Postharvest Biology and Technology 161 (2020) 111081

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Postharvest Biology and Technology

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

Effect of cutting and storage temperature on sucrose and organic acids T

metabolism in postharvest melon fruit
Zhangfei Wua, Mingmei Tua, Xingping Yangb, Jinhua Xub, Zhifang Yua,*
a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China
b
Institute of Vegetable Science, Jiangsu Academy of Agricultural Sciences, Jiangsu, 210095, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: In order to reveal the molecular information of soluble sugars and organic acids both in stored fresh-cut and
Fresh-cut melon whole melon fruits, the enzymes and genes involved in the metabolism of sucrose and organic acids in melon
Sucrose fruit (cv. Xizhoumi-17) were investigated. Cutting of fruit and storage temperature significantly affected the
Organic acid content of sugars, citric acid (CA) and malic acid (MA), and the change was controlled by enzymes involved in
Metabolism
the metabolism of sucrose and organic acids. Fresh-cut melon had higher content of hexose (fructose and glu-
Temperature
cose) and organic acids when stored at 15 °C for a short period of time, and this was correlated with higher
activities of acid invertase (AI), neutral invertase (NI), sucrose synthase-cleavage (SS-c), phosphoenolpyruvate
carboxylase (PEPC), citrate synthase (CS), aconitase (ACO), malate dehydrogenase (MDH) and malic enzyme
(ME) and lower activity of isocitrate dehydrogenase (IDH). These patterns were due to the up-regulation of
CmAI1/2, CmNI3, CmPEPC3, CmCS1, CmACO1/2, CmMDH1/3 and CmME3/4 and down-regulation of CmIDH1 in
fresh-cut melon. Compared with fresh-cut melon stored at 15 °C, lower temperature (5 °C) significantly extended
the shelf life and reduced the quality loss as indicated by higher content of sucrose, CA and MA in fresh-cut fruit
stored at 5 °C. These differences resulted from the up-regulation of CmPEPC2/3 and down-regulation of CmAI1/
2, CmNI3, CmCS1, CmACO1/2 and CmME3/4 during the storage period.

1. Introduction
Melon (Cucumis melo L.) is one of the most popular fruits, and is
cultivated around the world. Fresh-cut melon is a common product
found in stores, where the fruit is washed, peeled, packaged, and sold in
ready-to-eat packages, though this process leads to shorter shelf life
(Aguayo et al., 2008; Lester and Saftner, 2008). The sensory qualities of
fresh-cut melon including firmness, flesh color, etc. can quickly dete-
riorate due to increased respiratory rate and ethylene release (Lester
and Saftner, 2008; Saftner et al., 2006). However, lower temperature
can prolong the storage life of fresh-cut fruits and vegetables by redu-
cing respiration and metabolic activity, water loss and microbial
growth (Lamikanra and Richard, 2002).
Melon contains bioactive substances that are important for human
health, including carotenoid, ascorbic acid, vanillic acid, trans-cin-
namic acid, and benzoic acid (Falah et al., 2015; Kolaylı et al., 2010).
The sugar and organic acid content of melon fruit determines its taste
(Mccollum, 1988). For this reason, the accumulation and changes in
sugars and organic acids in the fruit is an important predictor of fruit
sensory quality. Previous studies on melon (Huber and Pharr, 1989),
tomato (Kortstee et al., 2007), strawberry (Souleyre et al., 2010) and
peach (Yu et al., 2017) showed that fruit sugar content was regulated by
sucrose metabolism.
Although there are many organic acid components in fruit, such as
citric acid (CA), malic acid (MA), and tartaric acid, most fruits are
usually dominated by one or two organic acids, while others are only
present in small amounts. The main organic acid in melon is CA, fol-
lowed by MA and succinic acid (Burger et al., 2003), while the pre-
dominant organic acid in orange-flesh cantaloupes (cv. Athena and Sol
Real) is succinic acid (Beaulieu et al., 2003). The acid content of the
fruit is determined by the equilibrium of acid synthesis and degrada-
tion. While many studies have identified the enzymes involved in the
metabolism of organic acids, it is still unclear how these proteins and
genes are regulated in response to cutting of the fruit.
In melon fruit, sucrose phosphate synthase (SPS) activity determines
final sucrose concentration during fruit development and ripening
(Huber and Pharr, 1989), and a single recessive gene controls the ac-
tivity of SPS (Burger et al., 2003). Similar to sucrose concentration,
high acidity in melon fruit is attributed to related genes, but there is no
single enzyme that correlates with CA accumulation in melon fruit

⁎
Corresponding author.
E-mail address: yuzhifang@njau.edu.cn (Z. Yu).

