Scientia Horticulturae 277 (2021) 109806

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Improvement of fruit quality and pedicel color of cold stored sweet cherry
in response to pre-storage 1-methylciclopropene and chlorine
dioxide treatments
Combination treatment of 1-MCP plus ClO2 improves post-harvest quality of sweet
cherry fruit
Handong Zhao a, Maorun Fu a, Yamin Du a, Fei Sun a, Qingmin Chen b, *, Tong Jin a, Qian Zhang c,
Bangdi Liu d
a
Collage of Food Science and Technology, Qilu University of Technology Shandong Academy of Sciences, Jinan, 250353, China
b
Department of Food Science and Engineering, Shandong Agriculture and Engineering University, Jinan, 250100, China
c
Shandong Institute of Pomology, Taian, 271000, China
d
Academy of Agricultural Planning and Engineering, Ministry of Agriculture and Rural Affairs, Beijing, 100125, China

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

Keywords: Sweet cherry fruit is perishable which quality loss greatly influences consumers preference. To improve post­
Sweet cherry harvest quality of sweet cherry, the effect of the combination of 1-methylciclopropene (1-MCP) plus chlorine
Fruit1-MCP dioxide (ClO2) was estimated by analyses of postharvest characteristics, bioactive compounds, antioxidant ca­
Chlorine dioxide
pacity and pedicel quality changing. The fruit was treated separately or in combination with 1-MCP and ClO2,
Treatment
Postharvest quality
which were stored at 4 ◦ C for 25 d. In comparison with individual treatment, the combination treatment of 1-
Pedicel chlorophyll degradation MCP and ClO2 delayed senescence by suppressing decay incidence, softening and the reduction of ascorbic
acid, total phenolic as well as titratable acid. In addition, combination of 1-MCP + ClO2 inhibited the decreasing
of pedicel browning and pedicel force, maintained the higher content of chlorophyll more effectively. Moreover,
1-MCP or ClO2 individual treatment remarkably inhibited the expression of genes, including pheophytin pheo­
phorbide hydrolase, chlorophyllase, pheophorbide a oxygenase and red chlorophyll catabolite reductase, asso­
ciated to chlorophyll catabolism in cherry pedicel. Additionally, compared to the individual treatment, the
combination of 1-MCP + ClO2 contributed the maximum efficacy, especially in the middle duration of storage.
Overall, our preliminary study suggested that the combined treatment of 1-MCP + ClO2 might be a promising
method to preserve postharvest quality in sweet cherry fruit.

1. Introduction
Sweet cherry, with its desirable taste, attractive appearance and
nutritional value, has become one of the most popular non-climacteric
fruits worldwide (Zhao et al., 2019a). Previous researches have
proved that the bioactive compounds in sweet cherry were benefit to a
lessening of several diseases involving cardiovascular, diabetes, cancer
and inflammatory diseases (Mccune et al., 2010). However, due to high
respiration rate and high susceptibility of mechanical damage, sweet
cherry fruit deteriorate quickly during storage period even under strict
cold chain management (Wang et al., 2015). The deterioration,
including surface pitting, darkening of the skin and flavor loss, was most
commonly during cold storage, which dramatically influence fruit
storability and marketing acceptability (Yang et al., 2019). Moreover,
pedicel browning and dehydration are major symptoms of quality loss at
the retail stage, and therefore green pedicel are often used as indicators
of overall cherry fruit freshness (Linke et al., 2010). After harvest,
pedicel green loss in sweet cherry fruit would remarkably influence the
storage stability and market consumption. The chlorophyll breakdown
pathway involves in a series of enzymatic process, which includes the
reaction of pheophorbide a oxygenase (PAO), red chlorophyll catabolite
reductase (RCCR), chlorophyllase (CLH) and pheophytin pheophorbide

* Corresponding author.
E-mail address: 935226056@qq.com (Q. Chen).

