Journal of Food Composition and Analysis 106 (2022) 104353

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Spatial distribution and time-course of polyphenol accumulation in grape

berry (Vitis labruscana cv. ‘Kyoho’)
Ming Qi a, Zisheng Luo a, b, Bin Wu c, Lei Wang a, Mingyi Yang a, Xiaochen Zhang a, Xingyu Lin a, b,
Yanqun Xu a, b, Xihong Li d, Li Li a, b, *
a
Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs, College of Biosystems Engineering and Food Science, Zhejiang
University, Hangzhou, 310058, China
b
Ningbo Research Institute, Zhejiang University, Ningbo, 315100, China
c
Institute of Agro-products Storage and Processing, Xinjiang Academy of Agricultural Sciences, Urumqi, 830091, China
d
College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China

A R T I C L E I N F O A B S T R A C T

Keywords: The spatial distribution and time-course evolution of polyphenol compounds in response to exogenous light and
Kyoho temperature in grape berry (Vitis labruscana cv. ‘Kyoho’) were examined. Results demonstrated that the poly­
Flavanols phenol biosynthesis was induced via the up-regulated gene transcription levels during berry ripening. The
Anthocyanins
polyphenol biosynthesis was differentially observed in skins, stems, leaves and seeds, and the more polyphenols
Maturity stage
Spatial distribution
were accumulated in seeds; and the polyphenols were significantly distributed in bottom berries of grape clus­
Polyphenol ters. Additionally, the lower temperature (4 ℃) accelerated the polyphenol biosynthesis by 1.3 times after
storage of 28 days, whereas the light exposure slightly inhibited the polyphenol biosynthesis of berry during
storage at 25 ℃. In conclusion, the data presented in this study shed more light on deeper understanding of the
spatial accumulation and time-course evolution of polyphenols in grape berry.

1. Introduction
Grape vine is a highly profitable product rich in natural bioactive
compounds (Wang et al., 2020). Up to now, there are thousands of grape
varieties cultivated throughout the world, while numerous cultivars are
bred by crossing of different Vitis species (Guo et al., 2016). Vitis lab­
ruscana cv. ‘Kyoho’, firstly developed by Japanese scientists, is one of
the economically grape varieties due to its large berries (12− 14 g), rich
juice and pleasing taste (Xu et al., 2018; Deng et al., 2006). Its physio­
logical characters including strong adaptability, cold tolerance, and
disease resistance are also extremely attractive (Xu et al., 2018).
Polyphenols are the important bioactive secondary metabolites and
natural antioxidants in berry, which are outstandingly beneficial to
human health. As a class of phytochemicals, polyphenols mainly include
phenolic acids, stilbenes, and flavonoids. Flavonoids can be divided into
flavonols, flavanols, flavanones and anthocyanins (Askari-Khorasgani
and Pessarakli, 2019a). Anthocyanins, which generally exhibit
characteristic red, blue and purple colors, widely exist in grape tissues,
especially in berry skins (Askari-Khorasgani and Pessarakli, 2019a;
Zhang et al., 2015). The pathway of polyphenol biosynthesis has been
well studied, which has been proved through the phenylpropanoid and
flavonoid pathway. Firstly, the phenylalanine as the initial substrate of
phenylpropanoid pathway, and 4-coumaroyl-coA is formed by catalysis
of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H)
and 4-coumaroyl-coA synthase (4CL) in sequence. Phenolic acids and
precursors of stilbenes are mainly produced during phenylpropanoid
pathway. Subsequently, the flavanones, flavanols and flavonols are
produced in the beginning, which are further reduced and modified to
generate anthocyanidins in the flavonoid pathway. In the end, the an­
thocyanins are synthesized by glycosylation and methylation of antho­
cyanidins. In recent years, grape polyphenols have been studied by a
significant number of works due to an array of beneficial biological
properties such as antioxidant, anti-inflammatory, and anti-diabetes as
well as prevention of cancer, osteoporosis, and cardiovascular diseases

* Corresponding author at: Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs, College of Biosystems Engineering
and Food Science, Zhejiang University, Hangzhou, 310058, China.
E-mail addresses: qiming123@zju.edu.cn (M. Qi), luozisheng@zju.edu.cn (Z. Luo), xjuwubin0320@sina.com (B. Wu), wangley@zju.edu.cn (L. Wang), ymy008@
zju.edu.cn (M. Yang), xiaochen_zhang@zju.edu.cn (X. Zhang), xingyu@zju.edu.cn (X. Lin), xuyanqun@zju.edu.cn (Y. Xu), lixihong606@163.com (X. Li), lili1984@
zju.edu.cn (L. Li).

https://doi.org/10.1016/j.jfca.2021.104353
Received 22 June 2021; Received in revised form 22 November 2021; Accepted 11 December 2021
Available online 13 December 2021
0889-1575/© 2021 Elsevier Inc. All rights reserved.
M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Fig. 1. Morphological characteristics of grape clusters.

Fig. 2. Quality traits in ‘Kyoho’ berries at different maturity stages (A-F) and in berries differentially located in grape cluster (G-L). Error bars represent standard
deviations of three replicates. Different letters represent significant differences between groups (p < 0.05).

2
M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Table 1
Polyphenolic compounds identified in ‘Kyoho’ grape.
Peak No. Compound Wavelength (nm) Ion mode RT (min) m/z ration Product ions

Phenolic acids
1 Gallic acid 280 – 7.35 169.0160 169.02
2 Caftaric acid 280 – 13.91 311.0948 179.04; 135.04
3 Coutaric acid 280 – 19.70 295.0460 163.04

Flavonols
4 Myricetin-3-glucoside 280 – 37.89 479.0827 316.02
5 Quercetin-3-rhamnose-glucuronide 280 – 46.76 477.1400 301.04
6 Quercetin-3-rhamnoside 280 – 56.86 447.1699 301.03

Flavanols
7 (+)-Gallocatechin 280 – 11.13 305.0673 125.02
8 Proanthocyanidin dimer B3 280 – 15.11 577.1351 425.10; 407.08; 289.07
9 (-)-Epigallocatechin 280 – 15.41 305.0677 125.02
10 Proanthocyanidin tetramer-1 280 – 15.55 1153.2665 575.13; 287.06
11 Proanthocyanidin dimer B1 280 – 15.82 577.1872 425.09; 407.08; 289.07
12 (+)-Catechin 280 – 17.83 289.1073 245.08
13 Proanthocyanidin trimer-1 280 – 18.96 865.2004 577.14; 287.06
14 Proanthocyanidin tetramer-2 280 – 20.46 1153.2674 575.12; 287.06
15 Proanthocyanidin dimer B4 280 – 23.34 577.1860 425.09; 407.08; 289.07
16 (-)-Epicatechin 280 – 24.89 289.0721 245.08
17 Proanthocyanidin trimer-2 280 – 32.54 865.1995 577.14; 287.07
18 Proanthocyanidin trimer-3 280 – 33.77 865.2007 577.14; 287.06
19 Proanthocyanidin dimer B2 280 – 35.10 577.1356 425.09; 407.08; 289.07
20 Proanthocyanidin tetramer-3 280 – 35.96 1153.2655 575.12; 287.06
21 (-)-Epicatechin gallate 280 – 42.56 441.0830 289.07; 169.01

Anthocyanins
22 Malvidin-3,5-diglucoside 520 + 18.42 655.1864 331.08
23 Peonidin-3-glucoside 520 + 28.52 463.1235 301.07
24 Malvidin-3-glucoside 520 + 31.06 493.0982 331.08
25 Peonidin-3− 6′ ′ -p-coumaroyl-glucoside-5-glucoside 520 + 55.16 771.2134 301.07
26 Malvidin-3− 6′ ′ -p-coumaroyl-glucoside-5-glucoside 520 + 57.23 801.2247 331.08
27 Cyanidin-3-glucoside 520 + 61.27 449.1092 287.06
28 Petunidin-3-glucoside 520 + 62.10 479.1182 317.07
29 Cyanidin-3− 6′ ′ -p-coumaroyl-glucoside 520 + 62.49 595.1440 287.05
30 Petunidin-3− 6′ ′ -p-coumaroyl-glucoside 520 + 62.69 625.1557 317.07
31 Peonidin-3− 6′ ′ -p-coumaroyl-glucoside 520 + 63.21 609.1594 301.08
32 Malvidin-3− 6′ ′ -p-coumaroyl-glucoside 520 + 63.31 639.1706 331.08

