Scientia Horticulturae 283 (2021) 110113

Contents lists available at ScienceDirect

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

Combined application of hot water treatment and methyl salicylate

mitigates chilling injury in sweet pepper (Capsicum annuum L.) fruits
Rana Naveed Ur Rehman a, *, Aman Ullah Malik a, Ahmad Sattar Khan a, Mahmood Ul Hasan a,
Raheel Anwar a, Sajid Ali b, Muhammad Wasim Haider a
a
Postharvest Research and Training Centre, Institute of Horticultural Sciences, University of Agriculture, Faisalabad, 38040, Pakistan
b
Department of Horticulture, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, 60800, Pakistan

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

Keywords: Chilling injury (CI) is the major limitation in shelf life extension and quality conservation of sweet pepper fruits
Antioxidant enzymes subjected to below optimum storage temperature (<10 ◦ C). In the current work, the effect of hot water (HW)
Bell pepper treatment (45 ◦ C for 15 min), methyl salicylate (MS, 0.05 mmol L− 1), and the combined treatment (HW + MS) of
Chilling injury
HW and MS was investigated. Results exhibited that HW + MS treated fruits exhibited reduced chilling injury,
Hot water
mass loss, hydrogen peroxide, superoxide anion, and malondialdehyde content, in contrast with control. Like­
Oxidative stress
wise, ascorbic acid concentration, total phenolics content, and radical scavenging capacity were observed to be
markedly higher in HW + MS treated sweet pepper fruits, as compared to control. In addition to this, HW + MS
treatment showed markedly higher activity of peroxidase, superoxide dismutase, catalase, and ascorbate
peroxidase, in contrast with control. In conclusion, our study suggested that pre-storage treatment with HW +
MS could be considered as a potential approach for reducing chilling injury and maintaining a better quality of
sweet pepper fruits during shelf life following cold storage.

1. Introduction Chilling stress disrupts the physiological equilibrium between the

Sweet pepper is an imperative vegetable crop that is widely culti­
vated across the globe. It is admired for its unique taste in fast foods and
rich bioactive substances such as ascorbic acid, phenolic compounds,
and certain antioxidants (Rao et al., 2011; Endo et al., 2019). However,
the skin of sweet pepper makes it highly perishable after harvest. Hence,
it must be kept under low temperature to minimize decay and to
maintain its quality for an extended storage period. The fresh com­
modities including sweet peppers are stored under low-temperature
conditions to reduce the rate of ongoing metabolic processes and thus
their shelf life can be substantially prolonged (Ali et al., 2016). Sweet
generation of reactive oxygen species (ROS) and the enzymatic antiox­
idant system during metabolic processes (Nasef, 2018). The balance
between production and detoxification of ROS determines cell survival
during cold storage. Excessive accumulation of ROS causes oxidation of
polyunsaturated fatty acids and yield malondialdehyde (MDA), the toxic
product that is considered an indicator of the degree of oxidative stress
(Ali et al., 2016, 2020). For the running of metabolic processes smooth
and normal, ROS needs to be scavenged by the antioxidant system. This
antioxidant system comprises of various enzymes such as superoxide
dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and
catalase (CAT) that immediately quench the ROS such as H2O2 and O2–
•

pepper fruits, however, are very sensitive to rapid cooling and undergo
chilling injury (CI) when stored below 7 ◦ C depending upon the cultivar
type, maturity stage, and storage conditions (Ding and Wang, 2018). CI
is a physiological disorder that significantly affects the quality of pro­
duce, limits its storage potential and marketing period (Zhang et al.,
2016). CI symptoms are characterized by fruit surface pitting, sunken
lesions, water loss, water-soaked area, calyx discoloration, and quality
loss (Korkmaz et al., 2010; Hasan et al., 2019).
to overcome the membrane oxidative damage (Huan et al., 2017; Ling
et al., 2018).
Recently, various post-harvest treatments have been reported to
reduce the CI tolerance in sweet pepper and some of the avenues include
the application of certain chemicals (Fung et al., 2004; Korkmaz et al.,
2010; Ding and Wang, 2018), UV-C treatment (Vicente et al., 2005),
intermittent warming (Liu et al., 2015), and hot water (HW) treatment
(Endo et al., 2019). Methyl salicylate (MS) is a methyl ester of salicylic