https://doi.org/10.1016/j.scienta.2020.109806
Received 11 April 2020; Received in revised form 13 October 2020; Accepted 16 October 2020
Available online 19 November 2020
0304-4238/© 2020 Elsevier B.V. All rights reserved.
H. Zhao et al.
(PPH), all of them could be enrolled by stay-green1 protein in the
thylakoid membrane. At the end of chlorophyll degradation pathway,
fluorescent chlorophyll catabolite would be adjusted to liner tetra­
pyrrolic products stored in senescent cell (Wei et al., 2019). In order to
save postharvest loss of sweet cherry fruit, diverse strategies have been
explored recently.
Although sweet cherries are non-climacteric, these fruits may un­
dergo similar biochemical changes as in climacteric fruits, and some
evidence have indicated that ethylene might induce senescence meta­
bolism in cherry fruit (Ren et al., 2011). As a synthetic cyclic alkene,
1-methylciclopropene (1-MCP) regulates the tissue response to ethylene
by blocking the ethylene receptors and preventing binding of ethylene to
its receptors. In some non-climacteric fruit, such as strawberry, 1-MCP
treatment could improve postharvest quality and extended the
post-harvest life of treated fruit (Ren et al., 2011), but a single 1-MCP
application had no impact on pedicel browning in sweet cherry fruit
(Gong et al., 2002). Moreover, combination of hexanal and 1-MCP could
enhance the quality and shelf-life of sweet cherry (Sharma et al., 2010).
Therefore, it is possible that 1-MCP may be utilized to bring beneficial
effect on the storability and quality of sweet cherry fruit.
Chlorine dioxide (ClO2) gas, as a safe and broad sanitizer, could
reduce pathogenic microorganisms and inhibit fruit enzymatic brown­
ing by reducing the activities of polyphenol oxidase and peroxidase
(Saengnil et al., 2014), which did not react with organic compounds
produce toxic chlorinated by-products (Gómez-López et al., 2009). For
harvesting fruit and vegetables, ClO2 has been recently used for
reducing enzymatic browning and maintaining storage quality, such as
litchi (Wu et al., 2011) and blueberry (Xu et al., 2016). Currently, little
available data on pedicel color changes in sweet cherry fruit under the
effect of ClO2 treatment.
Although the treatment of 1-MCP or ClO2 alone could improve the
postharvest quality of sweet cherry fruit, their mechanism of action
might be different. Additionally, there are no information on the influ­
ence of the combination of ClO2 and 1-MCP on the quality of sweet
cherry fruit. Hongdeng sweet cherry (Prumus avium L.) is one of widely
distributed cultivars in north China (Zhang et al., 2007). This object of
our research was to investigate the efficiency of ClO2 gas in combination
with 1-MCP treatment for improving postharvst quality and the pedicel
chlorophyll metabolism of stored sweet cherry fruit, and to elucidate the
potential mechanism of the combination treatment.
2. Materials and methods
2.1. Plant materials and treatment
Sweet cherry (Prumus avium L. cv. Hongdeng) fruit (an early season
cultivar) were harvested at commercial maturity (values of flesh firm­
ness, soluble solids content (SSC) and titratable acid (TA) were about
33 N, 17 g 100 g− 1 and 0.75 g MA (malic acid) 100 g− 1 fresh weight,
respectively) from an orchard (36.4 ◦ N, 117.0 ◦ E, 380 m above sea level)
in Shandong Province, China. Sweet cherry trees, with 10-years-old,
were sustained with the standard fertilizer, pesticide practices, herbi­
cide and cultivation. After harvesting, fruit, with uniformity in shape,
color, size, physical integrity and no apparent defects, were randomly
transported to the laboratory immediately. All fruit were pro-cooled at
4 ◦ C for 12 h. After pro-cooling, fruit were randomly divided into
following four lots of 16 kg each. According to preliminary study (in
supplementary material, Figs. S1–S3), every lot was divide into four
treated groups (about 4 kg each) as following: control (without treat­
ment), 30 μL L-1 ClO2 (gas was released from 0.01 g solid ClO2, which
was purchased from Beijing Hualong Xingyu Science and Technology
Development Co., Ltd, China), 1 μL L-1 1-MCP (1-MCP was released from
a commercial powder formulation, which was purchased from Smart
Fresh, Agrofresh Ltd., Pliladelphia, Pa., U.S.A.) treatment and the
combination of 1-MCP + ClO2 (30 μL L-1 of ClO2 and 1 μL L-1 of 1-MCP in
a box), and all treatment groups were treated for 24 h at 4 ◦ C. For
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Scientia Horticulturae 277 (2021) 109806
removing 1-MCP and ClO2 residues, the plastic bags were open and
ventilated for one hour after treatment. All fruit were then stored in
polyethylene film bags (0.008 mm thick) at the condition of 4 ◦ C (to
simulate refrigerator temperature) and 90 % relative humidity for 25 d.
Every treatment included four subsamples, and there were 4 kg fruits in
each bag. Every five days, one subsample was used for testing decay
incidence and pedicel quality. For the other three subsamples, 500 g
fruits of each bag were removed to estimate the physiological and
biochemical attributes, and the collected fresh sample was immediately
used or frozen in liquid nitrogen and stored at − 80 ◦ C. Each treatment
group was conducted with three biological replicates at each sample
time.
Measurements of decay incidence, firmness, soluble solids content
(SSC) and titratable acid (TA) of sweet cherry fruit
One hundred fruits per replicate were evaluated for fruit decay