(Ferreyra et al., 2012; Silva et al., 2020). Furthermore, effects of biotic
and abiotic factors such as pathogens, temperature, light and drought
(Askari-Khorasgani and Pessarakli, 2019a; Li et al., 2016; Romero et al.,
2020), on the polyphenol profile and synthesis of grapevine are getting
more and more attention.
Recent studies indicated that the accumulation and biosynthesis of
grape polyphenols are very complex in dependence of berry variety,
maturity stage, genetic diversity, soil, environmental stress, and agro­
nomic management practices (Askari-Khorasgani and Pessarakli, 2019a;
Li et al., 2019a). A series of physicochemical and biochemical processes
take place as evolution of berry development and ripening, which
contributed to the presence of variation in polyphenol composition and
antioxidant capacities (Nedomova et al., 2017; Fuentes et al., 2016).
Previous studies showed that anthocyanins and flavonols increased as
berry ripening, while flavanols content remained constant or declined
from post-veraison to harvest time in ‘Nebbiolo’ and ‘BRS Vitoria’ grape
berry (Locatelli et al., 2016; Colombo et al., 2021). Additionally, the
spatial distribution of grapes directly or indirectly affects the poly­
phenolic accumulation in berry. The quality of the grape juice and wine
produced has partly been dependent on the polyphenolic profiles
(Biniari et al., 2020). Grape skins, stems, leaves and seeds are rich in
polyphenols, which provide valuable information for their antioxidant
potential and great commercial value as agricultural by-products
(Biniari et al., 2020; Perez-Navarro et al., 2019; Pastrana-Bonilla
et al., 2003). It is demonstrated that the flavan-3-ols and proanthocya­
nidins are predominant polyphenols in the grape seeds, while flavonols
and anthocyanins are abundant in the skins of 13 grapevine varieties
such as ‘Cabernet Sauvignon’, ‘Merlot’ and ‘Prokupac’ grapes (Pantelic
et al., 2016). Li et al. (2019a) reported that ‘Kyoho’ grape contained
higher concentration of polyphenols than other red grape varieties (Gold
Finger, Hakuho, White Olympia, Shine Muscat, Beni Fuji and Moldova).
In the vineyard, the weight, total soluble solids (TSS), titratable acidity
(TA), alcoholic degree, aromatic composition as well as polyphenolic
composition variations within grape cluster have been studied to
improve the cultivating quality and to provide useful information for
wine quality prediction (Rutan et al., 2018; Figueiredo-Gonzalez et al.,
2013; Noguerol-Pato et al., 2012). Furthermore, the altered microcli­
matic sunlight conditions were evident to influence the development
and composition of berries (Gao et al., 2021). Noguerol-Pato et al.
(2012) reported that there was presence of variability in the aromatic
composition between the shoulder and tip berries in grape clusters. As
for polyphenols, exposure to sunlight could promote the accumulation
of flavonols and modulate the concentration and composition of the
anthocyanins in berry skins (Downey et al., 2006).
It is worthwhile to mention the polyphenolic profiles in grapevine
were considerably affected by exogenous abiotic stress, such as tem­
perature and light. According to previous studies, there is no common
trend in the change of grape total polyphenol content in response to
postharvest preservation treatment (Azuma et al., 2012; Romero et al.,
2020; Šamec and Piljac-Žegarac, 2011). Šamec and Piljac-Žegarac
(2011) found that the contents of total phenols, total flavonoids and
total anthocyanins in red grapes increased at the end of storage at 4 ℃
and 25 ℃, and the increase might be due to a release of phenolic com­
pounds from their complexes with other components like proteins and
carbohydrates. In ‘Pione’ grape, the flavonoid biosynthesis was appar­
ently enhanced when grape clusters were exposed to light or low

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M. Qi et al.
Fig. 3. Identification of polyphenols in ‘Kyoho’ grape. HPLC chromatograms of the grape extract (skin, stem, leaf and seed) recorded at 280 nm, 320 nm and 520 nm.
The numbered identified peaks are listed in Table 1.
temperature, whereas the flavonoid biosynthesis was inhibited in the
dark (Azuma et al., 2012). Under specific environmental conditions,
grapevine receives information to activate or inhibit related gene
expression and metabolic pathways, such as producing more bioactive
compounds like flavonoids to activate defense mechanisms against UV
light and biotic stress stimuli (Azuma et al., 2012; Askari-Khorasgani
and Pessarakli, 2019b). It was indicated that the accumulation of fla­
vonoids hindered protein aggregation and relieved the stress injury
resulted from the electrolyte leakage, oxidative damage, and inactiva­
tion of photosynthesis to improve the plant survivability in cold stress
(Askari-Khorasgani and Pessarakli, 2019b).
Based on above, it is essential to investigate the polyphenols distri­
bution and evolution as berry development and ripening, in order to not
only perceive the repertoire of polyphenol compounds, but also provide
evidence for value-added utilization. Up to now, there has been very
limited information available about the polyphenol profiles in ‘Kyoho’
grape (Li et al., 2013; Yang et al., 2020). In the present study, both the
bio-distribution (in skins, stems, leaves, seeds and berries located on top,
middle and bottom of grape cluster) and spatial accumulation of poly­
phenol compounds in berry (Vitis labruscana cv. ‘Kyoho’) were studied;
4
Journal of Food Composition and Analysis 106 (2022) 104353
and the effect of exogenous light and temperature on endogenous
polyphenol biosynthesis was investigated. Additionally, the gene tran­
scriptions involved in berry polyphenol biosynthesis were determined.
Results from the present study shed new light on the comprehensive
understanding and efficient utilization of bioactive substance in berries.
2. Materials and methods
2.1. Chemicals
Catechin and quercetin standards were purchased from Aladdin
Reagent Co., Ltd. (Shanghai, China); cyanidin-3-glucoside standard was
obtained from Macklin Biochemical Co., Ltd., (Shanghai, China). Stan­
dards were used for high performance liquid chromatography with
diode array detector (HPLC-DAD) analysis to quantify the polyphenolic
compounds. Methanol and acetonitrile of HPLC grade were purchased
from Macklin Biochemical Co., Ltd., (Shanghai, China), while phos­
phoric acid, hydrochloric acid and ethanol of analytical-reagent grade
were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
China). Deionized water was prepared by water purification system
M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Table 2 Table 2 (continued )

1
The polyphenol concentrations in different grape tissues (mg kg− tissue FW)*. Compound Skins Stems Leaves Seeds
Compound Skins Stems Leaves Seeds
Peonidin-3− 6′ ′ -p- 9.36 ± 0.94 ± ND ND
Phenolic acids coumaroyl-glucoside 0.71a 0.02b
Gallic acid ND ND ND 3.94 ± 0.23 Malvidin-3− 6′ ′ -p- 21.26 ± 1.23 ± ND ND
Caftaric acid 9.52 ± 1.25 ± 3.34 ± 5.45 ± coumaroyl-glucoside 0.57a 0.13b
0.72a 0.07d 0.31c 0.14b Total anthocyanin 111.86 ± 5.00 ±
Coutaric acid 47.40 ± 20.95 ± 44.99 ± 1.07 ± content 4.68a 0.13b
0.76a 0.32c 1.45b 0.63d Total polyphenol 2158.37 11215.68 4930.76 13692.92
Total phenolic acid 56.92 ± 22.20 ± 48.33 ± 10.46 ± content ± 49.37d ± 77.65b ± 83.29c ± 63.33a
content 1.16a 0.30c 1.62b 0.33d
Data are shown in mean ± standard deviation (n = 3). Different letters of the
same component represent statistically significant difference, respectively (p <
Flavonols
Myricetin-3-glucoside 4.79 ± 0.67 ± 15.42 ± ND
0.05). ND: not detected.
*
0.46b 0.58c 0.50a The flavanols were quantified by catechin external standard, the phenolic
Quercetin-3-rhamnose- 42.58 ± 43.51 ± 729.84 ± 1.65 ± acids and flavonols were quantified by quercetin external standard and the an­
glucuronide 8.78b 1.31b 11.32a 0.06c thocyanins were quantified by cyanidin-3-glucoside external standard.
Quercetin-3-rhamnoside 33.46 ± 5.19 ± 150.33 ± 2.64 ±
3.34b 1.33c 1.27a 0.15c
Total flavonol content 80.83 ± 49.37 ± 895.59 ± 4.29 ±
(Milli-Q IQ 7000, Merck Millipore, Germany). All other chemicals
10.29b 1.54c 12.56a 0.12d (analytical-reagent grade) used in this study were obtained from Sino­
pharm Chemical Reagent Co. Ltd. (Shanghai, China).
Flavanols
(+)-Gallocatechin 18.23 ± 383.02 ± ND 39.17 ±
2.82b 22.65a 7.49b 2.2. Sample preparation and experimental design
Proanthocyanidin dimer 113.12 ± 751.90 ± 55.07 ± 392.02 ±
B3 1.83c 6.20a 3.52d 23.10b Grape clusters (Vitis labruscana cv. ‘Kyoho’) were manually collected
(-)-Epigallocatechin 3.32 ± 78.61 ± ND 11.60 ±
from a 10-year old local vineyard in Hangzhou, Zhejiang, China (120.2
0.67c 2.72a 2.22b
Proanthocyanidin 5.17 ± 111.90 ± 46.02 ± 29.18 ±
◦
E, 30.3 ◦ N) which is characterized as subtropical monsoon climate. The
tetramer-1 1.28d 1.77a 2.10b 9.54c plants were spaced at 2.5 × 2.0 m in the 2-hectare vineyard, rendering a
Proanthocyanidin dimer 1041.09 3055.12 ± 3417.68 779.96 ± planting density of 2000 plants/ ha, and the three maturity stage grapes
B1 ± 49.25c 26.64b ± 83.88a 35.54d were picked on 60, 75, 90 days after flowering (green, veraison and
(+)-Catechin 113.23 ± 2813.92 ± 25.31 ± 4868.97 ±
purple), respectively, with one cluster picked in each vine. Samples were
6.15c 20.66b 1.30d 27.07a
Proanthocyanidin 119.44 ± 939.67 ± 77.13 ± 365.59 ± randomly chosen without visual defects or damage (Fig. 1) and imme­
trimer-1 1.20c 10.61a 2.75d 8.10b diately transported to the laboratory within two hours. Four different
Proanthocyanidin 329.15 ± 613.96 ± 267.35 ± 448.22 ± tissues (leaves, seeds, stems and skins) and three different cluster parts
tetramer-2 7.03c 8.31a 5.63d 7.60b
(top, middle and bottom) in grape clusters were collected on 90 days
Proanthocyanidin dimer 21.96 ± 152.36 ± 4.91 ± 148.57 ±
B4 2.59b 3.47a 0.92c 1.36a
after flowering. The top, middle and bottom berries, seeds, stems and
(-)-Epicatechin 56.40 ± 405.81 ± 16.96 ± 3519.95 ± skins were randomly taken from the previously cut grape clusters, while
4.96c 5.14b 2.53c 75.29a the leaves were obtained from near the apex of the clusters. In addition,
Proanthocyanidin 38.39 ± 980.73 ± 12.39 ± 517.56 ± other grape clusters collected on 90 days after flowering were stored in
trimer-2 2.69c 15.28a 0.81d 18.56b
light/dark at 4 ◦ C and 25 ◦ C and samplings were carried out periodically.
Proanthocyanidin 24.55 ± 201.71 ± ND 787.77 ±
trimer-3 1.63c 7.52b 29.31a The grape berries were manually peeled with absence of residual water
Proanthocyanidin dimer 7.46 ± 7.92 ± 21.32 ± 140.57 ± and the skins were collected. After sampling, they were then immedi­
B2 0.97b 6.88b 0.42b 13.57a ately frozen in liquid nitrogen and kept at -80 ◦ C until used. Three
Proanthocyanidin 9.58 ± 34.34 ± 31.22 ± 10.16 ± biological replications were performed on each subsequent experiment.
tetramer-3 0.07c 2.56a 13.44ab 17.60c
(-)-Epicatechin gallate 7.67 ± 608.15 ± 11.48 ± 1631.94 ±
0.21c 23.90b 0.12c 42.45a 2.3. Determination of fruit quality
Total flavanol content 1908.76 11139.10 3986.85 13678.17
± 43.26d ± 77.21b ± 78.54c ± 63.45a
The determination of firmness, color index, total soluble solids (TSS)
Anthocyanins and titratable acidity (TA) were carried out. The firmness was evaluated
Malvidin-3,5- 11.28 ± ND ND ND using a TA-XT2i texture analyzer (Stable Micro Systems Ltd., Godalmin,
diglucoside 1.29 UK) equipped with a 5-mm diameter plunger. The maximum force was
Peonidin-3-glucoside 11.60 ± 0.60 ± ND ND
measured as the value of firmness. The color values were determined by
0.44a 0.03b
Malvidin-3-glucoside 7.72 ± ND ND ND a colorimeter (KONICA MINOLTA, CR-400, Japan) as L*, a* and b*
0.70 values, which were taken from four times around the equatorial region
Peonidin-3− 6′ ′ -p- 2.52 ± ND ND ND of each berry. The TSS and TA of the fruit juice were estimated by a Brix-
coumaroyl-glucoside- 1.03 Acidity Meter (Model PAL-BX/ACID 112 F5, Atago, Japan) at 25 ◦ C.
5-glucoside
Fifty berries from each treatment group were randomly sampled for the
Malvidin-3− 6′ ′ -p- 39.04 ± 1.94 ± ND ND
coumaroyl-glucoside- 1.76a 0.02b determination and three technical replications were carried out.
5-glucoside Three grape clusters in each group were examined for the berry
Cyanidin-3-glucoside 2.40 ± 0.16 ± ND ND abscission and rotten index and the stem browning rate during storage.
0.10a 0.02b
The berry abscission and rotten index were calculated as the percentages
Petunidin-3-glucoside 4.16 ± 0.13 ± ND ND
0.21a 0.01b
of the abscission berries weight and rotten berries weight to each clus­
Cyanidin-3− 6′ ′ -p- 0.60 ± ND ND ND ter’s total weight, respectively. The stem browning rate was calculated
coumaroyl-glucoside 0.06 using the following formula: Browning rate = 100 % × Σ[(stem
Petunidin-3− 6′ ′ -p- 1.94 ± ND ND ND browning scale) × (number of clusters with that stem browning scale)] /
coumaroyl-glucoside 0.14
[4 × total number of clusters evaluated]. The stem browning scale was
0 = 0 % stem surface area browning, 1 = 1–25 % area browning, 2 =