https://doi.org/10.1016/j.postharvbio.2019.111081
Received 26 April 2019; Received in revised form 22 November 2019; Accepted 26 November 2019
0925-5214/ © 2019 Elsevier B.V. All rights reserved.
Z. Wu, et al.
development (Tang et al., 2010). At present, most studies have focused
on sensory evaluation, physiological and biochemical changes and the
application of various storage techniques in melon fruit during storage.
Changes in content of soluble sugars and organic acids have been re-
ported in fresh-cut fruits (Cavaiuolo et al., 2015; Guerreiro et al., 2017).
However, the molecular mechanism underlying the changes in meta-
bolites in the melon fruit after cutting remains unclear. Consequently,
this study aimed to explore the effect of cutting and storage tempera-
ture on the metabolism of sucrose and organic acids in whole and fresh-
cut melon fruit.
2. Materials and methods
2.1. Fruit samples and experimental design
The melon fruit “Xizhoumi-17” (Cucumis melo L. var. reticulatus
Nand.) at commercially mature stage was picked on October 14, 2017
in Xinghua, Jiangsu Province, China, and was transported immediately
after harvesting to the laboratory at Nanjing Agricultural University.
Fruits were held at 21 °C for 24 h. Fruits that were uniform in size,
shape, and without mechanical damage were selected for the study and
divided into two groups. One group was used as whole melon sample,
and the other was used for fresh-cut samples. For the fresh-cut samples,
melons were manually peeled and cut into 2.0 × 2.0 × 2.0 cm cubes
and packaged into rigid plastic boxes (17.2 × 12.0 × 6.8 cm,
120 ± 5 g fresh-cut cubes per box).
Whole melon fruits were stored at 15 °C (WF 15 °C) and sampled on
days 2, 4, 8 and 12. Fresh-cut melon cubes were stored at 5 °C (FC 5 °C)
and sampled on days 1, 2, 4 and 8. Fresh-cut melon cubes were also
stored at 15 °C (FC 15 °C) and sampled on days 1, 2, 3 and 4. Lower
temperatures would be more suitable for melon fruit storage. However,
melon is very chill-sensitive and shows chilling injuries when stored at
4–6 °C (Fogelman et al., 2011). Thus, the storage temperature (15 °C)
used in this study is not recommended to protect the quality of fresh-cut
melon fruit in industry, as it only provides the shelf life of 4 days for
fresh-cut melon (Falah et al., 2015). It was applied only to expedite the
response to wounding stress (Fernando Reyes et al., 2007; Li et al.,
2017). At each time point, 10 fruit samples were taken for analysis from
each group, and all samples were collected in triplicate. The pulp from
the center of the melon tissue that was sampled was immediately frozen
in liquid nitrogen and samples were stored at -80 °C for further analysis.
2.2. Determination of total soluble solids (TSS), titratable acid (TA), pH
and firmness
Firmness was measured according to the procedure reported by
Chen et al. (2017) and expressed as Newton (N), where 10 melon cubes
were measured at the selected time points. Fresh 50 g samples from 10
melon cubes were ground and filtered with four layers gauze, and the
filtrate was used for determination of TSS, TA concentration and pH.
The pH was measured with a pH meter (METTLER TOLEDO FE 20). TSS
and TA were determined using a Pocket Brix-acidity Meter (Atago, PAL-
BX/ACID 5, Tokyo, Japan), and expressed as percentage of Brix and
percentage of MA (%), respectively.
2.3. Determination of soluble sugars and organic acids
Tissues (20.0 g) from 10 melon fruit were ground in liquid nitrogen,
and the resulting powder (2.0 g) was placed in a test tube and dissolved
with 20 mL of deionized water and then kept at 80 °C water bath for
30 min. After filtration, the supernatant was collected, and deionized
water was added to 25 mL. The diluted supernatant was passed through
a 0.45-μm filter and used to determine the soluble sugars and organic
acids content.
Fructose, glucose and sucrose content was measured following the
procedure given by Yu et al. (2017) with slight modifications.
2
Postharvest Biology and Technology 161 (2020) 111081
Quantitative measurements of sugars content were obtained using high
performance liquid chromatography (HPLC) system (Model 2695, Wa-
ters, USA) equipped with a InertSustain NH2 Column (5 μm,
4.6 × 250 mm, GL Sciences, Japan) and a refractive index detector
(Model 2414, Waters, USA). For each sample, 20 μL of the diluted su-
pernatant was injected into the HPLC system for analysis. Column
temperature was 30 °C and acetonitrile/water (80:20, v/v) was used as
the mobile phase at a flow rate of 1.0 mL min−1.
Organic acids content was measured using an HPLC system (Model
LC-20A, GL Sciences, Japan) equipped with a ZORBAX SB-Aq C18
column (5 μm, 4.6 × 250 mm, Shimadzu, Japan) following the method
of Covarrubias and Rombolà (2015). HPLC analysis was conducted
using 20 μL of the diluted supernatant. Phosphate buffer (25 mM, pH
2.4) was used as the mobile phase at a flow rate of 0.5 mL min−1 at
30 °C, and the organic acids were detected at a wavelength of 210 nm
by a photodiode array detector.
The soluble sugars and organic acids were identified and quantified
through comparison of retention times and peak areas with standard
substances, and the results were expressed as g kg−1 fresh weight.
2.4. Extraction and determination of sucrose and organic acids metabolism-
related enzymes
Enzymes involved in sucrose metabolism were extracted following
the procedure proposed by Zhang et al. (2013) with slight modifica-
tions. All procedures were conducted at 4 °C. Melon powder (2.0 g) was
homogenized with 5 mL of 100 mM Tris-HCl buffer solution (pH 7.2)
containing 5 mM MgCl2, 2 mM ethylenediaminetetraacetic acid
(EDTA), 2 % (v/v) ethylene glycol, 0.2 % (w/v) bovine serum albumin
(BSA), 2 % polyvinyl pyrrolidone and 5 mM dithiothreitol. The homo-
genate was centrifuged at 8000 g for 30 min. The supernatant was
dialyzed immediately with 10-fold volumes of the extraction buffer
without BSA and polyvinyl pyrrolidone for 24 h at 4 °C. The supernatant
(crude enzyme extract) was used to determine the activity of enzymes
involved in sucrose metabolism following the procedure given by Yu
et al. (2017) with slight modifications.
Acid invertase (AI) activity was determined in a reaction mixture
that consisted of 0.05 mL of the crude enzyme extract and 0.95 mL of
100 mM sodium citrate buffer (pH 4.7, containing 50 mM sucrose). The
mixture was incubated for 10 min at 37 °C and immediately placed in a
boiling water bath for 3 min to stop the reaction. After cooling the
samples down to room temperature, the production of glucose was
measured according to the method of Zhang et al. (2013). Neutral in-
vertase (NI) activity assay procedure was similar to that of AI, except
that 100 mM sodium phosphate buffer (pH 7.0) was used as the reaction
mixture. Sucrose synthase-cleavage (SS-c) activity was determined in a
reaction mixture consisting of 0.05 mL of the crude enzyme extract and
0.95 mL of 80 mM MES buffer (pH 5.5, containing 5 mM NaF, 100 mM
sucrose and 5 mM uridine diphosphate). Subsequent steps were as de-
scribed for AI. Enzymes that were inactivated by boiling were used as
controls for SS-c, NI and AI activity measurements. To determine su-
crose synthase-synthesis (SS-s) activity, the reaction mixture consisting
of 0.05 mL of crude enzyme extract, 0.5 mL of 100 mM Tris-HCl buffer
(pH 7.0, containing 10 mM uridine diphosphate glucose, 10 mM fruc-
tose, 5 mM MgCl2 and 5 mM dithiothreitol) and 0.45 mL of deionized
water was incubated for 10 min at 37 °C. The reaction mixture was then
immediately placed in a boiling water bath for 3 min to terminate the
reaction. After cooling the samples down to room temperature, the
production of sucrose was measured following the method of Zhang
et al. (2013). The SPS assay procedure was similar to that of SS-s, except
10 mM fructose-6-phosphate was used instead of fructose in the reac-
tion mixture. Deionized water was used as the control for SPS and SS-s
activity measurements instead of uridine diphosphate glucose in the
reaction mixture. One U was defined as the amount of enzyme that
produced 1 μmol glucose or sucrose per hour and activities of enzymes
were expressed as U mg−1 based on protein content.
Z. Wu, et al.
Melon powder (2.0 g) was homogenized using 3 mL of 200 mM Tris-