incidence. Any splitting and rot area, regardless of severity, were
recorded Zhao et al. (2018). Decay incidence (%) = (number of fruits
showing decay symptoms)/total number of fruits) × 100 %.
To measure the firmness and SSC, 25 fruits of each replicate were
randomly opted. Fruit firmness was tested independently in 25 fruit of
each lot using a FirmTech 2 Fruit Firmness instrument (BioWorks Inc.,
Wamego, KS) by determining the force requirement to compress on the
opposite sides of each individual peeled fruit. For each peeled fruit, a
penetrometer with a 3.5 mm probe was used to test firmness, and the
results were expressed in N. After testing the firmness values, the fruit
juice was used to evaluate SSC levels. A digital refractometer (PR-101,
Spectrum Technologies, Plainfield, IL) was used for estimating SSC of
fruit, and results were expressed in g 100 g− 1. TA was tested by titrating
20 mL of cherry juice to pH 8.2 with 0.1 mol L-1 NaOH, and results
expressed as malic acid (MA) 100 per gram fresh weight (g MA 100 g-1
FW). Every treatment included replicates, and all experiments were
carried out in triplicate.
2.2. Analysis of total phenolic (TP) and ascorbic acid of sweet cherry fruit
Frozen cherry flesh (4.0 g) was homogenized in 6 mL of methanol.
After centrifugation, the supernatant was used to test the level of TP
(Zhao et al., 2019b). Briefly, 0.5 mL supernatant was mixed with 2.0 mL
Folin-Ciocalteu reagent and 6.0 mL 1 mol L-1 sodium carbonate, and the
total volume of mixture was adjusted to 10 mL with distilled water. After
the mixture incubation at room temperature, the absorbance was
recorded at 740 nm. and results were expressed as gram gallic acid
equivalents per kilogram fresh weight (mg GA kg-1).
To evaluate the ascorbic acid changes, sample was extracted as the
previous method (Zhao et al., 2019a) with little modification. 1.0 g flesh
sample was mixed with 5 mL cold 20 mmol L− 1 NaH2PO4 buffer solution
(pH 2.1, containing 1 mmol L-1 ethylene diamine tetraacetic acid
(EDTA)). Then the sonicated extract was centrifuged at 12,000 g for
20 min at 4 ◦ C. 1.0 mL of the filtered extract was added to 300 μL of
dithiothreitol (DTT) (20 g L-− 1) as a reducing agent. Precautions were
made to avoid light exposure during the whole procedure. The con­
centration of ascorbic acid was determined by high-performance liquid
chromatography (HPLC) method using a C18 column (Shimpack VP-ODS
15 cm ×4.6 mm ID, 5 μm, Shimadzu Co., Japan) where 50 μL was
injected for each sample. The elusion was conducted isocratically using
a mixture of 90 % doubly deionised water, 10 % methanol and 0.1 %
formic acid (v/v/v) at a flow rate of 1.0 mL min− 1. The quantification
was carried from the peak areas recorded at 245 nm with reference to
the calibration curve obtained with ascorbic acid reference. Results were
expressed as milligram of ascorbic acid for kilograms (mg AA kg− 1).
2.3. Non-enzymatic antioxidant capacity assay of sweet cherry fruit
The non-enzymatic antioxidant capacity of pulp was investigated by
DPPH and reducing capacity. Fresh sample (2 g) was homogenized in
6 mL of methanol. After centrifugation, the supernatant was collected
H. Zhao et al.
and used to test DPPH scavenging activity. Sample extract (200 μL) was
mixed with 3.5 mL of DPPH radical (300 μmol L− 1). After incubated at
25 ◦ C for 60 min, the absorbance of mixture at 517 nm was recorded
(Ibrahim et al., 2012). Aqueous solution of Trolox was used for cali­
bration, and the results were expressed as mmol TE kg− 1 fresh weight
basis (Zhao et al., 2019b).
The reducing capacity of sweet cherry flesh was tested following
previous report with little modification (Mao et al., 2006). 200 μL
extract was mixed with 2.5 mL of 0.2 mol L− 1 potassium ferricyanide
[K3Fe(CN)6]. After incubating at 50 ◦ C for 20 min, 3 mL of 0.6 mol L− 1
trichloroacetic acid was added into the mixture. Then, the mixture was
centrifugated at 12,000 g for 15 min. 2.5 mL upper layer solution was
mixed with 3 mL of distilled water and 0.5 mL of 0.006 mol L-− 1 FeCl3,
and then the absorbance was recorded at 700 nm. Butylated hydrox­
yanisole, butylated hydroxytoluene and α-tocopherol were used as
positive standards. Increased absorbance of the reaction mixture indi­
cated increased reducing capacity.
2.4. Quality evaluation of sweet cherry pedicel
One hundred fruit of each replicate sample was used for pedicel
quality evaluation. Pedicel browning was expressed as the percentage of
whole surface browned. To confirm the degree of pedicel browning, the
pedicel was classified into the scale of 0–3 based on the browned area
covering the pedicel surface, where 0 no browning, 1, slight (≤ 25 % of
browning); 2, moderate (26～50 % of browning); 3, severe (≥ 50 % of
browning). The browning index was calculated with formula:
∑
(browning class × number of stem in each class)/3 × total number of
stem in each treatment. For pedicel traction force evaluation, a Texture
Analyzer GT2 (Brookfield, Massachusets, USA) was used in order the
pedicel to be removed with stretching force. Fruit was put down a plate
with a hold in the middle (about 10 mm diameter) through the pedicel
was directed to the grip. Then, the pedicels were weighted and dried at
80 ◦ C until the final weights were stable and recorded. The ratio of final
dry weight and initial fresh weight was used for calculating the pedicel
moisture content. Measure unit was expressed as “g 100 g− 1”.
2.5. Measurement of chlorophyll content in sweet cherry pedicel