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M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Fig. 4. Heatmap of relative expression profiles of 14 genes involved in polyphenolic biosynthesis. The log2 of relative expression levels are shown, measured by RT-
qPCR. Relative expression level in grape skin was set as control.

26–50 % area browning, 3 = 51–70 % area browning and 4 = 76–100 % DAD was set between 190 and 600 nm at steps of 2 nm. The polyphenols
area browning. were quantified at 280 nm (flavanols), 320 nm (phenolic acids and
flavonols) and 520 nm (anthocyanins). Quantitative determinations of
2.4. Extraction of polyphenolic compounds flavanols, phenolic acids and flavonols, and anthocyanins were carried
out with the calibration curves obtained for catechin (6.25− 400 mg
Frozen samples of each group were ground to powder in liquid ni­ L− 1), quercetin (6.25− 400 mg L− 1) and cyanidin-3-glucoside (6.25− 400
trogen using porcelain mortars and pestles. Polyphenols were extracted mg L− 1) external standards, respectively. Results were expressed as mg
by adding 1.0 g of sample powder to 10 mL of 1% HCl-methanol. The kg− 1 fresh weight of tissues. All values were obtained from three
homogenates were vortexed for 10 min and stored in dark at 4 ◦ C for 12 samples.
h. The mixtures were then centrifuged (4 ◦ C, 10,000×g) for 15 min and
the residues were re-extracted twice. The supernatants were subse­ 2.6. Real-time quantitative PCR (RT-qPCR) and gene expression analysis
quently filtered by 0.22 μm microporous membrane for following high
performance liquid chromatography quadrupole time of flight mass Genes involved in polyphenolic biosynthesis pathway were selected
spectrometry (HPLC-Q-TOF-MS) and HPLC-DAD analysis. based on the website of Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway. The details of primers are given in Table S1. RT-qPCR
experiment was carried out in three steps. Firstly, the total RNA was
2.5. HPLC-Q-TOF-MS and HPLC-DAD analysis
extracted using the rapid cetyl trimethyl ammonium aromide (CTAB)
method. CTAB method can remove impurities such as proteins, poly­
The identification of polyphenolic compounds was performed using
saccharides and phenolic compounds from plant tissues, and precipitate
HPLC system (LC-20AD, Shimadzu, Japan) coupled to Q-TOF-MS (Triple
RNA with ethanol. The RNA concentrations were measured using a
TOF™ 5600+, 130 SCIEX incorporation, United States) with an elec­
microplate reader (TECAN, Spark®, Switzerland). Then, mRNA was
trospray ionization source (ESI) system. An AQ-C18 column (water-
reversely transcribed into cDNAs using a PrimeScript™ RT reagent kit
based, 5 μm, 4.6 × 250 mm, Welch Materials incorporation, Shanghai,
(Code DRR047A, TaKaRa, Japan). Finally, RT-qPCR reaction was done
China) was used to separate individual polyphenolic compounds with a
in ABI Step One RT-PCR system, the reaction mixture was comprised of
flow rate of 1 mL min− 1. Polyphenols were eluted with the following
10.0 μl of SYBR Premix Ex Taq™ (RR820A, TaKaRa, Japan) in a total
gradient of solvent B (acetonitrile) in solvent A (1 % phosphoric acid in
volume of 20 μl. The genes transcript levels were calculated by the
water): 0− 10 min, 2 % B; 10− 55 min, 10 % B; 55− 65 min, 18 % B;
house-keeping gene glyceraldehyde-3-phosphate dehydrogenase
65− 68 min, 50 % B; 68− 69 min, 80 % B; 69− 79 min, 2 % B. Mass
(GAPDH) according to the 2− ΔΔCT method. Three biological replicates
spectra were performed in the ESI both negative ion and positive ion
were used.
modes in the 100− 1500 m z− 1 scan range under the following condi­
tions: source voltage, -4.5 kV and 5.5 kV; source temperature, 550 ◦ C;
2.7. Statistical analyses
Gas 1 (Air) and Gas 2 (Air), 50 psi; and curtain gas (N2), 35 psi. The
polyphenols were detected at 280 nm and 520 nm depending on their
Data were presented as mean ± standard deviation (SD) values. The
types and identified according to their retention time, rate of mass to
statistical significance analysis of measurement data was analyzed using
charge, and the fragment ion compared to references (Lambert et al.,
one-way analysis of variance (ANOVA), the mean comparisons were
2015; Colombo et al., 2021). Chromatographic system and mass spectra
performed by Duncan’s multiple rage test at the significance level of p <
data were managed and processed using the Peak View software version
0.05 with SPSS software 25.0.
1.2, and a maximum error of 5 ppm was accepted.
For quantifying polyphenols, HPLC equipped with a DAD (SPD-M
20A, Shimadzu, Japan) were used, and the HPLC conditions were the
same as those for HPLC-Q-TOF-MS analysis. The scanning range of the

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M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Table 3 Data are shown in mean ± standard deviation (n = 3). Different letters of the
The polyphenol concentrations in berries at different maturity stages (mg kg− 1 same component represent statistically significant difference, respectively (p <
skin FW). 0.05). ND: not detected.
Compound Berry in Berry in Berry in
green veraison purple

Phenolic acids
Gallic acid ND ND ND
Caftaric acid 5.41 ± 0.54b 7.75 ± 0.76a 7.99 ± 0.59a
Coutaric acid 32.54 ± 50.15 ± 50.38 ±
0.62b 0.55a 0.40a

Total phenolic acid content 37.95 ± 57.89 ± 58.37 ±

0.10b 1.31a 0.39a
Flavonols
Myricetin-3-glucoside 5.15 ± 0.21c 7.08 ± 0.36b 8.35 ± 0.23a
Quercetin-3-rhamnose- 13.03 ± 24.19 ± 45.19 ±
glucuronide 0.65c 1.02b 1.20a
Quercetin-3-rhamnoside 2.27 ± 0.07c 12.22 ± 33.31 ±
0.38b 1.76a
Total flavonol content 20.45 ± 43.48 ± 86.85 ±
0.73c 1.05b 0.47a

