VviABAGT and accumulation of inactive ABA-GE.
VviABABG has been shown to act as a functional
enzyme in ABA metabolism (Sun et al., 2015). How‐
ever, the functions of other enzymes possibly involved
in ABA metabolism, encoded by homologous genes
of other plants such as Arabidopsis, including
VviCYP707As and VviNCEDs, have not yet been eluci‐
dated. Therefore, it cannot yet be said that these genes
are involved in the control of the ABA level. Further
analysis is required for functional characterization of
the enzymes encoded by these genes and ABA transport
to clarify the different ABA accumulation patterns in
the late stage of maturation of these cultivars.
Endogenous IAA, thought to be an inhibitor of berry
maturation, decreased before véraison. Meanwhile,
inactive IAA-Asp increased during berry maturation
(Böttcher et al., 2010; Gouthu and Deluc, 2015). Active
auxin is inactivated by IAA-amido synthetase, known
as GH3-1, producing IAA-Asp from IAA (Böttcher
et al., 2010). The content of IAA-Asp increased after
véraison in both cultivars, and the final content was
higher in ‘Ruby Roman’ (Fig. 4). These results were
consistent with our previous study (Gao-Takai et al.,
2019). Although the content of IAA remained low after
véraison, the increase in IAA-Asp indicated that the
biosynthesis and catabolization (inactivation) of IAA
may occur simultaneously during maturation. Another
possibility is that IAA-Asp (or IAA) may be transported
to berries from other organs.
It has been proposed that iP, an active cytokinin,
affects the accumulation of sugars by maintaining sink
strength in ripening berries that is related to the expan‐
sion driven growth after véraison (Böttcher et al.,
2015). In this study, the iP content started to increase a
few days before véraison and continued until harvest.
The iP content in ‘Ruby Roman’ was two times lower
than that in ‘Aki Queen’ around 70 DAFB (Fig. 5). The
changes in ABA, IAA-Asp, and iP content during berry
development were different between the two cultivars.
Several reports have described the hormonal interac‐
tions. Griesser et al. (2020) reported that they detected a
high content of ABA-GE, and low IAA-Asp and iP
 Hort. J. Preview
contents in shriveling disorder berries that were artifi‐
cially induced by exogenous application of ACC
(aminocyclopropene-1-carboxylic acid). Li et al. (2021)
reported that exogenous ABA treatment at véraison
reduced the IAA and increased the iP content, and
mentioned a close relationship between iP and berry
enlargement. In our study, however, berry enlargement
of ‘Ruby Roman’ was still ongoing at the late stage of
maturation, while iP content was lower than of ‘Aki
Queen’. The results suggested that there is some inter‐
play between these phytohormones in terms of fruit
ripening at the later stage of maturation. Further analy‐
sis is needed to clarify the interaction in the berry matu‐
ration process. In subsequent studies, transcriptomic
analysis, including IAA inactivation and response, and
iP biosynthesis, may reveal the reason for the different
accumulation levels of phytohormones or their metabo‐
lites between the two cultivars and provide some infor‐
mation on berry maturation.
In conclusion, the coloration pattern and contents of
endogenous ABA, IAA, and cytokinins in two red
hybrid grape cultivars, which had the same haplotypes
at the coloring locus was investigated. The results
showed that the accumulation pattern of anthocyanins
in the two cultivar skins were different. Also, the phyto‐
hormone contents at different developmental stages
were not similar. Further clarification of the traits found
in ‘Ruby Roman’ may be useful in breeding grapes and
for a deeper understanding of grape berry ripening in
the future.
Acknowledgements
We thank Mariko Esaki and the other technicians of
Experimental Farm of Ishikawa Prefectural University,
for their assistance in experimentation and cultivation.
Literature Cited
Azuma, A. 2018. Genetic and environmental impacts on the
biosynthesis of anthocyanins in grapes. Hort. J. 87: 1–17.
Azuma, A., Y. Udo, A. Sato, N. Mitani, A. Kono, Y. Ban, H.
Yakushiji, Y. Koshita and S. Kobayashi. 2011. Haplotype
composition at the color locus is a major genetic determinant
of skin color variation in Vitis × labruscana grapes. Theor.
Appl. Genet. 122: 1427–1438.
Ban, Y., N. Mitani, T. Hayashi, A. Sato, A. Azuma, A. Kono and
S. Kobayashi. 2014. Exploring quantitative trait loci for
anthocyanin content in interspecific hybrid grape (Vitis
labruscana × Vitis vinifera). Euphytica 198: 101–114.
Böttcher, C., P. K. Boss and C. Davies. 2013. Increase in
cytokinin levels during ripening in developing Vitis vinifera
cv. Shiraz berries. Am. J. Enol. Vitic. 64: 527–531.
Böttcher, C., C. A. Burbidge, P. K. Boss and C. Davies. 2015.
Changes in transcription of cytokinin metabolism and signal‐
ing genes in grape (Vitis vinifera L.) berries are associated
with the ripening-related increase in isopentenyladenine.
BMC Plant Biol. 15: 223. DOI: 10.1186/s12870-015-0611-5.