Chlorophyll content was tested following the previous method with
some modifications (Wei et al., 2019). Chlorophyll was extracted from
1 g pedicel sample of each replicate in 15 mL 85 % (v/v) acetone in
permanent darkness at 25 ◦ C for 24 h. Absorbance of chlorophyll a and b
were tested by a spectrophotometer (V-1100D, Mapada Inc., Shanghai,
China) at 645 nm and 633 nm. The results of chlorophyll a, chlorophyll b
and chlorophyll a + b content were calculated by Arnon (1949).
RNA extraction and real-time PCR analysis of chlorophyll degrada­
tion for sweet cherry pedicel
Pedicel sample tissue (0.05 g) was used fro isolating total RNA
following with the manufacture’s instructions of TaKaRa MiniBEST
Plant Extraction Kit (Takara Bio, Shiga, Japan). RNA concentration was
tested by an MD2000 sepectrophotometer (BioFuture, Britain) and
deemed to be acceptable with a ratio of 260/280 nm between 1.8 and
2.2. The integrity of RNA was evaluated by electrophoresis in 1.2 % (w/
v) agarose gel. The first-stand of cDNA was synthesized from total RNA
(0.1 μg) using Primescript™ II RTase (Takara Bio Inc.) following the
manuscript instructions. RT-qPCR primer sequences for each gene were
well-designed in the light of nucleotide sequences obtained from GEN­
BANK database, using the Primer Expressing 5.0 software (Applied
Biosystems, USA), as shown in Table S1. Actin mRNA was used as
reference housekeeping gene to normalize transcript level for each
sample. qRT-PCR test was conducted using SYBR® Premix Ex Taq™
(Takara Bio Inc.) on FQD-96A (Bioer, China). The cycle processed for
PCR amplification as follows: 40 cycles of 95 ◦ C for 10 s, 55 ◦ C for 10 s,
and 72 ◦ C for 20 s. The relative expression levels of the target genes were
calculated using the 2－△△CT formula (Livak and Schmittgen, 2001).
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Scientia Horticulturae 277 (2021) 109806
Each RNA sample was run in triplicate and repeated in at least two in­
dependent sets of treatments for at total six replicate per gene per
sample.
2.6. Statistical analysis
All experimental analysis were performed in triplicate and data were
expressed as mean ± SD (standard deviation). The physio-chemical data
were subjected to analysis of variance (ANOVA) by SPSS version 20
windows (SPSS Inc., Chicago, Illinois, USA), and the Spearman’s cor­
relations (P < 0.05 and P < 0.01) were used for estimating the correla­
tions among characteristic parameters (Table S2). In all cases,
examinations were completely randomized with three replicates, and
significant differences were performed by Duncan’s multiple range tests
(P < 0.05).
3. Results
3.1. Decay incidence and firmness of sweet cherry fruit
Changes in decay incidence and firmness are important parameters
of quality and senescence for fresh sweet cherry fruit. As shown in
Fig. 1A, decay incidence of sweet cherry was reduced in all groups
throughout the storage period. Compared to untreated fruit, the decay
incidences of 1-MCP and ClO2 treatment were alleviated significantly
(P < 0.05) and showed 23.3 % and 38.9 % decrease, respectively, at the
end of storage. There was not significant (P > 0.05) changes between 1-
MCP and ClO2 treatment during the earlier storage stage, and the 1-MCP
showed higher than ClO2 at the end of storage. Moreover, on day 25, the
decay incidence of 1-MCP + ClO2 showed 11.7 % and remained at the
lowest level.
The firmness of control and ClO2 groups declined rapidly from 5 d to
20 d, then followed by the slower decrease after 25 d, respectively
(Fig. 1B). Moreover, the firmness of fruit in 1-MCP + ClO2 group, with
about 23 N, was approximately 1.5-fold higher than that of the control
fruit on 25 d. Furthermore, firmness of both 1-MCP and 1-MCP + ClO2
groups maintained higher than those of no 1-MCP treated fruit, but there
was no remarkable difference between 1-MCP and 1-MCP + ClO2.
3.2. SSC and TA changes of sweet cherry fruit
As shown in Fig. 1C, there were not significant (P > 0.05) differences
in SSC between 1-MCP and ClO2 during the whole storage. However,
compared to control and ClO2 groups, 1-MCP + ClO2 significantly
(P < 0.05) delayed SSC reduction, and effectively maintained the initial
quantity. Additionally, TA content decreased gradually during storage
for both treated and untreated fruit (Fig. 1D). Compared to other groups,
however, 1-MCP + ClO2 treatment remained the initial level, remark­
ably. Fig. 1E is the appearance of treated and untreated fruit on 25 day.
As the picture shown, both control and 1-MCP groups appeared the
symptom microbial infection, while ClO2 treatment could obviously
reduce the microbial population. Compared to the ClO2 treated fruit, the
combination of 1-MCP + ClO2 treatment showed the better appearance
and improved consumer acceptance.
3.3. Ascorbic acid contents and TP accumulation of sweet cherry fruit
With respect to the content of ascorbic acid, level at harvest (about
150 mg kg− 1) increased significantly in both treated and untreated
cherries until day 10 of storage and thereafter decreased until the end of
storage (Fig. 2A). However, content of ascorbic acid were significantly
higher (P < 0.05) in treated fruit than in control, especially for the 1-
MCP + ClO2 treatment.
As shown in Fig. 2B, the ascorbic acid and phenolics content
exhibited similar trends, and there were significant differences among
the groups of 1-MCP, ClO2 and control. Initial TP content in sweet cherry
H. Zhao et al. Scientia Horticulturae 277 (2021) 109806