Flavanols
(+)-Gallocatechin 3.76 ± 0.25c 13.90 ± 17.37 ±
0.65b 0.56a
Proanthocyanidin dimer B3 76.00 ± 121.79 ± 99.25 ±
1.70c 0.56a 1.73b
(-)-Epigallocatechin 1.80 ± 0.79b 1.38 ± 0.29b 3.67 ± 0.20a
Proanthocyanidin tetramer-1 0.98 ± 0.03c 3.27 ± 0.13b 5.59 ± 0.37a
Proanthocyanidin dimer B1 954.71 ± 1124.59 ± 958.58 ±
39.46b 2.31a 16.17b
(+)-Catechin 41.95 ± 106.90 ± 121.26 ±
1.91c 1.05b 2.62a
Proanthocyanidin trimer-1 48.65 ± 98.74 ± 114.84 ±
2.01c 1.98b 1.23a Fig. 5. Correlation matrix analysis of polyphenols and quality indexes in
Proanthocyanidin tetramer-2 172.19 ± 335.99 ± 416.09 ± ‘Kyoho’ berries at different maturity stages. * values are significant at p < 0.05.
7.90c 1.41b 4.80a
Proanthocyanidin dimer B4 6.80 ± 0.18c 17.82 ± 20.22 ±
0.13b 0.36a 3. Results and discussion
(-)-Epicatechin 28.23 ± 48.73 ± 48.84 ±
1.85b 0.97a 0.23a 3.1. Berry color index, firmness, TSS, TA and morphology
Proanthocyanidin trimer-2 12.57 ± 32.22 ± 39.55 ±
0.98c 0.50b 0.70a
Proanthocyanidin trimer-3 7.03 ± 0.41c 20.28 ± 24.37 ±
The color index L* value was continuously decreased, the value of a*
0.12b 0.55a increased while the value of b* decreased, which indicated the berry
Proanthocyanidin dimer B2 3.40 ± 0.29c 6.12 ± 0.06b 7.28 ± 0.61a surface became darker as grape ripening (Fig. 2A-C). Similarly, previous
Proanthocyanidin tetramer-3 3.37 ± 0.41c 7.25 ± 0.15b 13.33 ± study reported that the color tonality, indicating the proportion between
0.18a
yellow and red colors increased during berry development (Locatelli
(-)-Epicatechin gallate 1.14 ± 0.99c 5.07 ± 1.28b 7.27 ± 0.15a
Total flavanol content 1362.59 ± 1944.07 ± 1897.52 ± et al., 2016). Firmness and TA of the berries gradually decreased, while
56.77b 7.04a 19.07a TSS rapidly increased during grapes ripening (Fig. 2D-F). The noticeable
changes were in agreement with the previous studies (Fuentes et al.,
Anthocyanins 2016; Walker et al., 2001). Based on the above results, the ripening
Malvidin-3,5-diglucoside ND 3.31 ± 0.16b 10.45 ±
process of ‘Kyoho’ grape was accompanied by the color purpling, berry
4.21a
Peonidin-3-glucoside ND 4.77 ± 0.21b 11.35 ± softening, and sugar accumulation, and ‘Kyoho’ grape reached the
0.21a maturity stage approximately 15 days after veraison, which is faster
Malvidin-3-glucoside ND 2.34 ± 0.08b 5.37 ± 0.30a than other grape varieties (Colombo et al., 2021). Furthermore, artificial
Peonidin-3− 6′ ′ -p-coumaroyl- ND 0.50 ± 0.02b 1.14 ± 0.02a sensing systems such as electronic noses (EN), electronic tongues (ET)
glucoside-5-glucoside
Malvidin-3− 6′ ′ -p-coumaroyl- ND 9.85 ± 0.43b 22.26 ±
and electronic eyes (EE) were recently developed to monitor grape
glucoside-5-glucoside 0.35a ripening based on the analysis of physico-chemical traits (Orlandi et al.,
Cyanidin-3-glucoside ND 1.35 ± 0.09b 2.93 ± 0.08a 2019; Gliszczynska-Swiglo and Chmielewski, 2017).
Petunidin-3-glucoside ND 1.12 ± 0.04b 2.62 ± 0.01a There were obvious difference in firmness, TA and TSS values of
Cyanidin-3− 6′ ′ -p-coumaroyl- ND 0.29 ± 0.03b 0.95 ± 0.02a
grape berry according to different parts of the grape clusters. It was
glucoside
Petunidin-3− 6′ ′ -p-coumaroyl- ND 0.47 ± 0.04b 1.48 ± 0.03a interesting that bottom berries exhibited the lowest firmness and the
glucoside highest TA values, while top berries had the lowest TSS value (Fig. 2J-L).
Peonidin-3− 6′ ′ -p-coumaroyl- ND 4.00 ± 0.33b 9.37 ± 0.19a In addition, the berries located in middle of cluster had the highest L*
glucoside and b* values and the lowest a* value. Results demonstrated that the
Malvidin-3− 6′ ′ -p-coumaroyl- ND 5.59 ± 0.23b 15.21 ±
bottom berries had the lowest firmness and the highest TSS/TA ratio as
glucoside 0.29a
Total anthocyanin content 33.59 ± 83.14 ± well as the closest color to purple, which provided evidence that bottom
0.76b 3.98a berries are the ripest berries in grape clusters.
Total polyphenol content 1420.99 ± 2079.04 ± 2125.87 ± The morphology of grape clusters was influenced by exogenous light
57.48b 9.73a 15.46a
and temperature (Fig. 1). During storage at 25 ◦ C, the abscission rate,
the rotten index of berries and the browning rate of the grape stems

7
M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Table 4 Table 4 (continued )

The polyphenol concentrations in berries differentially located in grape cluster Compound Top berry Middle berry Bottom
(mg kg− 1 skin FW). berry
Compound Top berry Middle berry Bottom Total polyphenol content 2080.16 ± 2082.43 ± 2337.33 ±
berry 29.15b 18.64b 52.64a
Phenolic acids
Data are shown in mean ± standard deviation (n = 3). Different letters of the
Gallic acid ND ND ND
same component represent statistically significant difference, respectively (p <
Caftaric acid 10.64 ± 9.76 ± 1.26a 7.00 ± 0.82b
1.56a 0.05). ND: not detected.
Coutaric acid 44.96 ± 47.99 ± 50.41 ±
0.58c 0.13b 1.14a rapidly increased and depicted higher levels than storage at 4 ◦ C.
Total phenolic acid content 55.60 ± 57.74 ± 57.41 ±
Similarly, the grapes exposure to the light showed higher browning rate
1.98a 1.38a 1.08a
than the grapes exposure to the dark. After storage for 3 days, at 25 ◦ C
Flavonols and storage for 14 days, at 4 ◦ C, the stored grape clusters under light
Myricetin-3-glucoside 6.33 ± 0.37b 6.16 ± 0.19b 7.56 ± 0.26a completely browned, whereas the stored grape clusters under dark
Quercetin-3-rhamnose- 21.06 ± 40.84 ± 39.38 ± conditions partially browned. The results demonstrated that the low
glucuronide 0.54b 0.59a 2.38a
temperature and dark storage inhibited the decay development of
Quercetin-3-rhamnoside 28.40 ± 26.18 ± 32.87 ±
0.74b 0.55b 1.87a ‘Kyoho’ grape. Xu et al. (2018) revealed that the low temperature
Total flavonol content 55.79 ± 73.18 ± 79.82 ± reduced the abscission and decay rate of berries and maintained the
1.15c 0.95b 4.00a grape flavor and texture quality. Low temperature maintains the quality
of fruit and vegetables by effectively reducing of respiration rate as well
Flavanols as inhibiting the microbial activity. For example, the strawberries
(+)-Gallocatechin 17.55 ± 16.90 ± 17.44 ±
1.35a 0.04a 1.12a
treated with light for 7 days were also found to be visibly darker than the
Proanthocyanidin dimer B3 111.53 ± 123.96 ± 128.19 ± strawberries stored in the dark (Fu et al., 2017). The light accelerated
2.61b 2.32a 1.28a the postharvest senescence of fruit and vegetables probably by
(-)-Epigallocatechin 3.14 ± 0.77a 2.09 ± 0.34b 2.68 ± enhancing the respiration rate and accelerating water and gas diffusion
0.14ab
on surface of the produce.
Proanthocyanidin tetramer-1 6.64 ± 0.34a 5.68 ± 0.27a 6.17 ±
0.51ab
Proanthocyanidin dimer B1 1063.33 ± 1060.86 ± 1173.72 ± 3.2. Polyphenolic profiles
26.80b 16.27b 16.01a
(+)-Catechin 107.90 ± 102.31 ± 112.00 ±
2.42ab 1.79c 4.36a Polyphenols contribute to the crucial attributes of table grapes and
Proanthocyanidin trimer-1 110.30 ± 105.79 ± 112.89 ± wines, and are also positively correlated with the antioxidant capacity
0.88a 2.88a 5.76a with health-promoting properties (Askari-Khorasgani and Pessarakli,
Proanthocyanidin tetramer-2 294.64 ± 285.70 ± 360.31 ± 2019a; Silva et al., 2020). Thirty-two phenolic compounds were detec­
6.24b 6.07b 12.92a
Proanthocyanidin dimer B4 22.87 ± 20.72 ± 23.63 ±
ted in the tested samples, including three phenolic acids, three flavonols,
0.31a 0.23b 1.80a fifteen flavanols and eleven anthocyanins using HPLC-Q-TOF-MS
(-)-Epicatechin 60.20 ± 63.61 ± 74.29 ± coupled system. The retention time, molecular ion and product ions of
2.19b 2.19b 3.41a these compounds are shown in Table 1, and the chromatograms at UV
Proanthocyanidin trimer-2 35.67 ± 35.10 ± 36.08 ±
280 nm, 320 nm and 520 nm are shown in Fig. 3. Phenolic acids include
1.22a 0.03a 1.96a
Proanthocyanidin trimer-3 22.80 ± 21.66 ± 22.04 ± hydroxybenzoic acid (gallic acid) and hydroxycinnamic acids (caftaric
0.40a 0.04a 1.04a acid and coutaric acid), while flavonols mainly include derivatives of
Proanthocyanidin dimer B2 6.31 ± 0.39a 5.63 ± 0.04b 5.18 ± 0.05b myricetin and quercetin. Concerning the flavanols, five monomers, four
Proanthocyanidin tetramer-3 11.18 ± 12.02 ± 15.23 ± dimers, three trimers and three tetramers belonging to this class of
0.25c 0.24b 0.60a
(-)-Epicatechin gallate 6.43 ± 0.21a 5.13 ± 0.08c 5.93 ± 0.18b
compounds were detected, and five monomers include (+)-galloca­
Total flavanol content 1880.47 ± 1867.14 ± 2095.77 ± techin, (-)-epigallocatechin, (+)-catechin, (-)-epicatechin and (-)-epi­
27.89b 21.23b 43.50a catechin gallate. Proanthocyanidin structures vary depending upon the
nature (stereochemistry and hydroxylation pattern) of the flavan-3-ol
Anthocyanins starter and extension units, the position and stereochemistry of the
Malvidin-3,5-diglucoside 8.51 ± 0.16b 11.68 ± 8.29 ± 0.65b
linkage to the ‘lower’ unit, the degree of polymerization, and the pres­
0.71a
Peonidin-3-glucoside 10.45 ± 11.35 ± 13.72 ± ence or absence of modifications such as esterification of the 3-hydroxyl
0.18c 0.21b 0.43a group (Dixon et al., 2005). The anthocyanins are comprised of cyanidin,
Malvidin-3-glucoside 6.52 ± 0.13b 5.37 ± 0.30c 8.72 ± 0.58a delphinidin, petunidin, peonidin and malvidin 3-monoglucosides (or 3,
Peonidin-3− 6′ ′ -p-coumaroyl- 1.43 ± 0.06b 1.14 ± 0.02c 2.38 ± 0.12a
5-diglucosides) along with the corresponding acetyl, p-coumaroyl, and
glucoside-5-glucoside
Malvidin-3− 6′ ′ -p-coumaroyl- 28.49 ± 22.26 ± 29.21 ±
caffeoyl derivatives in grape berry skins (Liang et al., 2008). In the
glucoside-5-glucoside 0.80a 0.35b 1.57a present study, the 3-glucosides of cyanidin, petunidin, peonidin and
Cyanidin-3-glucoside 2.50 ± 0.07c 2.93 ± 0.08b 3.67 ± 0.16a malvidin, the -3− 6′′ -p-coumaroyl-glucosides of cyanidin, petunidin,
Petunidin-3-glucoside 3.43 ± 0.08b 2.62 ± 0.01c 4.49 ± 0.24a peonidin and malvidin, the -3− 6′′ -p-coumaroyl-glucoside-5-glucosides
Cyanidin-3− 6′ ′ -p-coumaroyl- 0.57 ± 0.01c 0.95 ± 0.02a 0.86 ± 0.03b
of peonidin and malvidin as well as the malvidin-3,5-diglucoside were
glucoside
Petunidin-3− 6′ ′ -p-coumaroyl- 1.69 ± 0.04b 1.48 ± 0.03c 1.83 ± 0.10a detected.
glucoside
Peonidin-3− 6′ ′ -p-coumaroyl- 6.97 ± 0.29c 9.37 ± 0.19b 10.16 ±
3.3. Polyphenol content and gene expression involved in polyphenol
glucoside 0.52a
Malvidin-3− 6′ ′ -p-coumaroyl- 17.73 ± 15.21 ± 21.02 ± biosynthesis
glucoside 0.44b 0.29c 1.24a
Total anthocyanin content 88.29 ± 84.37 ± 104.34 ± 3.3.1. Polyphenols and gene expression analysis in different berry tissues
1.89b 1.82b 5.17a
Polyphenols distributed in different berry tissues were detected in
the present study. Perez-Navarro et al. (2019) suggested that the