Böttcher, C., R. A. Keyzers, P. K. Boss and C. Davis. 2010.
Sequestration of auxin by the indole-3-actic acid synthase
GH301 in grape berry (Vitis vinifera L.) and the proposed
9
role of auxin conjugation during ripening. J. Exp. Bot. 61:
3615–3625.
Castellarin, S. D., G. A. Gambetta, H. Wada, M. N. Krasnow,
G. R. Cramer, E. Peterlunger, K. A. Shackel and M. A.
Matthews. 2016. Characterization of major ripening events
during softening in grape: turgor, sugar accumulation,
abscisic acid metabolism, colour development, and their
relationship with growth. J. Exp. Bot. 67: 709–722.
Chiwocha, S. D. S., S. R. Abrams, S. J. Ambrose, A. J. Cutler, M.
Loewen, A. R. S. Ross and A. R. Kermode. 2003. A method
for profiling classes of plant hormones and their metabolites
using liquid chromatography-electrospray ionization tandem
mass spectrometry: an analysis of hormone regulation of
thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant
J. 35: 405–417.
Corso, M., A. Vannozzi, F. Ziliotto, M. Zouine, E. Maza, T.
Nicolato, N. Vitulo, F. Meggio, G. Valle, M. Bouzayen, M.
Müller, S. Munné-Bosch, M. Lucchin and C. Bonghi. 2016.
Grapevine rootstocks differentially affect the rate of ripening
and modulate auxin-related genes in Cabernet Sauvignon
berries. Front. Plant Sci. 7: 69. DOI: 10.3389/fpls.2016.
00069.
Costantini, L., G. Malacarne, S. Lorenzi, M. Troggio, F. Mattivi,
C. Moser and M. S. Grando. 2015. New candidate genes for
the fine regulation of the colour of grapes. J. Exp. Bot. 66:
4427–4440.
Cramer, G. R., N. Cochetel, R. Ghan, A. Destrac-Irvine and S.
Delrot. 2020. A sense of place: transcriptomics identifies
environmental signatures in Cabernet Sauvignon berry skins
in the late stage of ripening. BMC Plant Biol. 20: 41. DOI:
10.1186/s12870-020-2251-7.
Fortes, A. M., R. T. Teixeira and P. Agudelo-Romero. 2015.
Complex interplay of hormonal signals during grape berry
ripening. Molecules 20: 9326–9343.
Gao-Takai, M., A. Katayama-Ikegami, K. Matsuda, H. Shindo, S.
Uemae and M. Oyaizu. 2019. Low temperature promotes
anthocyanin biosynthesis but does not accelerate endoge‐
nous abscisic acid accumulation in red-skinned grapes. Plant
Sci. 283: 165–276.
Gouthu, S. and G. Deluc. 2015. Timing of ripening initiation in
grape berries and its relationship to seed content and peri‐
carp auxin levels. BMC Plant Biol. 15: 46. DOI: 10.1186/
s12870-015-0440-6.
Gouthu, S., J. Morre, C. S. Maier and L. G. Deluc. 2013. An ana‐
lytical method to quantify three plant hormone families in
grape berry using liquid chromatography and multiple reac‐
tion monitoring mass spectrometry. p. 19–36. In: D. R. Gang
(ed.). Phytochemicals, Plant Growth, and the Environment.
Recent Advances in Phytochemistry 42. Springer, New
York.
Griesser, M., S. Savoi, S. Supapvanich, P. Dobrev, R. Vankova
and A. Forneck. 2020. Phytohormone profiles are strongly
altered during induction and symptom development of the
physiological ripening disorder berry shrivel in grapevine.
Plant Mol. Biol. 103: 141–157.
Jeong, S. T., N. Goto-Yamamoto, S. Kobayashi and M. Esaka.
2004. Effects of plant hormones and shading on the accumu‐
lation of anthocyanins and the expression of anthocyanins
biosynthetic genes in grape berry skins. Plant Sci. 167: 247–
252.
Jia, H., C. Zhang, T. Pervaiz, P. Zhao, Z. Liu, B. Wang, C. Wang,
L. Zhang, J. Fang and J. Qian. 2016. Jasmonic acid involves
in grape fruit ripening and resistant against Botrytis cinerea.
Funct. Integr. Genomics 16: 79–94.
Katayama-Ikegami, A., M. Gao-Takai, R. Shimada, K. Matsuda
 10 A. Katayama-Ikegami, Y. Sugiyama, T. Katayama, A. Sakamoto, R. Shimada, C. Miyazaki and M. Gao-Takai
and T. Sakamoto. 2017. Difference of coloration between
‘Aki Queen’ and ‘Ruby Roman’ grapes treated with abscisic
acid-containing fertilizer at late stage of maturation. Hort.
Res. (Japan) 16: 317–324 (In Japanese with English
abstract).
Katayama-Ikegami, A., T. Sakamoto, T. Katayama and M. Gao-
Takai. 2016a. Reference gene validation for gene expression
studies using quantitative RT-PCR during berry development
of ‘Aki Queen’ grapes. Vitis 55: 157–160.