Fig. 1. Effect of different treatments on decay incidence (A), firmness (B), soluble solid content (C) and titratable acid (TA) (D) of sweet cherry fruit. Data are
expressed as the mean ± SD (n = 3). Vertical bars represent the standard deviation of the means (P < 0.05). E means the visual appearance of different treatment fruit
stored for 20 days at 4 ℃. Duncans test letters represent the difference among the different treatments.

fruit at 0 d was 245.0 ± 3.5 mg GA kg− 1, and the maximum levels pulp tissue of sweet cherry fruit during storage, which was 6.5 % higher
attained at 10 d. In all treatment group, TP content showed an initial than control at the end of storage.
increase, followed by a decrease during cold storage, while 1-
MCP + ClO2 treatment remarkably increased the accumulation of TP in

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H. Zhao et al. Scientia Horticulturae 277 (2021) 109806

Fig. 2. Effect of different treatments on nutritional attributes of sweet cherry fruit. Ascorbic acid (A) and total phenolics (TP) (B) of sweet cherry fruit. Data are
expressed as the mean ± SD (n = 3). Vertical bars represent the standard deviation of the means (P < 0.05). Duncans test letters represent the difference among the
different treatments.

3.4. Changes in the antioxidant properties of sweet cherry fruit
The property of non-enzymatic antioxidants was showed in Fig. 3.
Reducing capacity and DPPH radical scavenging capacity of fruit pulp
exhibited the similar changes during the storage, with an initial increase
to the peak levels, and followed by decrease. At the end of storage, the
combination of 1-MCP + ClO2 keep remarkably (P < 0.05) higher level
of antioxidant capacity than those of control and individual treatment
and roughly remained the initial antioxidant property.
Changes in browning index, moisture content, stretch force and
chlorophyll content of sweet cherry pedicel
Compared to untreated fruit, both 1-MCP and ClO2 treatments
reduced the percentage of pedicel browning (P < 0.05) after fifteen days
(Fig. 4A). Moreover, the combination of 1-MCP + ClO2 reduced pedicel
browning index of fruit treated with 1-MCP and ClO2 alone up to 6 %
and 30 %, respectively, at the end of storage. Pedicel moisture content
had a negative relationship with the pedicel browning index for all of the
sweet cherry fruit (Table S2 and Fig. 6). Pedicel moisture content at
harvest was 60 g 100 g− 1 and significantly decreased during storage
reaching final values of 37 g 100 g− 1 and 35 g 100 g− 1 in 1-MCP and
ClO2, respectively, and significantly higher, 48 g 100 g− 1 in combination
of 1-MCP + ClO2 treatment. The Fig. 4E also exhibited 1-MCP + ClO2
treatment could remain pedicel with the better moisture content and
inhibit browning incidence.