8
M. Qi et al. Journal of Food Composition and Analysis 106 (2022) 104353

Table 5
1
The time-course accumulation of polyphenol concentrations in ‘Kyoho’ berries (mg kg− skin FW).
Total phenolic acid content Total flavonol content Total flavanol content Total anthocyanin content Total polyphenol content
c c a e
0 day 56.92 ± 1.16 80.83 ± 10.29 1908.76 ± 43.26 111.86 ± 4.68 2158.37 ± 49.37bc
3 days 62.03 ± 0.62b 89.32 ± 8.44bc 1787.42 ± 38.62b 137.21 ± 3.28d 2075.99 ± 41.10b
Light
25 ℃ 6 days 45.41 ± 0.98d 118.31 ± 4.21a 1417.66 ± 5.82c 208.74 ± 8.05a 1790.12 ± 4.63c
3 days 64.40 ± 2.19a 98.45 ± 4.12b 1847.38 ± 114.45ab 151.38 ± 3.62c 2161.61 ± 108.71bc
Dark
6 days 64.86 ± 0.94a 88.42 ± 1.31bc 1913.04 ± 24.96a 171.17 ± 4.24b 2237.49 ± 21.82a
0 day 46.60 ± 0.64b 72.99 ± 1.37a 2089.69 ± 26.79e 121.75 ± 2.68h 2331.02 ± 27.98d
7 days 37.96 ± 1.99c 42.38 ± 0.34e 1854.78 ± 65.08f 134.74 ± 0.66g 2069.86 ± 65.26e
14 days 33.70 ± 1.16d 42.42 ± 0.63e 1829.29 ± 73.22f 173.22 ± 1.06f 2078.63 ± 72.86e
Light
21 days 43.86 ± 2.92b 59.29 ± 0.78c 2351.60 ± 74.32d 244.91 ± 2.48c 2699.66 ± 69.30c
4℃ 28 days 46.60 ± 0.64b 72.99 ± 1.37a 2606.15 ± 61.70bc 278.66 ± 6.88b 3004.39 ± 63.85b
7 days 40.72 ± 0.91c 35.88 ± 1.67f 2327.64 ± 23.97d 194.99 ± 5.21e 2599.24 ± 27.90c
14 days 51.59 ± 3.11a 53.40 ± 0.39d 3236.36 ± 117.01a 206.08 ± 4.82d 3547.43 ± 111.25a
Dark
21 days 45.03 ± 0.40b 61.70 ± 1.26b 2529.72 ± 70.60c 290.70 ± 3.61a 2927.15 ± 73.67b
28 days 50.90 ± 1.29a 53.80 ± 1.79d 2673.32 ± 63.34b 251.82 ± 7.30c 3029.83 ± 59.10b

Data are shown in mean ± standard deviation (n = 3). Different letters of the same component represent statistically significant difference (p < 0.05). The data of 25 ℃
and 4 ℃ are compared separately.

phenolic composition profiles become an available tool for grape variety
differentiation, principally anthocyanins, flavonols and flavan-3-ols
located in grape skins and seeds. Highest levels of total polyphenols
were found in seeds, followed by stems, leaves and finally skins, and the
total polyphenol content of seeds were approximately 6.3 times rela­
tively to the total polyphenol content of skins, even though only flava­
nols were most abundant in seeds (Table 2). Among flavanols group,
(+)-catechin showed the highest concentration in the seeds (4868 ± 27
mg kg− 1 seed FW), while skins, leaves and stems were rich in proan­
thocyanidin dimer B1 (1041 ± 49 mg kg− 1 skin FW, 3417 ± 83 mg kg− 1
leaf FW and 3055 ± 26 mg kg− 1 stem FW). Previous studies also showed
that the maximum concentration of total polyphenols was found in seeds
among different grape tissues (Pastrana-Bonilla et al., 2003; Souquet
et al., 2000). Besides, the proanthocyanidins and flavan-3-ols were the
most abundant in the grape seeds, while the skins of red grapes were rich
with anthocyanins (Pantelic et al., 2016). Notably, few flavanol mono­
mers were detected in leaves (Table 2). The results suggested the low
and high condensation degree of flavan-3-ols in seeds and leaves,
respectively. Gallic acid was only identified in seeds, which was in
contrast to a previous report that gallic acid was one of the major phe­
nols in seeds and leaves of ‘Muscadine’ grape (Pastrana-Bonilla et al.,
2003). Flavonols in grapes were mainly myricetin-3-glucoside, querce­
tin-3-rhamnose-glucuronide and quercetin-3-rhamnoside, while
myricetin-3-glucoside was not presented in seeds.
Quercetin-3-rhamnose-glucuronide showed the highest concentration.
The leaves were rich with quercetin-3-rhamnose-glucuronide content,
11.1 times more than skins (Table 2). Nevertheless, several studies
showed that quercetin-3-glucoside and quercetin-3-glucuronide were
the major flavonols in other grape cultivars (Blancquaert et al., 2019;
Colombo et al., 2021). The difference indicated that the flavonol profiles
could be potential approach to distinguish ‘Kyoho’ grape, which is
especially abundant in quercetin-3-rhamnose-glucuronide. In addition,
there were eleven anthocyanin compounds identified and quantified in
the skins and six in stems, whereas none of them was determined in the
leaves and seeds. The skins exhibited the highest concentrations in the
total anthocyanins content (111 ± 4 mg kg− 1 skin FW) and each indi­
vidual anthocyanin with a significant difference in comparison to other
tissues. The most abundant anthocyanins in skins were malvidin de­
rivatives, most of which was malvidin-3− 6′′ -p-coumar­
oyl-glucoside-5-glucoside, accounted for more than 34.9 % of total
anthocyanins in skins followed by malvidin-3− 6′′ -p-coumaroyl-gluco­
side. Similarly, Li et al. (2013) reported that malvidin-3-(­
trans)-coumaroyl-5-diglucoside and malvidin-3-glucoside were the most
abundant anthocyanins in fresh skins of ‘Kyoho’ and ‘Cabernet Sau­
vignon’ grapes, respectively.
Moreover, the genes expression involved in phenylpropanoid and
flavonoid pathways were investigated to elucidate the molecular
mechanism of polyphenol metabolism. Phenylpropanoids are a diverse
group of plant secondary metabolites, and the flavonoid pathway is the
last step of flavonoid synthesis, which is directly linked to the qualitative
and quantitative characteristics of the flavonoids (Li et al., 2019b). The
transcriptional profiles of polyphenol biosynthesis genes using RT-qPCR
are shown in Fig. 4. The transcript levels of chalcone isomerase (VvCHI),
dihydroflavonol 4-reductase (VvDFR), leucoanthocyanidin reductase
(VvLAR), anthocyanidin reductase (VvANR) and flavonol synthase
(VvFLS) in stems and leaves, as well as VvANR and VvFLS in seeds were
higher than those of skins, which was consistent with our results that
there were more flavanols in stems, leaves, and seeds than in skins. ANR
and LAR are both specific enzymes for the generation of flavan-3-ols.
ANR is a key enzyme in the biosynthesis of proanthocyanidin, and
plays a key role in the downstream of this biosynthetic pathway (Liang
et al., 2021).
3.3.2. Polyphenols and gene expression analysis in different maturity stages
Several studies demonstrated that the berry ripeness and the accu­
mulation of polyphenols have a significant correlation. Generally, the
content of polyphenols in grape skins shows a rising trend during
ripening (Zhou et al., 2019). Table 3 shows that two phenolic acids,
three flavonols, and fifteen flavanols were found in grapes at different
maturity stages, while eleven anthocyanins only exist in the veraison
and purple berries. Additionally, the results indicated that almost all
identified polyphenol compounds increased during ripening, the total
polyphenol contents of green, veraison and purple berries were 1420 ±
57, 2079 ± 9 and 2125 ± 15 mg kg− 1 skin FW, respectively. Moreover,
the total contents of polyphenols, phenolic acids, flavonols, flavanols
and anthocyanins were positively correlated with each other (r =
0.72− 0.99), and they were positively correlated with a* value and TSS,
while negatively correlated with L* value, b* value, firmness and TA
(Fig. 5). The correlation matrix analysis reflects positive correlation
between berry ripeness and polyphenol accumulation. On the other
hand, the total contents of flavonols and anthocyanins displayed sig­
nificant negative correlations with b* value, respectively (p < 0.05),
which indicated a strong relationship between color and total flavonol
content and total anthocyanin content (Fig. 5). However, proantho­
cyanidin B3 and proanthocyanidin B1 showed a trend from rising to
decline, and displayed the highest value at the veraison stage (Table 3).
The results were consistent with the previous study, where the content of
proanthocyanidin dimers in the skins decreased in the course of matu­
ration (Colombo et al., 2021). Furthermore, there were no significant
changes in phenolic acid content after veraison stage. It suggested
polyphenol content could remain constant or declined when grapes
reached a certain level of ripeness. Locatelli et al. (2016) reported that
during ripening, the caftaric acid content drastically decreased and the
highest value of coutaric acid was exhibited at veraison completion.The