* Corresponding author.
E-mail address: naveed138@gmail.com (R.N.U. Rehman).

https://doi.org/10.1016/j.scienta.2021.110113
Received 5 August 2020; Received in revised form 24 February 2021; Accepted 2 March 2021
Available online 18 March 2021
0304-4238/© 2021 Elsevier B.V. All rights reserved.
R.N.U. Rehman et al.
acid that has been extensively reported to regulate the chilling stress
response in different crops including loquat, pomegranate, and cherry
tomatoes (Cai et al., 2006; Zhang et al., 2011; Sayyari et al., 2011).
Salicylic acid is biosynthesized in the chloroplast of the cell via the
shikimate pathway and transported to the cytoplasm where it plays an
important role in the regulation of the enzymatic antioxidant system
(Khan et al., 2015). Møller (2001) suggested that the plant responds to
oxidative stress in two lines of defense and first-line comprises of ROS
avoidance that includes alternative oxidase (AOX), and the second line
of defense includes antioxidative enzymes such as POD, APX, SOD, CAT,
and others. Fung et al. (2004) found the AOX transcript levels induced
by MS and methyl jasmonate in sweet pepper correlated with the inci­
dence of chilling injury. Sayyari et al. (2011) reported that MS appli­
cation alleviated CI in pomegranate by up-regulating the hydrophilic
and lipophilic antioxidant activities. Zhang et al. (2011) reported that
MS-induced arginine catabolism is associated with the improvement of
chilling tolerance in cherry tomatoes. It has been reported that
MS-induced up-regulation of the enzymatic system and improvement of
chilling tolerance by reducing the oxidative damage in grapes and
apricot fruits (Khan et al., 2015; Wang et al., 2015). However, the role of
the enzymatic antioxidant system in MS-treated sweet pepper has not
been clearly understood yet.
Pre-storage heat exposure can induce an adaptive response in hor­
ticultural crops to bear low temperature and ameliorate the incidence of
CI (Huan et al., 2017). Intermittent warming by hot air can effectively
improve the postharvest quality of sweet pepper (Liu et al., 2015);
however hot water (HW) is considered a more efficient heat transfer
medium than that of hot air (Huan et al., 2017). In this regard, HW
treatment can be effectively used to hinder the ripening process and
improve CI tolerance in sweet pepper (Endo et al., 2019).
MS is a GRAS (generally recognized as safe) chemical (Giménez
et al., 2016) and it has been reported to ameliorate the negative impacts
of temperature stress on various crops (Cai et al., 2006; Sayyari et al.,
2011; Giménez et al., 2016). Besides this, HW treatment is a physical,
safe, and effective technique to reduce the physiological disorders in
horticultural produce because it maintains the quality for the extended
storage period (Nasef, 2018; Endo et al., 2019). However, the influence
of the combined application of HW and MS to mitigate chilling
injury-related deleterious effects on sweet pepper fruits is still lacking
and needs to be thoroughly investigated. Hence, the objective of this
study was to explore the effect of the combined application of HW and
MS in inducing chilling injury tolerance in sweet pepper fruits.
2. Materials and methods
2.1. Plant materials
Physiologically mature green sweet peppers (Capsicum annuum L. cv.
‘Winner’) were harvested from the greenhouse of MA Agri Farms, Fai­
salabad. Peppers were immediately transported to the laboratory on a
reefer van (15 ± 1 ◦ C). Fruits were sorted to eliminate those with dis­
eases, defects, or over-ripe, and those with uniform shape and size were
selected, washed, air-dried, and used in the experiments.
2.2. Treatments
A total of 900 fruits were used in the experiment. The sweet peppers
were divided into four lots. The first lot was control and simply dipped in
tap water and air-dried at room temperature. The second lot was treated
with HW treatment by dipping the sweet peppers at 45 ◦ C for 15 min and