Fig. 3. Changes of non-enzymatic antioxidant capacity in untreated, 1-MCP, ClO2 and 1-MCP + ClO2 treated sweet cherry fruit during storage. Reducing capacity
(A), DPPH scavenging (B). Data are expressed as the mean ± SD (n = 3). Vertical bars represent the standard deviation of the means (P < 0.05). Duncans test letters
represent the difference among the different treatments.

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H. Zhao et al. Scientia Horticulturae 277 (2021) 109806

Fig. 4. Effect of different treatments on pedicel browning index (A), pedicel moisture content (B), pedicel traction force (C) and pedicel chlorophyll content (D) of
sweet cherry fruit. Data are expressed as the mean ± SD (n = 3). Vertical bars represent the standard deviation of the means (P < 0.05). E means pedicel appearance
changes of different treatment fruit stored 20 days at 4 ℃. Duncans test letters represent the difference among the different treatments.

Similarly, pedicel traction force decreased also during storage from
initial levels of 7.7 ± 0.2 N to 2.1 ± 0.22 N in control fruit, the reduction
being significantly less in 1-MCP and combination of 1-MCP + ClO2,
without significant differences between 1-MCP and the combination
treatment (Fig. 4C). Pedicel chlorophyll content reduced in untreated
and treated fruit during cold storage (Fig. 4D), however, this decrease
was apparently inhibited by three treatments. On day 25, pedicel chlo­
rophyll content had decreased by 66.8 %, 49.1 %, 57.9 %, and 42.3 %
under control, 1-MCP, ClO2, and 1-MCP + ClO2 treatment, respectively,
while the pedicel chlorophyll content of 1-MCP + ClO2 combination
declined more slowly than 1-MCP and ClO2 treatments alone during
storage.
3.5. Expression pattern of PaCLH, PaPPH, PaPAO and PaRCCR of
cherry pedicel
Four expressions of related genes, involving the chlorophyll break­
down pathway, were investigated. The expression of chlorophyll
degradation genes were tested and normalized to that in the control on
0 d. The PaCLH relative expression of untreated fruit was comparatively
higher during storage, and no significant difference was noted between
1-MAC and ClO2 treatments (Fig. 5A). The PaCLH relative expression
decreased first and then increased in 1-MCP + ClO2 combination, and
continuously maintained a lower level compared with other groups. The
PaPPH relative expression of untreated fruit increased significantly until
day 15 of storage and thereafter decreased until the end of storage
(Fig. 5B). Moreover, the PaPPH relative expression of treated fruit was