9
M. Qi et al.
synthesis of hydroxycinnamates occurred mainly before veraison and
their concentration decreased while fruit size increased and the solutes
diluted due to their accumulation occurred prevalently in the pulp
(Locatelli et al., 2016). Previous study demonstrated that there were
strong correlations between total polyphenol content and antioxidant
capacity (Fuentes et al., 2016; Ban et al., 2020). The present work also
provided the evidence that ‘Kyoho’ grapes could be potentially used as a
promising functional source due to the high content of polyphenols.
In the present study, RT-qPCR result shows almost all genes were up-
regulated during ripening, except VvANR and VvFLS, as they exhibited
higher transcript levels in green berries (Fig. 4). In flavonoid biosyn­
thesis, the flavonols produced by FLS, and anthocyanidin serves as the
substrate for the synthesis of epicatechin by ANR (Liu et al., 2013), thus
the decrease of VvFLS and VvANR gene transcription levels may
contribute to the accumulation of anthocyanins. These results indicated
the notably up-regulated gene transcription of polyphenol metabolism
which contributed to the rapid accumulation of polyphenols.
3.3.3. Polyphenols and gene expression analysis in different cluster parts
Most of polyphenolic components in bottom berries had a higher
concentration than top and middle berries except caftaric acid, (-)-epi­
gallocatechin, (-)-epicatechin gallate, proanthocyanidin tetramer-1,
proanthocyanidin dimer B2, malvidin-3,5-diglucoside, and cyanidin-
3− 6′′ -p-coumaroyl-glucoside (Table 4). The total polyphenol content in
bottom berries was 12.4 % higher than that in top berries and 12.2 %
higher than that in middle berries, while there was no significant dif­
ference between total polyphenol contents in middle and top berries.
Correspondingly, almost all genes in bottom berries had higher
expression levels than that in top and middle berries while the expres­
sion levels of these genes in top and middle berries were approximately
equal. These results were in agreement with the study by Figueir­
edo-Gonzalez et al. (2013), which found that total anthocyanins and
total flavonols exhibited higher upward trends in tops than that in
shoulders within the ‘Mouratón’ grape clusters during ripeness. How­
ever, Figueiredo-Gonzalez et al. (2012) reported that no significant
difference in anthocyanin and flavonol contents collected from both
positions of ‘Gran Negro’ grape. Grapes located inside the top and
middle positions receive less sunlight radiation than bottom berries,
which could explain the different accumulations of polyphenols
observed for different grape cluster parts. In ‘Pinot noir’ grape, the
significant changes in flavonols and anthocyanins were found between
shaded and light-exposed berries (Cortell and Kennedy, 2006). Never­
theless, in ‘Cabernet franc’ and ‘Chardonnay’ grapes, the interior clus­
ters had lower total flavonol concentrations than the exterior clusters,
while the position of clusters in the canopy had no major impacts on the
composition of anthocyanins and flavan-3-ols (Gao et al., 2021). The
results indicated that the distribution of polyphenolic compounds was
affected by sunlight conditions around grape clusters. It was previously
studied that the different distribution of polyphenols in grape cluster
could be attributed to the blockage of phloem elements into the berry
and as a consequence, different influx of solutes (sugars, e.g.) among
top, middle and bottom berries (Figueiredo-Gonzalez et al., 2013). It
was also demonstrated that the chemical compounds in berries are not
only sensitive to microclimate (especially sunlight radiation), but also
influenced by intrinsic factors.
3.3.4. Polyphenols and gene expression analysis in response to light and
temperature
During storage period, the phenolic acid content in grapes declined
stored at 25 ℃/light, while it increased stored at 25 ℃/dark (Table 5).
The variations of phenolic acid content during the first 14 days of grapes
stored at 4 ℃/light and 4 ℃/dark were similar to those in grapes stored
at 25 ℃/light and 25 ℃/dark, respectively. However, the contents of
phenolic acids were recovered later (Table 5). Besides, the total flavonol
content in grapes stored at 25 ℃ were found to be increased, whereas it
decreased in grapes stored at 4 ℃. Polyphenols have a strong ability for
10
Journal of Food Composition and Analysis 106 (2022) 104353
eliminating the free radicals, as the phenol hydroxyl is easily oxidized.
The environmental stress can lead to lots of reactive oxygen and free
radicals of fruit, and polyphenols are consumed when they are involved
in scavenging of these free radicals (Xia et al., 2015; Radovanovic et al.,
2009). Additionally, Table 5 reflects that total flavanols were nearly
25.7 % lost after storage at 25 ℃/light, while it was essentially main­
tained after storage at 25 ℃/dark. On the contrary, increasing trends of
the total flavanol content were exhibited in grapes stored at 4 ℃ light
and dark, from 2089 ± 26 mg kg− 1 skin FW to 2606 ± 61 and 2673 ± 63
mg kg− 1 skin FW, respectively. The considerably higher levels of fla­
vanols in grapes stored at 4 ℃ indicated that flavan-3-ols underwent
more marked polymerization and condensation reactions. The higher
accumulation of flavan-3-ols derivatives in the grapes grown under a
cold climate were also observed by Serratosa et al. (2014). In present
study, the concentrations of total anthocyanins were increased in four
storage groups, whereas anthocyanins rose more sharply under light
than that in dark. Correspondingly, Romero et al. (2020) reported that
low temperature postharvest storage and application of light irradiation
could increase anthocyanin levels. Moreover, total polyphenol contents
varied similar to total flavanol contents in four storage groups, respec­
tively, since the flavanol accounted for a large proportion in polyphenol
content.
The metabolic regulation of polyphenol biosynthesis pathway is
partially dependent on the transcriptional level, including transcrip­
tional regulation in response to various abiotic stresses, such as the light
and temperature. Polyphenols, especially flavonoids, are closely related
to plant resistance (Ferreyra et al., 2012). The expression level of most of
genes increased during the storage at 25 ℃, while chalcone synthase
(VvCHS), anthocyanidin-3-glucosyltransferase (Vv3GT) and VvLAR were
down-regulated. Furthermore, most of the genes were inhibited during
the storage at 4 ℃, whereas the expression of VvCHS, VvCHI, flavanone
3β-hydroxylase (VvF3H) and VvDFR increased. Nevertheless, it is
notable that CHS, which is the first enzyme in the flavonoid pathway and
provides the precursors for various flavonoids (Ghasemzadeh et al.,
2014), was down-regulated at 25 ℃ but up-regulated at 4 ℃, which was
in good agreement with changing trend of polyphenol concentrations.
Previous studies showed that low temperature promoted the accumu­
lation of polyphenols by up-regulating the gene expression in the poly­
phenol biosynthesis pathway, while the effect of high temperature on
polyphenol metabolism was controversial (Azuma et al., 2012; Zhang
et al., 2015). High temperature may reduce the polyphenol content via
suppressing the gene expression or accelerated degradation of poly­
phenols (Azuma et al., 2012; Zhang et al., 2015; Mori et al., 2007).
Moreover, RT-qPCR result shows that VvCHI, VvF3H, flavonoid 3′ -hy­
droxylase (VvF3′ H), flavonoid 3′ 5′ -hydroxylase (VvF3′ 5′ H), VvDFR,
leucoanthocyanidin dioxygenase (VvLDOX), anthocyanin 3′ -methyl­
transferase (VvOMT), VvFLS and flavonoid 3′ ,5′ -methyltransferase
(VvAOMT) in grapes stored at 25 ℃/dark were more expressed than 25
℃/light, while the difference of gene expression had no obvious regu­
larity between grapes stored at 4 ℃/dark or light. Previous studies
showed that light had a dual effect on polyphenol metabolism. The
prolonged and high intensity light can cause different degrees of adverse
effects on plants (Wei et al., 2013; Interdonato et al., 2011; Savitch et al.,
2001). Azuma et al. (2012) studied the interrelationships between light
and temperature effects on flavonoid biosynthesis of grape berries in
early stages of veraison, they found that the accumulation of anthocy­
anins was dependent on both light and low temperature. Our result