HCl buffer (pH 8.2, containing 10 mM isoascorbic acid, 600 mM su-
crose), and enzymes involved in organic acids metabolism were ob-
tained following the method reported by Tang et al. (2010). Enzymes
involved in organic acids metabolism, including phosphoenolpyruvate
carboxylase (PEPC), citrate synthase (CS), isocitrate dehydrogenase
(IDH), malic enzyme (ME) and malate dehydrogenase (MDH) were
measured according to the method reported by Tang et al. (2010) and
aconitase (ACO) was measured following the method of Yang et al.
(2011). The activities of enzymes were expressed as U mg−1 based on
protein content. One U was defined as the amount of enzyme that
produced 1 nmol oxidation or reduction product per minute.
The protein content of crude enzyme extract was determined using a
Bradford assay (Bradford, 1976), with BSA as the standard.
2.5. RNA isolation and mRNA expression analysis
Genes involved in sucrose and organic acids metabolism were
identified from CuGenDB (http://cucurbitgenomics.org/). Total RNA
was extracted from each sample using the RNAprep Pure Plant Kit
(DP441, TIANGEN, China) following the manufacturer’s instructions.
First-strand cDNA was synthesized using the PrimeScript™ RT Master
Mix (RR036A, TaKaRa, Japan) following the manufacturer’s instruc-
tions. Gene-specific primers (Table S1) were designed with Primer 5.0.
Real-time quantitative reverse transcription PCR (qRT-PCR) was used to
analyze expression with the Applied Biosystems 7500 Fast Real-Time
PCR System (Applied Biosystems, USA) using the SYBR® Premix Ex
Taq™ (RR420A, TaKaRa, Japan) in a 20 μL system. qRT-PCRs were
performed using a thermo cycling method that was set with an initial
denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and
60 °C for 1 min. Gel-electrophoretic and melting curves were used to
confirm the specificity of primers. Relative gene expression was cal-
culated using the “Comparative 2−ΔΔCT” method with β-actin as the
reference gene (Livak and Schmittgen, 2001). Data were double-nor-
malized, first against the expression levels of β-actin and then to values
at day 0, the gene expression level of melon at day 0 was set as 1 (Chen
et al., 2017; Mitalo et al., 2019).
2.6. Statistical analysis
Data were expressed as means ± standard error (SE) of three re-
plicates and analyzed by the one-way analysis of variance (ANOVA)
using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) with cutting, storage
temperature and storage period as the factors. The mean separations
were analyzed using Duncan’s multiple range test and differences were
considered to be statistically significant when p < 0.05. Data were
analyzed by Pearson correlation analysis using SPSS 17.0 and analyzed
by heatmap using Origin Pro 2018 (Origin Lab, USA).
3. Results
3.1. Firmness, pH, TSS and TA
Firmness of whole melon at 15 °C and fresh-cut melon at 5 °C re-
mained similar during the storage period, though it declined gradually
(Fig. S1A). The firmness of fresh-cut melon stored at 15 °C declined
sharply after two days and was significantly lower than the other two
groups after longer storage periods (Fig. S1A, p ＜ 0.05).
The pH of whole melon stored at 15 °C changed slightly with storage
time. The pH of fresh-cut melon at 5 °C increased after a day of storage,
followed by a decrease and then remained at a similar level as whole
melon. However, the pH of fresh-cut melon at 15 °C was significant
lower than that of the other two samples after day 3 (Fig. S1B, p ＜
0.05).
TSS concentration of whole melon changed slightly during storage.
However, TSS concentration of fresh-cut melon at 15 °C was
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Postharvest Biology and Technology 161 (2020) 111081
significantly lower than that of the other two groups after day 2 (Fig.
S1C, p ＜ 0.05).
Significant decrease in TA concentration was observed in whole
melon during the storage period, the decline was rapid earlier in sto-
rage, and the change was less dramatic after day 2. Unlike whole melon,
fresh-cut melon showed an initial increase in TA concentration followed
by a rapid decrease at 5 °C and 15 °C. The TA concentration of fresh-cut
melon stored at 15 °C declined earlier and quicker than at 5 °C and was
higher during the initial two days than that of whole melon (Fig. S1D, p
＜ 0.05).
3.2. Changes in soluble sugars and organic acids content
To understand how cutting and storage temperature affect the so-
luble sugars content of melon fruit, sucrose, fructose and glucose levels
were measured. Fructose content in whole melon increased during the
initial eight days of storage and then declined sharply. Moreover,
fructose content increased at day 1 in fresh-cut melon stored at 5 and
15 °C, followed by a significant decrease after longer storage time.
However, fructose content in fresh-cut melon stored at 15 °C was higher
than that in whole fruit at the earlier stage of storage and had no sig-
nificant change compared to samples stored at 5 °C (Fig. 1A, p ＜ 0.05).
Glucose content in the three groups showed similar trends as fructose
(Fig. 1B). Interestingly, changes in sucrose content of stored whole and
fresh-cut melon were opposite of the changes in hexose content (fruc-
tose and glucose) (Fig. 1C, p ＜ 0.05).
The determination of organic acid content showed that the principal
organic acid components in the melon fruit “Xizhoumi-17” were CA and
MA. CA and MA content in whole melon declined during the initial 2
days of storage, and then their levels remained steady. Compared with
whole melon, fresh-cut melon stored at 15 °C had similar changes in MA
and CA content during storage, and their levels remained higher during
shorter storage periods. Interestingly, in the later stage of storage, CA
and MA content of fresh-cut melon stored at 5 °C was higher than that of
the other two groups (Fig. 2, p ＜ 0.05).
3.3. Activities of key enzymes involved in sucrose and organic acids
metabolism
Activities of enzymes involved in sucrose and organic acids meta-
bolism were altered in whole and fresh-cut melon fruit by cutting and
storage temperature (Figs. 3 and 4).
All three groups showed similar changes in AI activity during the
storage (Fig. 3A). AI activity of whole melon significantly increased at
first, and then decreased. Compared to whole melon, AI activity of
fresh-cut melon stored at 15 °C was higher earlier during storage, and
lower at the end of storage. In addition, the increase in AI activity in
fresh-cut melon was suppressed by lower temperature and AI activity in
fresh-cut melon stored at 5 °C was consistently lower than that stored at
15 °C.
The activity of NI in whole melon declined gradually during storage.
In comparison, cutting enhanced the NI activity in melon fruit during
storage, while lower temperature suppressed the increase in NI activity
of fresh-cut melon (Fig. 3B, p ＜ 0.05).
SS-c activity increased and then decreased in whole and fresh-cut
melon during storage (Fig. 3C). In comparison with whole melon, SS-c
activity in fresh-cut melon stored at 15 °C was higher at the earlier stage
of storage, and was lower after longer periods of storage. However, the
decrease in SS-c activity after longer storage was greater at 5 °C com-
pared to 15 °C.
SS-s activity in whole melon declined at the beginning of storage
and remained stable over longer storage periods, and similar changes
were observed in fresh-cut melon stored at 5 °C and 15 °C. SS-s activity
was generally similar across the three groups during the storage
(Fig. 3D, p ＜ 0.05).
Changes in SPS activity were similar for the three groups (Fig. 3E).
Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 1. Effect of cutting and storage temperature on the content of fructose (A), glucose (B) and sucrose (C) in melon fruit during storage. FC 5 °C: fresh-cut melon
fruit stored at 5 °C; FC 15 °C: fresh-cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. The values are expressed as means ± SE of three
replicates. Values with different letters indicate statistically significant differences (p ＜ 0.05). Lowercase letters indicated significant difference among FC 5 °C, FC
15 °C and WF 15 °C groups at the same time point, capital letters represented significant difference among storage time factors.