6
H. Zhao et al.
Fig. 5. Effect of different treatments on relative expression associated with chlorophyll breakdown pathway genes of sweet cherry pedicel. PaCLH (A), PaPPH (B),
PaPAO (C) and PaRCCR (D). Data are expressed as the mean ± SD (n = 3). Vertical bars represent the standard deviation of the means (P < 0.05). Duncans test letters
represent the difference among the different treatments.
significantly lower, especially in 1-MCP + ClO2 combination, and the
combination treatment dramatically downregulated the expression of
PaPPH at 15 and 20 d (P < 0.05).
Compared to the harvest condition, the expression levels of PaPAO
and PaRCCR were remarkably higher in the untreated fruit during
storage (Fig. 5C and D), and there was no difference on relative
expression of the PaRCCR gene between untreated and treated fruit at
the end of storage. However, all of the treatments suppressed the
expression of PaPAO at 15 and 20 d with respect to that in control fruit
and alleviated the expression of PaRCCR at 10 and 15 d. Above results
indicated that the combination of 1-MCP + ClO2 treatment could alle­
viate the chlorophyll breakdown in sweet cherry pedicel by regulating
genes expression involved in the chlorophyll breakdown pathway.
4. Discussion
Quality deterioration caused by senescence considerably influences
the postharvest storability and consumer acceptance of various crop
products such as fruits and vegetables. Combination of 1-MCP + ClO2
has become a new strategy and drawn extensive attention in postharvest
field recently for its function of oxidative stress regulation (Yang et al.,
2018). In this research, we checked the effect of 1-MCP and ClO2
treatments on the postharvest storage quality and pedicel browning of
sweet cherry fruit and preliminarily investigated the potential mecha­
nism of chlorophyll degradation in fruit pedicel from transcriptional
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Scientia Horticulturae 277 (2021) 109806
levels. Chlorophyll degradation is extensive during fruit senescence, and
the prerequisite for chlorophyll breakdown is occurring on the thylakiod
membrane (Hortensteiner and Krautler, 2011). Sweet cherry fruit is very
susceptible to postharvet decay caused by some pathogenic fungi, such
as Alternaria alternata Keissl, Rhizopus stolonifer Vuill and Penicillium
expansum Link, which are the main postharvest sweet cherry fungal
pathogens causing significantly economic losses (Gatto et al., 2016).
Results exhibited that combination of 1-MCP + ClO2 effectively reduced
the decay incidence and maintained the initial firmness. As Table S1
shown, decay was negatively correlated with firmness (r = 0.931,
P < 0.01), and the changes of decay and firmness were exhibited the
contrary trend with the storage period increasing. Decay symptom of
sweet cherry was caused by fungal pathogens (Ippolito et al., 2005), and
the softening was related to the enzymatic degradation of the middle
lamella and cell walls (Ippolito et al., 2005). A single 1-MCP application
(1 μL L-1) could stabilize the cell membrane by blocking ethylene
perception (Serradilla et al., 2019), and ClO2 has the effective antifungal
activity (Wang et al., 2014a). Our results indicated the combination of
1-MCP + ClO2 the dual effect.
After initial fluctuations, the decreases of SSC level might be attrib­
uted to respiration and sugars conversion (Petriccione et al., 2018).
Flavour loss caused by a reduction fruit acid content shortens the po­
tential storage life of sweet cherry, so reducing the acidity loss is an
important objective for improving the potential marketing period (Wang
et al., 2014b). Additionally, the colors of TA and SSC in treated by
H. Zhao et al.
Fig. 6. Heatmap of metabolic changes and chlorophyll degradation related genes expressions in sweet cherry fruit and pedicel tissues exposed to different treatments
following storage period. The graph depicts the relative level of each parameter compared to the initial quality (0 day). Fold change values war shown on a color
scale, which is proportional to the mean of each identified metabolite. The heatmap are shown on a log2 scale for each parameter (n = 3).
combination of 1-MCP + ClO2 changed little (Fig. 6), which indicated
that 1-MCP + ClO2 treatment could improve postharvest quality of
sweet cherry, and the similar result had also reported in strawberry fruit
(Yang et al., 2018).
A wide range of compounds (such as ascorbate, anthocyanins and
phenolic acids), naturally present in the fruit tissues may help in scav­
enging ROS. All these compounds may be globally determined by
measuring the fruit antioxidant capacity (Giné-Bordonaba et al., 2017).
Sweet cherry is thought to be a healthy fruit because of its bioactive
compounds, such as ascorbic acid and phenols. There was a significantly
negative correlation between ascorbic acid content and DPPH capacity
(r = 0.962, P < 0.01) (Table S2), which indicated the ascorbic acid might
have some contribution to the DPPH property. Ascorbic acid, acting as
an antioxidant, could maintain cell membrane integrity in fruit (Hodges
et al., 1999), and in non-enzymatic antioxidant system ascorbic acid
played an important role in detoxification of reactive oxygen species
(Shewfelt and Del Rosario, 2000). Several researches have reported that
both ClO2 and 1-MCP treatments cloud maintain the ascorbic acid in the
higher level (Karagiannis et al., 2018; Zhong et al., 2006). Moreover,
1-MCP treatment had similar efficacy in keeping TP content of sweet
cherry fruit as treated by ClO2 (Fig. 2A). Recent researches have re­
ported the application of could promote TP content of pitaya fruit (Liu
et al., 2019) and apple fruit (Ma et al., 2019). TP content has the positive
correlation with both DPPH (r = 0.901, P < 0.01) and reducing capacity
(r = 0.914, P < 0.01). and the similar changes in the content of ascorbic
acid and phenols may have affected the antioxidant property. Plants,
with high levels of antioxidants, have been considered as having greater
resistance to oxidative damage (Blokhina et al., 2003), and efficient
antioxidant is essential to maintain ROS at appropriate level. DPPH
radical scavenging activity, in fruit and vegetables, is associated with