indicated that light had a negative effect on total polyphenols at 25 ℃,
and gene expression results supported this hypothesis.
4. Conclusions
In the present study, it was demonstrated that the higher polyphenol
concentration was accumulated in seeds than that in stems, leaves and
skins, whereas phenolic acids, flavonols, and anthocyanins were rare or
absent. Polyphenol biosynthesis was significantly enhanced as berry
M. Qi et al.
ripening, and the polyphenols were most accumulated and bio­
synthesized in berries located in bottom of each cluster. Furthermore,
the results indicated that the light had a negative effect on polyphenol
accumulation in berry stored at 25 ℃, whereas the low temperature
increased polyphenol levels during storage. Our study provided evi­
dence for elucidating the polyphenol performance and biosynthesis in
‘Kyoho’ grape in dependence of berry ripeness, berry position, and berry
response to exogenous light and temperature stresses; and laid a foun­
dation for subsequent studies on the improvement of polyphenol
biosynthesis and on providing new insights for deeper elucidation and
efficient utilization of bioactive compounds in berry.
CRediT authorship contribution statement
Ming Qi: Conceptualization, Data curation, Formal analysis, Meth­
odology, Writing – original draft, Writing – review & editing. Zisheng
Luo: Conceptualization, Writing – review & editing, Supervision,
Funding acquisition. Bin Wu: Methodology, Validation. Lei Wang:
Methodology, Validation. Mingyi Yang: Formal analysis, Software.
Xiaochen Zhang: Software. Xingyu Lin: Conceptualization. Yanqun
Xu: Formal analysis. Xihong Li: Resources. Li Li: Conceptualization,
Writing – review & editing, Supervision, Project administration, Fund­
ing acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The research was funded by the National Natural Science Foundation
of China (U2003213; 32072271).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jfca.2021.104353.
References
Askari-Khorasgani, O., Pessarakli, M., 2019a. Fruit quality and nutrient composition of
grapevines: a review. J. Plant Nutr. 42 (17), 2133–2150. https://doi.org/10.1080/
01904167.2019.1643369.
Askari-Khorasgani, O., Pessarakli, M., 2019b. Relationship between signaling and
metabolic pathways of grapevines under temperature and light stresses-a review.
J. Plant Nutr. 42 (17), 2164–2175. https://doi.org/10.1080/
01904167.2019.1643368.
Azuma, A., Yakushiji, H., Koshita, Y., Kobayashi, S., 2012. Flavonoid biosynthesis-related
genes in grape skin are differentially regulated by temperature and light conditions.
Planta 236 (4), 1067–1080. https://doi.org/10.1007/s00425-012-1650-x.
Ban, Z.J., Zhang, J.L., Li, L., Luo, Z.S., Wang, Y.J., Yuan, Q.P., Zhou, B., Liu, H.D., 2020.
Ginger essential oil-based microencapsulation as an efficient delivery system for the
improvement of Jujube (Ziziphus jujuba Mill.) fruit quality. Food Chem. 306, 125628
https://doi.org/10.1016/j.foodchem.2019.125628.
Biniari, K., Xenaki, M., Daskalakis, I., Rusjan, D., Bouza, D., Stavrakaki, M., 2020.
Polyphenolic compounds and antioxidants of skin and berry grapes of Greek Vitis
vinifera cultivars in relation to climate conditions. Food Chem. 307, 125518 https://
doi.org/10.1016/j.foodchem.2019.125518.
Blancquaert, E.H., Oberholster, A., Ricardo-da-Silva, J.M., Deloire, A.J., 2019. Grape
flavonoid evolution and composition under altered light and temperature conditions
in Cabernet Sauvignon (Vitis vinifera L.). Front. Plant Sci. 10. https://doi.org/
10.3389/fpls.2019.01062.
Colombo, R.C., Roberto, S.R., da Cruz, M.A., de Carvalho, D.U., Yamamoto, L.Y.,
Nixdorf, S.L., Perez-Navarro, J., Gomez-Alonso, S., Shahab, M., Ahmed, S., Azeredo
Goncalves, L.S., de Souza, R.T., Hermosin-Gutierrez, I., 2021. Characterization of the
phenolic ripening development of’ BRS Vitoria’ seedless table grapes using HPLC-
DAD-ESI-MS/MS. J. Food Compos. Anal. 95. https://doi.org/10.1016/j.
jfca.2020.103693.
Cortell, J.M., Kennedy, J.A., 2006. Effect of shading on accumulation of flavonoid
compounds in (Vitis vinifera L.) pinot noir fruit and extraction in a model system.
J. Agric. Food Chem. 54, 8510–8520. https://doi.org/10.1021/jf0616560.
Deng, Y., Wu, Y., Li, Y.F., 2006. Physiological responses and quality attributes of’ Kyoho’
grapes to controlled atmosphere storage. LWT-Food Sci. Technol. 39 (6), 584–590.
https://doi.org/10.1016/j.lwt.2005.05.001.
11
Journal of Food Composition and Analysis 106 (2022) 104353
Dixon, R.A., Xie, D.Y., Sharma, S.B., 2005. Proanthocyanidins - a final frontier in
flavonoid research? New Phytol. 165 (1), 9–28. https://doi.org/10.1111/j.1469-
8137.2004.01217.x.
Downey, M.O., Dokoozlian, N.K., Krstic, M.P., 2006. Cultural practice and environmental
impacts on the flavonoid composition of grapes and wine: a review of recent
research. Am. J. Enol. Vitic. 57 (3), 257–268. https://doi.org/10.1016/j.
scienta.2005.07.007.
Ferreyra, M.L.F., Rius, S.P., Casati, P., 2012. Flavonoids: biosynthesis, biological
functions, and biotechnological applications. Front. Plant Sci. 3, 222. https://doi.
org/10.3389/fpls.2012.00222.
Figueiredo-Gonzalez, M., Simal-Gandara, J., Boso, S., Martinez, M.C., Santiago, J.L.,
Cancho-Grande, B., 2012. Flavonoids in Gran Negro berries collected from shoulders
and tips within the cluster, and comparison with Brancellao and Mouraton varieties.
Food Chem. 133 (3), 806–815. https://doi.org/10.1016/j.foodchem.2012.01.095.
Figueiredo-Gonzalez, M., Cancho-Grande, B., Boso, S., Santiago, J.L., Martinez, M.C.,
Simal-Gandara, J., 2013. Evolution of flavonoids in Mouraton berries taken from
both bunch halves. Food Chem. 138 (2–3), 1868–1877. https://doi.org/10.1016/j.
foodchem.2012.11.083.
Fu, X.M., Cheng, S.H., Zhang, Y.Q., Du, B., Feng, C., Zhou, Y., Mei, X., Jiang, Y.M.,
Duan, X.W., Yang, Z.Y., 2017. Differential responses of four biosynthetic pathways of
aroma compounds in postharvest strawberry (Fragaria x ananassa Duch.) under
interaction of light and temperature. Food Chem. 221, 356–364. https://doi.org/
10.1016/j.foodchem.2016.10.082.
Fuentes, L., Valdenegro, M., Gomez, M.G., Ayala Raso, A., Quiroga, E., Martinez, J.P.,
Vinet, R., Caballero, E., Figueroa, C.R., 2016. Characterization of fruit development
and potential health benefits of arrayan (Luma apiculata), a native berry of South
America. Food Chem. 196, 1239–1247. https://doi.org/10.1016/j.
foodchem.2015.10.003.
Gao, X.T., Sun, D., Wu, M.H., Li, H.Q., Liu, F.Q., He, F., Pan, Q.H., Wang, J., 2021.
Influence of cluster positions in the canopy and row orientation on the flavonoid and
volatile compound profiles in Vitis vinifera L. Cabernet franc and Chardonnay berries.
Food Res. Int. 143. https://doi.org/10.1016/j.foodres.2021.110306.
Ghasemzadeh, A., Nasiri, A., Jaafar, H.Z.E., Baghdadi, A., Ahmad, I., 2014. Changes in
phytochemical synthesis, chalcone synthase activity and pharmaceutical qualities of
sabah snake grass (Clinacanthus nutans L.) in Relation to Plant Age. Molecules 19
(11), 17632–17648. https://doi.org/10.3390/molecules191117632.
Gliszczynska-Swiglo, A., Chmielewski, J., 2017. Electronic nose as a tool for monitoring
the authenticity of food. A review. Food Anal. Methods 10 (6), 1800–1816. https://
doi.org/10.1007/s12161-016-0739-4.
Guo, D.L., Yu, Y.H., Xi, F.F., Shi, Y.Y., Zhang, G.H., 2016. Histological and molecular
characterization of grape early ripening bud mutant. Int. J. Genomics 2016,
5620106. https://doi.org/10.1155/2016/5620106.
Interdonato, R., Rosa, M., Nieva, C.B., Gonzalez, J.A., Hilal, M., Prado, F.E., 2011. Effects
of low UV-B doses on the accumulation of UV-B absorbing compounds and total
phenolics and carbohydrate metabolism in the peel of harvested lemons. Environ.
Exp. Bot. 70 (2–3), 204–211. https://doi.org/10.1016/j.envexpbot.2010.09.006.
Lambert, M., Meudec, E., Verbaere, A., Mazerolles, G., Wirth, J., Masson, G.,
Cheynier, V., Sommerer, N., 2015. A high-throughput UHPLC-QqQ-MS method for
polyphenol profiling in rose wines. Molecules 20 (5), 7890–7914. https://doi.org/
10.3390/molecules20057890.
Li, Y., Ma, R.J., Xu, Z.Z., Wang, J.H., Chen, T., Chen, F., Wang, Z.F., 2013. Identification
and quantification of anthocyanins in Kyoho grape juice-making pomace, Cabernet
Sauvignon grape winemaking pomace and their fresh skin. J. Sci. Food Agric. 93 (6),
1404–1411. https://doi.org/10.1002/jsfa.5907.