SPS activity in whole melon decreased gradually during storage.
However, SPS activity was consistently lower in fresh-cut melon stored
at 15 °C compared to whole melon, and loss of SPS activity of fresh-cut
melon was accelerated at lower temperature (p ＜ 0.05).
PEPC activity of whole melon remained stable during the storage
period (Fig. 4A). However, PEPC activity of fresh-cut melon stored at
15 °C increased earlier during storage and then decreased. Moreover,
PEPC activity was higher in fresh-cut melon compared to whole melon
at the earlier stage of storage. In addition, the pattern of change in PEPC
activity was similar for fresh-cut melon samples stored at 5 °C and 15 °C.

Fig. 2. Effect of cutting and storage temperature on the content of citric acid (A) and malic acid (B) in melon fruit during storage. FC 5 °C: fresh-cut melon fruit stored
at 5 °C; FC 15 °C: fresh-cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. The value are expressed as means ± SE of three replicates. Values
with different letters indicate statistically significant differences (p ＜ 0.05). Lowercase letters indicated significant difference among FC 5 °C, FC 15 °C and WF 15 °C
groups at the same time point, capital letters represented significant difference among storage time factors.

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Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 3. Effect of cutting and storage temperature on the activities of (A) acid invertase (AI), (B) neutral invertase (NI), (C) Sucrose synthase-cleavage (SS-c), (D)
sucrose synthase-synthesis (SS-s) and (E) sucrose phosphate synthase (SPS) in melon fruit during storage. FC 5 °C: fresh-cut melon fruit stored at 5 °C; FC 15 °C: fresh-
cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. The values are expressed as means ± SE of three replicates. Values with different letters
indicate statistically significant differences (p ＜ 0.05). Lowercase letters indicated significant difference among FC 5 °C, FC 15 °C and WF 15 °C groups at the same
time point, capital letters represented significant difference among storage time factors.