then cooled to 20 ◦ C as mentioned by Endo et al. (2019). The third lot
was treated with 0.05 mmol L− 1 methyl salicylate (MS). Briefly, fruits
were packed in a hermetically airtight container and MS was spotted
onto filter paper at a vapor concentration of 0.05 mmol L-1. The incu­
bation period was 12 h in darkness at 20 ± 1 ◦ C. The concentration of MS
was optimized based on a preliminary study in which sweet peppers
2
Scientia Horticulturae 283 (2021) 110113
were treated with 0.01 mmol L-1, 0.05 mmol L-1, and 0.1 mmol L-1 of MS.
The results revealed that 0.05 mmol L-1 MS was most effective to reduce
the CI in sweet pepper (data not shown) and hence this optimized
concentration was used in the current study. The fourth lot was treated
with the combined application of HW (former) and MS (later). Sweet
peppers of all lots with corresponding replicates were kept in cold
storage at 5 ± 1 ◦ C with 85–90 % RH for 20 days. Each treatment con­
tained three replicates and each biological replicate had 15 sweet pep­
pers. Following the evaluation of CI, weight loss assessment, overall
visual quality evaluation and relative electrolyte leakage of fruit, a
composite sample of 15 fruit per replicate was diced, frozen, and
powdered with liquid nitrogen to study the rest of the biochemical and
enzymatic parameters. For this, samples were dipped in liquid nitrogen,
packed in aluminum foil and stored at − 80 ◦ C until analyzed. The sweet
pepper fruits were sampled after every five days interval and held for
2-days of conditioning at 20 ± 1 ◦ C as 0, 5 + 2, 10 + 2, 15 + 2, and 20 +
2 days of storage period.
2.3. Evaluation of CI, weight loss, and overall visual quality
CI was visually assessed based on the prevalence of sunken lesions
and surface pitting. The incidence of CI was estimated as given below
and expressed as a percentage (Endo et al. (2019).
CI incidence (%) =
Number of CI fruits
× 100
Total number of fruits
The fresh weight of sweet peppers was measured on each removal of
fruit. The weight loss was measured using the given formula:
Initial weight − Final weight
Weight loss (%) = × 100
Initial weight
The overall visual quality of sweet pepper was assessed by the
methodology reported by Ali et al. (2016). A hedonic scale was used that
comprised of 9 = extremely like, 5 = neither like nor dislike, and 1 =
extremely dislike.
2.4. Relative electrolyte leakage, MDA, H2O2, and O2– contents
•
Relative electrolyte leakage was carried out using methods of Nasef
(2018). The discs from ten fruits were made to obtain a composite
sample and kept in 50 mL double distilled water in the glass beaker at
ambient temperature. After one hour, initial conductivity (EC1) was
measured by digital electric conductivity meter followed by boiling for
10 min to get final electrolyte leakage (EC2). Finally, it was calculated as
(EC1/EC2) × 100.
The malondialdehyde (MDA) content was measured according to
methods described by Rehman et al. (2018) with minor changes and
expressed as μmol Kg− 1 fresh weight. The 1 g frozen sample was thor­
oughly milled in 80 % ethanol (v/v) and centrifuged (Z326 K, Hermle,
Germany) at 12,000 × g for 10 min. The subsequent supernatant (1 mL)
was mixed with 0.65 % trichloroacetic acid (TCA) buffer and heated for
8 min in boiling water and promptly ice-cooled. The assay was centri­
fuged again at 10,000 × g for 5 min and finally, absorbance was
recorded at 600, 450, and 532 nm using a UV–vis spectrophotometer
(UV–1800, Shimadzu, Japan). MDA content was determined using the
following formula:
[ ]
MDA content (mmol Kg − 1) = 6.45 × (A532 − A600 ) − 0.56 × A450
[ ]
Vt × Vr
×
Vs × m
Where, Vt = volume of extract solution; Vr = volume of reaction
mixture; Vs = volume of extraction solution containing reaction mixture
and m = mass of sample.
H2O2 content was assayed according to the methodology of Rehman
R.N.U. Rehman et al. Scientia Horticulturae 283 (2021) 110113