Li, D., Limwachiranon, J., Li, L., Du, R.X., Luo, Z.S., 2016. Involvement of energy
metabolism to chilling tolerance induced by hydrogen sulfide in cold-stored banana
fruit. Food Chem. 208, 272–278. https://doi.org/10.1016/j.foodchem.2016.03.113.
Li, F.X., Li, F.H., Yang, Y.X., Yin, R., Ming, J., 2019a. Comparison of phenolic profiles and
antioxidant activities in skins and pulps of eleven grape cultivars (Vitis vinifera L.).
J. Integr. Agric. 18 (5), 1148–1158. https://doi.org/10.1016/s2095-3119(18)
62138-0.
Li, D., Zhang, X.C., Li, L., Aghdam, M.S., Wei, X.X., Liu, J.Q., Xu, Y.Q., Luo, Z.S., 2019b.
Elevated CO2 delayed the chlorophyll degradation and anthocyanin accumulation in
postharvest strawberry fruit. Food Chem. 285, 163–170. https://doi.org/10.1016/j.
foodchem.2019.01.150.
Liang, Z.C., Wu, B.H., Fan, P.G., Yang, C.X., Duan, W., Zheng, X.B., Liu, C.Y., Li, S.H.,
2008. Anthocyanin composition and content in grape berry skin in Vitis germplasm.
Food Chem. 111 (4), 837–844. https://doi.org/10.1016/j.foodchem.2008.04.069.
Liang, C.M., Yang, B., Wei, Y., Zhang, P.F., Wen, P.F., 2021. SA incubation induced
accumulation of flavan-3-ols through activated VvANR expression in grape leaves.
Sci. Hortic. 287. https://doi.org/10.1016/j.scienta.2021.110269.
Liu, Y., Shi, Z., Maximova, S., Payne, M.J., Guiltinan, M.J., 2013. Proanthocyanidin
synthesis in Theobroma cacao: genes encoding anthocyanidin synthase,
anthocyanidin reductase, and leucoanthocyanidin reductase. BMC Plant Biol. 13,
202. https://doi.org/10.1186/1471-2229-13-202.
Locatelli, M., Travaglia, F., Coisson, J.D., Bordiga, M., Arlorio, M., 2016. Phenolic
composition of Nebbiolo grape (Vitis vinifera L.) from Piedmont: characterization
during ripening of grapes selected in different geographic areas and comparison with
Uva Rara and Vespolina cv. Eur. Food Res. Technol. 242 (7), 1057–1068. https://
doi.org/10.1007/s00217-015-2610-z.
Mori, K., Goto-Yamamoto, N., Kitayama, M., Hashizume, K., 2007. Loss of anthocyanins
in red-wine grape under high temperature. J. Exp. Bot. 58 (8), 1935–1945. https://
doi.org/10.1093/jxb/erm055.
Nedomova, S., Kumbar, V., Pavlousek, P., Pytel, R., Lampir, L., Buchar, J., 2017. Effect of
harvest date on composition and geometry of grape berries. Eur. J. Hortic. Sci. 82
(1), 21–30. https://doi.org/10.17660/eJHS.2017/82.1.3.
M. Qi et al.
Noguerol-Pato, R., Gonzalez-Barreiro, C., Cancho-Grande, B., Santiago, J.L., Martinez, M.
C., Simal-Gandara, J., 2012. Aroma potential of Brancellao grapes from different
cluster positions. Food Chem. 132 (1), 112–124. https://doi.org/10.1016/j.
foodchem.2011.10.042.
Orlandi, G., Calvini, R., Foca, G., Pigani, L., Simone, G.V., Ulrici, A., 2019. Data fusion of
electronic eye and electronic tongue signals to monitor grape ripening. Talanta 195,
181–189. https://doi.org/10.1016/j.talanta.2018.11.046.
Pantelic, M.M., Dabic Zagorac, D., Davidovic, S.M., Todic, S.R., Beslic, Z.S., Gasic, U.M.,
Tesic, Z.L., Natic, M.M., 2016. Identification and quantification of phenolic
compounds in berry skin, pulp, and seeds in 13 grapevine varieties grown in Serbia.
Food Chem. 211, 243–252. https://doi.org/10.1016/j.foodchem.2016.05.051.
Pastrana-Bonilla, E., Akoh, C.C., Sellappan, S., Krewer, G., 2003. Phenolic content and
antioxidant capacity of muscadine grapes. J. Agric. Food Chem. 51 (18), 5497–5503.
https://doi.org/10.1021/jf030113c.
Perez-Navarro, J., Miguel Izquierdo-Canas, P., Mena-Morales, A., Martinez-Gascuena, J.,
Luis Chacon-Vozmediano, J., Garcia-Romero, E., Hermosin-Gutierrez, I., Gomez-
Alonso, S., 2019. Phenolic compounds profile of different berry parts from novel Vitis
vinifera L. Red grape genotypes and Tempranillo using HPLC-DAD-ESI-MS/MS: a
varietal differentiation tool. Food Chem. 295, 350–360. https://doi.org/10.1016/j.
foodchem.2019.05.137.
Radovanovic, A., Radovanovic, B., Jovancicevic, B., 2009. Free radical scavenging and
antibacterial activities of southern Serbian red wines. Food Chem. 117 (2),
326–326331. https://doi.org/10.1016/j.foodchem.2009.04.008.
Romero, I., Vazquez-Hernandez, M., Maestro-Gaitan, I., Escribano, M.I., Merodio, C.,
Sanchez-Ballesta, M.T., 2020. Table Grapes during postharvest storage: a review of
the mechanisms implicated in the beneficial effects of treatments applied for quality
retention. Int. J. Mol. Sci. 21 (23) https://doi.org/10.3390/ijms21239320.
Rutan, T.E., Herbst-Johnstone, M., Kilmartin, P.A., 2018. Effect of cluster thinning Vitis
vinifera cv. Pinot Noir on wine volatile and phenolic composition. J. Agric. Food
Chem. 66 (38), 10053–10066. https://doi.org/10.1021/acs.jafc.8b04062.
Šamec, D., Piljac-Žegarac, J., 2011. Postharvest stability of antioxidant compounds in
hawthorn and cornelian cherries at room and refrigerator
temperatures—comparison with blackberries, white and red grapes. Sci. Hortic. 131
(1), 15–21. https://doi.org/10.1016/j.scienta.2011.09.021.
Savitch, L.V., Pocock, T., Krol, M., Wilson, K.E., Greenberg, B.M., Huner, N.P.A., 2001.
Effects of growth under UVA radiation on CO2 assimilation, carbon partitioning, PSII
photochemistry and resistance to UVB radiation in Brassica napus cv. Topas. Aust. J.
Plant Physiol. 28 (3), 203–212. https://doi.org/10.1071/pp99116.
Serratosa, M.P., Marquez, A., Moyano, L., Zea, L., Merida, J., 2014. Chemical and
morphological characterization of Chardonnay and Gewurztraminer grapes and
12
Journal of Food Composition and Analysis 106 (2022) 104353
changes during chamber-drying under controlled conditions. Food Chem. 159,
128–136. https://doi.org/10.1016/j.foodchem.2014.02.167.
Silva, A.S., Reboredo-Rodriguez, P., Suntar, I., Sureda, A., Belwal, T., Loizzo, M.R.,
Tundis, R., Sobarzo-Sanchez, E., Rastrelli, L., Forbes-Hernandez, T.Y., Battino, M.,
Filosa, R., Daglia, M., Nabavi, S.F., Nabavi, S.M., 2020. Evaluation of the status quo
of polyphenols analysis: part I-phytochemistry, bioactivity, interactions, and
industrial uses. Compr. Rev. Food Sci. Food Saf. 19 (6), 3191–3218. https://doi.org/
10.1111/1541-4337.12629.
Souquet, J.M., Labarbe, B., Le Guerneve, C., Cheynier, V., Moutounet, M., 2000. Phenolic
composition of grape stems. J. Agric. Food Chem. 48 (4), 1076–1080. https://doi.
org/10.1021/jf991171u.
Walker, T.L., Morris, J.R., Threlfall, R.T., Main, G.L., Lamikanra, O., Leong, S., 2001.
Density separation, storage, shelf life, and sensory evaluation of’ Fry’ muscadine
grapes. Hortscience 36 (5), 941–945. https://doi.org/10.21273/HORTSCI.36.5.941.
Wang, L., Luo, Z.S., Yang, M.Y., Li, D., Qi, M., Xu, Y.Q., Abdelshafy, A.M., Ban, Z.J.,
Wang, F.Z., Li, L., 2020. Role of exogenous melatonin in table grapes: first evidence
on contribution to the phenolics-oriented response. Food Chem. 329, 127155
https://doi.org/10.1016/j.foodchem.2020.127155.
Wei, Z.F., Luo, M., Zhao, C.J., Li, C.Y., Gu, C.B., Wang, W., Zu, Y.G., Efferth, T., Fu, Y.J.,
2013. UV-Induced Changes of active components and antioxidant activity in
postharvest pigeon pea Cajanus cajan (L.) Millsp. Leaves. J. Agric. Food Chem. 61
(6), 1165–1171. https://doi.org/10.1021/jf304973f.
Xia, X.J., Zhou, Y.H., Shi, K., Zhou, J., Foyer, C., Yu, J.Q., 2015. Interplay between
reactive oxygen species and hormones in the control of plant development and stress
tolerance. J. Exp. Bot. 66 (10), 2839–2856. https://doi.org/10.1093/jxb/erv089.
Xu, H.J.L., Wu, P.W., Chen, R.C., Liu, Y.M., Chen, C.K., Tian, H.Q., Zhu, B.Z., 2018. The
influence of storage temperature on post-harvest physiology and storage quality of
Kyoho grape. Food Res. Dev. 39 (21), 192–197. https://doi.org/10.3969/j.
issn.1005-6521.2018.21.032.
Yang, M.Y., Wang, L., Belwal, T., Zhang, X.C., Lu, H.Y., Chen, C.K., Li, L., 2020.
Exogenous melatonin and abscisic acid expedite the flavonoids biosynthesis in grape
berry of Vitis vinifera cv. Kyoho. Molecules 25 (1). https://doi.org/10.3390/
molecules25010012.
Zhang, C., Jia, H.F., Wu, W.M., Wang, X.C., Fang, J.G., Wang, C., 2015. Functional
conservation analysis and expression modes of grape anthocyanin synthesis genes
responsive to low temperature stress. Gene 574 (1), 168–177. https://doi.org/
10.1016/j.gene.2015.08.003.
Zhou, Y., Su, P., Yin, H., Dong, Z., Yang, L., Yuan, C., 2019. Effects of different harvest
times on the maturity of polyphenols in two red wine grape cultivars (Vitis vinifera
L.) in Qingtongxia (China). South Afr. J. Enol. Vitic. 40 (1), 1–12. https://doi.org/
10.21548/40-1-2770.