PEPC activity of fresh-cut melon stored at 5 °C was higher compared to
15 °C after day 1.
CS activity in the three groups declined with storage time (Fig. 4B).
Throughout the entire storage period, CS activity of fresh-cut melon
stored at 15 °C was higher than that of whole melon, and lower than
that of fresh-cut melon stored at 5 °C.
ACO activity decreased at first and remained stable during the later
storage periods. Cutting the melon fruit reduced the decrease in ACO
activity earlier during storage, and led to accelerated decline in ACO
activity later in storage. In addition, storage at 5 °C for two days re-
duced ACO activity of fresh-cut melon compared to the samples stored
at 15 °C (Fig. 4C).
IDH activity was reduced during storage in the three groups. IDH
activity of fresh-cut melon stored at 15 °C was lower than that of whole
melon and higher than that of fresh-cut melon stored at 5 °C until the
end of storage (Fig. 4D, p ＜ 0.05).
MDH activity of whole melon remained stable during storage, while
cutting and lower temperature enhanced the MDH activity of melon
fruit. In addition, over longer storage periods, MDH activity of fresh-cut
melon stored at 5 °C was higher than that of the samples stored at 15 °C
(Fig. 4E, p ＜ 0.05).
ME activity increased slowly in whole melon during the storage, and
cutting of melon enhanced ME activity at the earlier stage of storage.
However, over longer storage periods, ME activity was lower in fresh-
cut melon stored at 15 °C compared to whole melon. Moreover, ME
activity was lower in fresh-cut melon at the earlier stage of storage of
5 °C compared to samples stored at 15 °C, and no significant difference
was found at the end of storage between both groups (Fig. 4F, p ＜
0.05).
3.4. Expression of genes involved in sucrose and organic acids metabolism
To understand the molecular mechanisms behind the changes in
sucrose and organic acid levels in melon fruit during storage, the ex-
pression of genes involved in sucrose and organic acids metabolism was
measured (Figs. 5 and 6).
The expression pattern of CmAI1/2 was similar across the three
groups. However, compared to whole melon, CmAI1/2 expression in
fresh-cut melon at 15 °C was up-regulated during the early stage of
storage and was down-regulated at the end of storage. Moreover,
CmAI1/2 expression in fresh-cut melon was inhibited by storage at 5 °C
(Fig. 5, p ＜ 0.05).
Fig. 5 shows that cutting stimulated the expression of CmNI3 when
melon fruit was stored at 15 °C. Compared to whole fruit, fresh-cut
melon also had higher expression level of CmNI1 at day 2 of storage,
despite CmNI2 level being similar at this stage. Storage of fresh-cut
melon at lower temperature affected the expression of CmNI1/2/3
differently. However, CmNI3 was the only gene that was down-regu-
lated in response to storage at lower temperature.
Cutting showed varying effects on the expression of SS genes
(CmSS1/2/3) in melon fruit during storage at 15 °C. The expression of
CmSS1 in fresh-cut melon was increased, while cutting of melon de-
creased the CmSS3 expression and slightly affected the CmSS2 expres-
sion. Lower temperature increased the expression of CmSS1 in fresh-cut
melon and reduced the expression of CmSS2/3.
The patterns of changes in the expression of SPS genes (CmSPS1/2/
3/4) were similar among the three groups. CmSPSs expression in fresh-
cut melon during storage at 15 °C was decreased in comparison with
whole melon. Compared with fresh-cut melon stored at 15 °C, expres-
sion of CmSPS1/3 in samples stored at 5 °C was only lower during the

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Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 4. Effect of cutting and storage temperature on the activities of (A) phosphoenolpyruvate carboxylase (PEPC), (B) citrate synthase (CS), (C) aconitase (ACO), (D)
isocitrate dehydrogenase (IDH), (E) malate dehydrogenase (MDH) and (F) malic enzyme (ME) in melon fruit during storage. FC 5 °C: fresh-cut melon fruit stored at
5 °C; FC 15 °C: fresh-cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. The values are expressed as means ± SE of three replicates. Values
with different letters indicate statistically significant differences (p ＜ 0.05). Lowercase letters indicated significant difference among FC 5 °C, FC 15 °C and WF 15 °C
groups at the same time point, capital letters represented significant difference among storage time factors.