Katayama-Ikegami, A., T. Sakamoto, K. Shibuya, T. Katayama
and M. Gao-Takai. 2016b. Effects of abscisic acid treatment
on berry coloration and expression of flavonoid biosynthesis
genes in grape. Amer. J. Plant Sci. 7: 1325–1336.
Kobayashi, S., N. Goto-Yamamoto and H. Hirochika. 2004.
Retrotransposon-induced mutations in grape skin color.
Science 304: 982.
Kobayashi, S., N. Goto-Yamamoto and H. Hirochika. 2005.
Association of VvmybA1 gene expression with anthocyanin
production in grape (Vitis vinifera) skin-color mutants. J.
Japan. Soc. Hort. Sci. 74: 196–203.
Koyama, K., K. Sadamatsu and N. Goto-Yamamoto. 2010.
Abscisic acid stimulated ripening and gene expression in
berry skins of the Cabernet Sauvignon grape. Funct. Integr.
Genomics 10: 367–381.
Kuhn, N., L. Guan, Z. W. Dai, B. H. Wu, V. Lauvergeat, E.
Gomès, S. H. Li, F. Godoy, P. Arce-Johnson and S. Delrot.
2014. Berry ripening: recently heard through the grapevine.
J. Exp. Bot. 65: 4543–4559.
Li, J., B. Liu, X. Li, D. Li, J. Han, Y. Zhang, C. Ma, W. Xu, L.
Wang, S. Jiu, C. Zhang and S. Wang. 2021. Exogenous
abscisic acid mediates berry quality improvement by altered
endogenous plant hormones level in “Ruiduhongyu”
grapevine. Front. Plant Sci. 12: 739964. DOI: 10.3389/
fpls.2021.739964.
Matsuda, K., A. Katayama-Ikegami, N. Higashi, K. Sakai, S.
Nakano, S. Tamamura, T. Hayakawa, A. Date and M. Gao-
Takai. 2020. Effects of temperature on skin coloration at
different developmental stages of potted ‘Ruby Roman’
grape berries. Hort. Res. (Japan) 19: 29–38 (In Japanese
with English abstract).
Owen, S. J., M. D. Lafond, P. A. Bowen, C. P. Bogdanoff, K. B.
Usher and S. R. Abrams. 2009. Profiles of abscisic ands and
View publication stats
its catabolites in developing Merlot grape (Vitis vinifera)
berries. Am. J. Enol. Vitic. 60: 277–284.
Pilati, S., G. Bagagli, P. Sonego, M. Moretto, D. Brazzale, G.
Castorina, L. Simoni, C. Tonelli, G. Guella, K. Engelen, M.
Galbiati and C. Moser. 2017. Abscisic acid is a major regu‐
lator of grape berry ripening onset: new insights into ABA
signaling network. Front. Plant Sci. 8: 1093. DOI: 10.3389/
fpls.2017.01093.
Shiraishi, M., M. Yamada, N. Mitani and T. Ueno. 2007. A rapid
determination method for anthocyanin profiling in grape
genetic resources. J. Japan. Soc. Hort. Sci. 76: 28–35.
Sugiyama, Y., A. Gotoh, T. Katoh, Y. Honda, E. Yoshida, S.
Kurihara, H. Ashida, H. Kumagai, K. Yamamoto, M.
Kitaoka and T. Katayama. 2016. Introduction of H-antigens
into oligosaccharides and sugar chains of glycoproteins
using highly efficient 1,2-α-L fucosynthase. Glycobiology
26: 1235–1247.
Sun, J. H., Y. H. Dong, C. L. Li and Y. Y. Shen. 2015. Transcrip‐
tion and enzymatic analysis of beta-glucosidase VvBG1 in
grape berry ripening. Plant Growth Regul. 75: 67–73.
Sun, L., M. Zhang, J. Ren, J. Qi, G. Zhang and P. Leng. 2010.
Reciprocity between abscisic acid and ethylene at the onset
of berry ripening and after harvest. BMC Plant Biol. 10: 257.
DOI: 10.1186/1471-2229-10-257.
Walker, A. R., E. Lee, J. Bogs, D. A. J. McDavid, M. R. Thomas
and S. P. Robinson. 2007. White grapes arose through the
mutation of two similar and adjacent regulatory genes. Plant
J. 49: 772–785.
Wan, C. Y. and T. A. Wilkins. 1994. A modified hot borate
method significantly enhances the yield of high-quality RNA
from cotton (Gossypium hirsutum L.). Anal. Biochem. 223:
7–12.
Yamada, M. and A. Sato. 2016. Advances in table grape breeding
in Japan. Breed. Sci. 66: 34–45.
Yamane, T. and K. Shibayama. 2006. Effects of changes in the
sensitivity to temperature on skin coloration in ‘Aki Queen’
grape berries. J. Japan. Soc. Hort. Sci. 75: 458–462.
Ziliotto, F., M. Corso, F. Rizzini, A. Rasori, A. Botton and C.
Bonghi. 2012. Grape berry ripening delay induced by a pre-
véraison NAA treatment is paralleled by a shift in the
expression pattern of auxin-and ethylene-related genes.
BMC Plant Biol. 12: 185. DOI: 10.1186/1471-2229-12-185.