8
Scientia Horticulturae 277 (2021) 109806
antioxidant antioxidant capacity (Blokhina et al., 2003). Some re­
searches have regarded various pre- or post-harvest treatments could
influence antioxidant capacity of fruit and vegetable (Zhang et al., 2013;
Wang et al., 2016). Therefore, the combination effected in delaying the
declines of ascorbic acid and TP was preeminent as always, and the
differences reached the significant level (P < 0.05) in comparison with
the individual treatments.
The characteristics of the fruit pedicel is an important indicator of
sweet cherry fruit quality by the consumer (Schick and Toivonen, 2002).
Cherry pedicel browning generally is due to the loss of membrane
integrity that permits polyphenol oxidase and polyphenol substances to
involve in the damage cells inducing tissue browning (Wang et al.,
2014b). Some other researches showed 1-MCP treatment alone could
induce senescence symptoms in cherry pedicel (Karagiannis et al., 2018;
Wani et al., 2014), while the individual ClO2 treatment might enhance
the green maintaining (Wei et al., 2019). Data indicated cherry pedicel
exposed to both 1-MCP and ClO2 treatments alone displayed senescence
symptoms as illustrating by higher pedicel browning and lower pedicel
removal force as well as moisture content while combination application
substantially improved these features.Transpiration could be minmized
by decreasing the driving force for transpiration during storage, besides,
pedicel surface area and stomatal number might also influence water
vapor transfer (Knoche et al., 2015). Loss of green pedicel color in sweet
cherry affects fruit quality and reduces the market value, which is one of
obvious traits that accompanies chlorophyll degradation (Wang et al.,
2017). Researches have showed that 1-MCP application on pears and
broccolis can delay chlorophyll degradation by inhibiting chlor­
ophyllase activity and alleviating the expression of chlorophyll catabolic
genes (Gómez-Lobato et al., 2012; Xie et al., 2017). The expression of
RCCR and PAO could induce the the loss of green color during
H. Zhao et al.
chlorophyll degradation, and the PPH would catalyze this reaction to
generate the substrate (Hörtensteiner, 2004). Compared to the untreated
fruit, in our present research, 1-MCP led to a lower expression values of
PaPPH and PaRCCR within 15-day storage duration and decreased the
expression levels of PaCLH and PaPAO at the whole storage period.
Therefore, 1-MCP treatment reduced gene expression of chlorophyll
degradation, which was consistent with ‘Comice’ pear (Zhao et al.,
2020).
Generally, ethylene is the main cause of chlorophyll degradation and
fading of the green color, and ClO2 treatment can reduce the respiration
and ethylene production rate (Wei et al., 2019). Previous studies have
reported that ClO2 treatment alone could delaying the chlorophyll
degradation in green pepper (Wei et al., 2019) and bamboo shorts (Yang
et al., 2015). In this present study that combination of 1-MCP + ClO2
treatment always maintained the higher level of chlorophyll than other
groups. Generally, CLH gene is identified the first step to regulate the
chlorophyll pathway, and previous showed that the CLH expression
level did not up-regulate (Wei et al., 2019), which is not consistent with
our study where the PaCLH expression level in 1-MCP + ClO2 combi­
nation was significantly (P < 0.05) lower than control group during
whole storage, while those of PaPPH, PaPAO and PaRCCR did not
change remarkably at the end of storage. In this research, the combi­
nation application exhibited better effects on sweet cherry fruit quality
as compared with the effects observed with 1-MCP and ClO2 alone.
5. Conclusion
By contrast to 1-MCP and ClO2 treatment alone, our results show that
the combination application (1-MCP + ClO2) could better improve the
postharvest quality of sweet cherry fruit, delaying decay incidence and
softening development was well as maintaining the SSC, TA, TP and
ascorbic acid level. Moreover, this combination of 1-MCP and ClO2
treatment not only improved pedicel quality, but also reduced the
chlorophyll degradation in fruit pedicel by regulating the relative genes
expression, which might help to elucidate the mechanism for pedicel
browning treated with 1-MCP + ClO2. Nevertheless, it should be
recognized more sweet cheery cultivars would be considered in further
study to ensure the extensive application of this combination. Thus, all
the findings indicated the combination of 1-MCP and ClO2 treatment
might be a promising storage method to improve quality and storability
in sweet cherry.
CRediT authorship contribution statement
Handong Zhao: Conceptualization, Methodology, Software, Visu­
alization, Data curation, Writing - original draft, Writing - review &
editing. Maorun Fu: Funding acquisition, Project administration.
Yamin Du: Methodology, Data curation, Software, Validation. Fei Sun:
Methodology, Software, Validation. Qingmin Chen: Project adminis­
tration, Visualization. Tong Jin: Methodology, Data curation. Qian
Zhang: Supervision. Bangdi Liu: Funding acquisition.
Declaration of Competing Interest
The authors declared that they have no conflicts of interest to this
work. We declare that we do not have any commercial or associative
interest that represents a conflict of interest in connection with the work
submitted.
Acknowledgements
The authors would like to thank the Outstanding Youth Science
Foundation in Shandong Province (Project No. ZR2019YQ16), the Youth
Science and Technology Innovation Team of Shandong Province (Proj­
ect No. 2019KJF01), the Special Fund for Innovation Teams of Fruit
Trees in Agricultural Technology System of Shandong Province (SDAIT-
9
Scientia Horticulturae 277 (2021) 109806
06-13), the Youth Fund of Shandong Institute of Pomology (2018KY08),
the International Cooperation Project (2019GHZD07) of Qilu University
of Technology (Shandong Academy of Sciences), and the Major Inno­
vation Project of Shandong Biotechnology Technology Innovation
Center (2019JSWGCCXZX001) in China, for financial support to this
research.
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.scienta.2020.109806.
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