early stage of storage. Moreover, expression of CmSPS4 was down-
regulated after longer storage periods, while CmSPS2 expression was
higher throughout the entire storage period.
Expression of PEPC genes (CmPEPC1/2/3) in whole melon changed
slightly during the storage period. CmPEPC3 expression in fresh-cut
melon was increased and higher than that in whole melon during the
storage period at 15 °C. Unlike CmPEPC3, the CmPEPC1 expression was
decreased in fresh-cut melon compared to whole melon, and CmPEPC2
expression remained at a similar level during the storage. Interestingly,
storage at 5 °C increased CmPEPC2/3 expression and decreased
CmPEPC1 expression in fresh-cut melon (p ＜ 0.05).
CmCS1 expression was consistently higher in fresh-cut melon stored
at 15 °C compared to whole melon, and storage at 5 °C led to a decrease
in CmCS1 expression in fresh-cut melon until day 2. Compared to whole
melon, CmACO1/2 expression in fresh-cut melon was higher during the
earlier stage of storage and CmACO1/2 expression was lower when
fresh-cut melon was stored for a longer period of time. However, lower
temperature reduced the expression of CmACO1/2 in fresh-cut melon at
the early stage of storage (p ＜ 0.05). The expression of CmIDH1 de-
clined with storage time in the three groups. Furthermore, cutting and
lower temperature led to more dramatic reduction in CmIDH1 expres-
sion in melon fruit.
Transcript levels of MDH genes (CmMDH1/2/3) of whole melon
showed different expression patterns during storage. CmMDH1/3 ex-
pression in fresh-cut melon stored at 15 °C increased earlier during
storage and then decreased, and the higher expression levels were
maintained during storage compared to whole melon, while CmMDH2
was down-regulated during the storage. The expression of CmMDH1/3
in fresh-cut melon stored at 5 °C increased over longer storage periods.
Compared to fresh-cut melon stored at 15 °C, CmMDH1/3 expression in
fresh-cut melon stored 5 °C was lower during the earlier stage of sto-
rage, and its expression was higher in the later stage of storage.
However, the expression patterns of CmMDH2 and CmMDH1/3 showed
opposite patterns in samples stored at 5 °C (Fig. 6, p ＜ 0.05).
In whole melon, CmME1/2 expression decreased during storage,
and CmME3/4 expression declined during the initial 4 days of storage
and increased afterwards, while CmME5 expression increased and then
decreased during storage. Cutting the melon fruit enhanced the ex-
pression of CmME1/2/3/4, and reduced CmME5 expression. However,
CmMEs expression in fresh-cut melon was significantly suppressed at
5 °C compared with 15 °C (p ＜ 0.05).
3.5. Heatmap analysis of full-data in melon fruit
The heatmap and cluster analysis of full-data showed that the
quality of whole melon changed slightly during storage, even at 15 °C
(Fig. 7). However, the good quality of fresh-cut melon stored at 15 °C
was only maintained in the initial 2 days of storage, and the fruit
quality declined after day 3. Placing fresh-cut melon at lower tem-
perature maintained the quality of fruit for much longer. The heatmap
and cluster analysis showed that sucrose, CA, MA and PEPC exhibited
similar patterns of change during storage. The results suggest that
cutting may result in sucrose decomposition and hexose production in
melon fruit, further leading to high PEPC activity and higher levels of
organic acid. Storing fruit at 5 °C suppressed sucrose decomposition in
fresh-cut melon, but enhanced PEPC activity, which may have led to the
high organic acid content observed at lower temperature.

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Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 5. Effect of cutting and storage temperature on the relative expression profiles of acid invertase (CmAI1 and CmAI2), neutral invertase (CmNI1, CmNI2 and
CmNI3), sucrose synthase (CmSS1, CmSS2 and CmSS3) and sucrose phosphate synthase (CmSPS1, CmSPS2, CmSPS3 and CmSPS4) in melon fruit during storage. FC 5
°C: fresh-cut melon fruit stored at 5 °C; FC 15 °C: fresh-cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. Transcript levels were normalized
with respect to β-actin and were expressed relative to the value of each gene at day 0, which were set to 1. The values are expressed as means for three biological
replicates, with vertical bars indicating standard errors. Values with different letters indicate statistically significant differences (p ＜ 0.05). Lowercase letters
indicated significant difference among FC 5 °C, FC 15 °C and WF 15 °C groups at the same time point, capital letters represented significant difference among storage
time factors.

4. Discussion
4.1. Changes in sucrose and organic acids metabolism of melon fruit by
cutting
Cutting is a critical point in a processing line that divides intact
fruits and vegetables into smaller cubes or pieces and induces a number
of physiological changes which resist cut-wounding (Li et al., 2017).
However, very little is understood on how cutting affects different
molecular mechanisms in the fruit regarding sugars and organic acids.
In this study, we explored whether cutting and storage temperature
altered the function of enzymes and genes involved in sucrose and
organic acids metabolism, and ultimately how these changes may lead
to differences in sucrose and organic acid content of melon fruit “Xiz-
houmi-17”. Based on our findings, there was low commercial value for
fresh-cut melon after two days of storage at 15 °C, as these samples had
lower firmness, TSS and pH, suggesting that cutting led to tissue
breakdown and changes in metabolic, physicochemical and textural
aspects of the fruit.
Plants synthesize and accumulate various substances to retain cell
osmotic potential, maintain cell pressure and membrane integrity under
stress (Bartels and Nelson, 2010; Sicher, 2011). The accumulation of
carbohydrates is one of the most important responses of plants to
abiotic stress (Bhaskar et al., 2010; Yu et al., 2017). Bogaart et al.

7
Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 6. Effect of cutting and storage temperature on the relative expression profiles of phosphoenolpyruvate carboxylase (CmPEPC1, CmPEPC2 and CmPEPC3), citrate
synthase (CmCS1), aconitase (CmACO1 and CmACO2), isocitrate dehydrogenase (CmIDH1), malate dehydrogenase (CmMDH1, CmMDH2 and CmMDH3) and malic
enzyme (CmME1, CmME2, CmME3, CmME4 and CmME5) in melon fruit during storage. FC 5 °C: fresh-cut melon fruit stored at 5 °C; FC 15 °C: fresh-cut melon fruit
stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. Transcript levels were normalized with respect to β-actin and were expressed relative to the value of each
gene at day 0, which were set to 1. The values are expressed as means for three biological replicates, with vertical bars indicating standard errors. Values with
different letters indicate statistically significant differences (p ＜ 0.05). Lowercase letters indicated significant difference among FC 5 °C, FC 15 °C and WF 15 °C
groups at the same time point, capital letters represented significant difference among storage time factors.