et al. (2017). Briefly, 1 g frozen sample of sweet pepper was ground in

cold TCA buffer and centrifuged (Z326 K, Hermle, Germany) at 12,500
× g for 15 min under pre-cooled condition. The supernatant was sepa­
rated and neutralized to pH = 7.8 with NH4OH and samples were
divided into two parts (A and B). Twenty units of catalase (EC 1.11.1.6)
were added to A and following 20 min incubation, 500 μL colorimetric
reagent was added to A and B. Colorimetric reagent was prepared by
mixing 0.6 mM 4,2-pyridylazo-resorcinol monosodium, 0.6 mM
potassium-titanium oxalate, and water with 1:1:2 ratio. Both assay
mixture A and B were pre-heated at (42 ± 1 ◦ C) for 20 min before taking
absorbance at 508 nm using a UV–vis spectrophotometer (UV–1800,
Shimadzu, Japan), and results were expressed as μmol Kg− 1 fresh
weight.
O2– content was evaluated according to the methods of Ali et al.
•

(2019) with minor modifications and expressed as nmol min− 1 Kg− 1

fresh weight. The 1 g of sample was extracted using pre-chilled 5 mL of
phosphate buffer (pH 7.8; 50 mmol L− 1) containing poly­
vinylpyrrolidone (1%, w/v). The mixture was centrifuged (Z326 K,
Hermle, Germany) at 12,500 × g for 12 min. The supernatant (1 mL) was
mixed with 0.5 mL hydroxylamine hydrochloride (10 mM) and 1 mL of
sodium phosphate buffer (pH 7.6; 100 mM) and incubated for 25 min at
room temperature. Then the reaction mixture (1 mL) was mixed in 1.5
mL of 4-aminobenzene sulphonic acid (12 mM) and 1.5 mL of α-naph­
thylamine (5 mM). After incubation for 25 min, 2.5 mL n-butanol was
added to the reaction mixture, and finally absorbance was immediately
measured at 530 nm.