(2007) reported that sucrose can act as a signal against biotic and similar to previous work on fresh-cut apple (Guerreiro et al., 2017).
abiotic stress, and prevent cell membrane damage. In the present study, These results indicate that sucrose and hexose in melon fruit can act as a
cutting significantly decreased sucrose content and increased hexose signal against the cut-wounding, and that changes in sugar content in
content at the earlier stage of storage (Fig. 1), and these findings are fresh-cut melon stored at 15 °C may be controlled by enzymes that are

8
Z. Wu, et al.
Fig. 7. Heatmap cluster analysis of full-data during storage period in whole and fresh-cut melon fruit. FC 5-1, 2, 4 and 8: fresh-cut melon fruit stored at 5 °C at days 1,
2, 4, and 8 respectively; FC 15-1, 2, 3 and 4: fresh-cut melon fruit stored at 15 °C at days 1, 2, 3 and 4 respectively; WF 15-0, 2, 4, 8 and 12: whole melon fruit stored at
15 °C at days 0, 2, 4, 8 and 12 respectively.
involved in sucrose decomposition. Pearson correlation analysis re-
vealed that AI, NI and SS-c activities in fresh-cut melon were positively
correlated with hexose content and negatively correlated with sucrose
content (Table S2, p ＜ 0.05). These findings indicate that AI, NI, SS-c
were involved in sucrose metabolism in stored fresh-cut melon; similar
results have been reported for stored loquat (Shifeng et al., 2013). Table
S2 also displays that AI and NI activities were correlated with the ex-
pression of CmAI1/2 and CmNI3, respectively (p < 0.05), suggesting
that CmAI1/2 and CmNI3 were involved in the regulation of sucrose
metabolism of fresh-cut melon by regulating the activities of AI and NI
(Figs. 3 and 5). These were also affirmed during melon fruit develop-
ment, as indicated by the increased sucrose content and the reduced
expression of CmAI1 and CmNI3 (Dai et al., 2011).
Organic acids can preserve quality of fruit (Huang et al., 2016; Liu
et al., 2016a) and defend plants against abiotic stress (Sweetman et al.,
2014) in fruits and vegetables. CA and MA content in fresh-cut melon
stored at 15 °C remained at higher levels in comparison with whole
melon at the earlier stage of storage (Fig. 2). Although MA and CA
content was not significantly correlated to PEPC activity during the
storage period of melon fruit (Table S3 and 4, p < 0.05), PEPC activity
in melon fruit was enhanced by cutting in our experiment, also in-
dicating that PEPC contributed to the synthesis of CA and MA (Sadka
et al., 2000). CS may play a primary role in CA biosynthesis of melon
fruit, as indicated by its positive correlation with CA (Table S3, p <
0.05), and the results are consistent with a study examining melon
development (Tang et al., 2010). CA is degraded by ACO in the TCA
cycle. Thus, CA content is expected to increase as ACO activity de-
creases, and our results were in accordance with this pattern. Compared
with ACO, IDH showed a similar relationship to CA in this experiment.
It was found that cutting can alter CS, ACO and IDH activities, and then
9
Postharvest Biology and Technology 161 (2020) 111081
affect CA biosynthesis in fresh-cut melon. According to the correlation
analysis (Table S3, p < 0.05), this was likely due to the increased
expression of CmPEPC3 and CmCS1 during storage and reduced ex-
pression of CmIDH1. Moreover, cutting stimulated the expression of
ACO1/2 transiently and then suppressed their expression. In short,
cutting prevented the decrease in CA content in melon fruit. It can be
inferred that the CA accumulation was coordinated by a cascade of gene
response, rather than by a single gene (Chen et al., 2012). Cutting in-
creased the activities of MDH and ME during the initial stage of storage
of melon fruit, and subsequently led to a decrease in MDH and ME
activities (Fig. 4). Therefore, the higher MA content of fresh-cut melon
may be caused by the combined function of MDH and ME, which is
consistent with the previous study on the growth and development of
apple (Yao et al., 2009). In addition, positive correlations were ob-
served between CmMDH1/3 expression and MDH activity, as well as
CmM3/4 expression and ME activity (Table S4, p < 0.05). Similarly,
1-MCP increased MA biosynthesis in apple fruit via up-regulating the
MdPEPC and MdcyMDH expression with higher activities of PEPC and
MDH (Liu et al., 2016b). Together with the results of this work, it is
proposed that the higher MA content in fresh-cut melon may be regu-
lated by the expression of CmPEPC3, CmMDH1/3 and CmM3/4.
As sugars and organic acids are the main substrates of primary
metabolism, higher hexose and organic acids content may indicate that
cutting enhanced the glycolytic pathway and tricarboxylic acid cycle
pathway in melon fruit, and further induced higher production of es-
sential precursors that serve as substrates for secondary metabolism
(Becerra-Moreno et al., 2012).
In conclusion, cutting accelerated the quality deterioration of melon
fruit, but fresh-cut melon still maintained good quality in the earlier
stage of storage at 15 °C, as indicated by higher hexose and organic
Z. Wu, et al. Postharvest Biology and Technology 161 (2020) 111081

Fig. 8. Model for effect of cutting and storage temperature on sucrose and organic acids metabolism in melon fruit during storage. FC 5 °C: fresh-cut melon fruit
stored at 5 °C; FC 15 °C: fresh-cut melon fruit stored at 15 °C; WF 15 °C: whole melon fruit stored at 15 °C. FC 15 °C VS WF 15 °C: Group FC 15 °C was compared with
Group WF 15 °C at days 2 and 4 respectively. FC 5 °C VS FC 15 °C: Group FC 5 °C was compared with Group FC 15 °C at days 1, 2 and 4 respectively. Red color means
the significant up-regulation for metabolite content, enzyme activity or gene expression (p < 0.05). Green color means the significant down-regulation for me-
tabolite content, enzyme activity or gene expression (p < 0.05). White color means no significant difference (p < 0.05). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).

acids content. The higher hexose levels mainly resulted from higher
activities of the enzymes involved in sucrose decomposition, while
higher organic acid levels likely resulted from changes in PEPC, CS,
ACO, IDH, MDH and ME activities. These may be due to the expression
of CmAI1/2, CmNI3, CmPEPC3, CmCS1, CmACO1/2, CmIDH1,
CmMDH1/3 and CmME3/4 in fresh-cut melon.
4.2. Changes in sucrose and organic acids metabolism of fresh-cut melon
fruit when stored at low temperature (5 °C)
In this study, it was found that storing fresh-cut melon at lower
temperature led to lower hexose levels and higher sucrose, CA and MA
levels. These results are in agreement with previous reports on potato
and myrtle fruits (Bhaskar et al., 2010; Mulas et al., 2012). At lower
temperature, physiological and biochemical functions are slowed down,
which was apparent by the reduction of NI, AI and SS-c activities ob-
served in fresh-cut melon stored at 5 °C in our experiment. The down-
regulation of CmAI1/2 and CmNI3 in samples stored at low temperature
further confirmed this. Several recent studies showed that invertase was
closely related to the cold response of plants (Wiberley-Bradford et al.,
2016). Therefore, it can be inferred that the regulation of sucrose in
fresh-cut melon was temperature-dependent.
The higher CA content observed in fresh-cut melon was accom-
panied by higher PEPC and CS activities and lower ACO and IDH ac-
tivities at 5 °C. In fresh-cut melon stored at 5 °C, genes and enzymes
involved in CA metabolism showed similar change patterns, except that
CmCS1 expression was down-regulated and CS activity was enhanced.
The inconsistency between CmCS1 expression and CS activity may be
due to post-translational regulation. The more interesting finding was
that expression of CmPEPC2/3 was more sensitive to low temperature
storage condition compared to cut-wounding stress, whereas cutting
only increased the CmPEPC3 expression in melon. Surprisingly, ME
activity in fresh-cut melon decreased with storage time at 5 °C, in-
dicating that lower temperature and cutting affected ME activity dif-
ferently. The relationship between MA content and activities of MDH
and ME in fresh-cut melon also revealed that changes in MA content
were related to the combined changes in MDH and ME activities. This
was further confirmed through changes in the expression of genes in-
volved in MA metabolism in fresh-cut melon stored at 5 °C. The effects
of cutting and storage temperature on CmME3/4 expression were dif-
ferent in our experiment. It is likely that cut-wounding activated the ME
gene promoter and lower temperature condition suppressed the effect
(Schaaf et al., 1995).
From the above results, it is evident that storing fruit at lower
temperature preserved the quality of fresh-cut melon and suppressed
sucrose decomposition. However, hexose content was not obviously
lower compared to the sample stored at 15 °C. These results indicated
that there was lower consumption of hexose, CA and MA in fresh-cut
melon stored at 5 °C, which likely resulted from the up-regulation of
CmPEPC2/3 and the down-regulation of CmAI1/2, CmNI3, CmCS1,
CmACO1/2 and CmME3/4 during storage, accompanied by the lower
activities of AI, NI, SS-c, ACO, IDH and ME and higher activities of
PEPC, CS and MDH. Li et al. (2017) reported that lower temperature
induced lower content of total soluble phenol in fresh-cut pitaya fruit,
which may have also resulted from lower consumption of sugars and
organic acids.
A summary of the changes in soluble sugars and organic acids and
the enzymes and genes involved in their metabolism after cutting and
storage are summarized in Fig. 8. Cutting led to the quality deteriora-
tion of melon fruit and induced higher hexose and organic acids content
at the earlier stage of storage. In addition, cutting also altered the ac-
tivity of enzymes and expression of genes that are involved in the
metabolism of sucrose and organic acids. However, lower temperature
significantly extended the shelf life of fresh-cut melon and maintained
higher content of sucrose and organic acids. At the same time, lower
temperature also led to decreases in AI, NI, SS-c, ACO, IDH and ME
activities and increases in PEPC, CS and MDH activities. These changes
were further confirmed at the transcriptional level by the observed up-
regulation of CmPEPC2/3 and the down-regulation of CmAI1/2, CmNI3,
CmCS1, CmACO1/2 and CmME3/4 during storage.

10
Z. Wu, et al.
5. Conclusion
This work investigated the molecular changes underlying the fluc-
tuations in content of soluble sugars and organic acids in whole and
fresh-cut melon fruit during storage. Cutting greatly accelerated quality
deterioration of melon fruit when stored at 15 °C, as indicated by lower
firmness, TSS and pH at the later stage of storage. However, fresh-cut
melon stored at 15 °C maintained its good quality at the earlier stage of
storage through higher hexose and organic acids content. This was
confirmed by the results that AI, NI and SS-c were the main regulators
of sucrose metabolism in fresh-cut melon while organic acids metabo-
lism was regulated by the combined function of PEPC, CS, ACO, IDH,
MDH and ME. These findings may be due to the up-regulation of
CmAI1/2, CmNI3, CmPEPC3, CmCS1, CmACO1/2, CmMDH1/3 and
CmME3/4 and down-regulation of CmIDH1 in fresh-cut melon. Storage
at 5 °C significantly extended the shelf life of fresh-cut melon and
maintained higher content of sucrose and organic acids, which resulted
from the up-regulation of CmPEPC2/3 and the down-regulation of
CmAI1/2, CmNI3, CmCS1, CmACO1/2 and CmME3/4 during storage.
This useful molecular information on the processing of melon will be
beneficial for protecting the sensory quality of fresh-cut melon. Cutting
of fruits accelerates their secondary metabolism, and these results will
be very helpful to better study how to maximize the nutritional value of
fresh-cut melon.
CRediT authorship contribution statement
Zhangfei Wu: Conceptualization, Methodology, Investigation,
Visualization, Data curation, Validation, Writing - original draft.
Mingmei Tu: Investigation, Visualization. Xingping Yang: Resources.
Jinhua Xu: Resources. Zhifang Yu: Project administration, Validation,
Funding acquisition, Writing - review & editing.
Declaration of Competing Interest
The authors have declared that no conflicts of interest exist.
Acknowledgment
This research was financially supported by the Innovation Project of
Jiangsu Agricultural Science (CX (15) 1018).
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.postharvbio.2019.
111081.
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