2.5. Ascorbic acid, TPC, and DPPH-RS activity

Ascorbic acid content was determined according to the titration

method, using 2, 6 dichlorophenol-indophenol indicator, and was
expressed as mg 100 g− 1 fresh weight (Ali et al., 2019).
TPC was assayed according to Vicente et al. (2005) method. The 1 g
of frozen sweet pepper powder was mixed with 5 mL ethanol and
centrifuged (Z326 K, Hermle, Germany) at 10,500 × g for 12 min at 4 ◦ C.
The resultant supernatant (200 μL) was mixed with 1.11 mL distilled
water and 200 μL of Folin–Ciocalteau reagent. After 5 min, a saturated
solution of Na2CO3 was added to the reaction mixer. After incubation for
1 h at room temperature, the absorbance was recorded at 765 nm. The
final results were expressed as mg GAE (gallic acid equivalent) Kg− 1
fresh weight following the standard curve; slope 0.0327 and R2 = 0.99.
The DPPH (1,1-diphenyl-2 picrylhydrazyl) scavenging capacity was
evaluated according to the proposed methodology of Ali et al. (2019) by
observing the variation in absorbance at 517 nm. The reaction mixer
contained free radical of DPPH (0.004 % in ethanol) mixed with 1 g of
sweet pepper milled with pre-chilled 2 mL ethanol and stored for 30 min
in darkness at room temperature. The results were determined according
to the following formula and expressed as DPPH (%):
1 − (the absorbance of sample)
DPPH (%) = × 100
the absorbance of DPPH (blank)
2.6. Assay of enzyme activities
Activities of ascorbate peroxidase (APX), peroxidase (POD), super­
oxide dismutase (SOD), and catalase (CAT) enzymes were analysed by
homogenizing 1 g frozen sample in pre-chilled 5 mL citrate buffer (pH
4.0) and centrifugation (Z326 K, Hermle, Germany) at 12,500 × g for 12
min as described by Ali et al. (2016). The supernatant was separated that
was used for the assessment of the following enzymes.
APX activity was assayed according to Nakano and Asada (1987)
with minor modifications. The reaction mixture contained 150 μL
enzyme extract, 60 mmol L− 1 phosphate buffer (pH 5.4, 300 μL), 0.5
mmol L− 1 L-ascorbate (150 μL), and 0.15 mmol L− 1 H2O2 (150 μL). The
activity was assessed by observing the reaction at 290 nm (oxidation of
L-ascorbate).
Fig. 1. Effect of alone or combined application of hot water (HW) and methyl
salicylate (MS) treatments on chilling injury (A), weight loss (B), and overall
visual quality (C) of sweet pepper fruits. Each bar is the mean ± SE (n = 3).
Results were analyzed using two-way analysis of variance (treatment × storage
period) and different letters show significant differences according to least
significant difference test at P ≤ 0.05.
For the measurement of POD and CAT activity, the methodology of
Ali et al. (2019) was followed. The reaction mixture for the POD activity
contained 300 μL enzyme extract, 0.8 mL guaiacol (2%), 1.4 mL of
phosphate buffer (pH 6.9; 50 mmol L− 1), and 0.5 mL of H2O2 (80 mmol
L− 1) and absorbance was noted at 470 nm on a spectrophotometer.
Similarly, the reaction mixture of CAT contained 2 mL of sodium
phosphate buffer (pH 7.3; 50 mmol L− 1), 0.5 mL of H2O2 (40 mmol L− 1),
0.3 mL enzyme extract and absorbance was observed at 240 nm.
For the measurement of SOD activity, the methodology of Rehman
et al. (2017) was opted. The reaction mixture contained 20 μL of the
enzyme extract, 100 μL of methionine (0.065 M), 200 μL of riboflavin
(20 μmol L− 1), 580 μL of Tris–HCl (pH 7.5; 0.1 M),100 μL of nitroblue
tetrazolium (50 μmol L− 1). Absorbance was noted at 560 nm and an
absorption coefficient of 2.89 mM cm− 1 was used for calculating SOD
activity. Protein contents were determined by using bovine serum al­
bumin as a standard (Bradford, 1976).
2.7. Data analysis
All data were subjected to two-way analysis of variance (treatment
and storage period) using Statistix 8.1 software (USA) and results are
explained as the relative contribution of treatment, storage period and
their interactions by calculating the percentage of total variance from

3
R.N.U. Rehman et al. Scientia Horticulturae 283 (2021) 110113

Table 1
Analysis of variance, percentage of total variance, means ± standard error (SE) and least significant difference (LSD) for factors (treatment, storage period and their
interaction) for the chilling injury (CI), weight loss (WL), overall visual quality (OVQ), electrolyte leakage (EL), malondialdehyde (MDA), ascorbic acid (AA) and total
phenolic content (TPC) in sweet pepper fruits treated with hot water (HW), methyl salicylate (MS) or combine (HW + MS) and stored for 5 + 2, 10 + 2, 15 + 2 and 20 +
2 days.
CI WL OVQ EL MDA AA TPC
Source of Variance
Percentage of total variance

Treatment (T) 24.64* 20.33* 17.21* 24.76* 26.24* 25.68* 24.08*

Storage period (SP) 63.45* 69.04* 69.42* 59.17* 60.31* 59.11* 61.22*
T × SP 8.73* 6.98* 6.15* 11.03* 7.89* 8.51* 8.71*
Error 3.16 3.52 7.20 5.02 5.56 6.69 5.98

Means ± SE
Factors
(Score) (%) (Score) (%) (μmol Kg− 1) (mg 100 g− 1) (mg GAE kg− 1)

T
Control 31.9 ± 2.0 a 5.1 ± 0.3 a 6.0 ± 0.3 c 38.7 ± 2.3 a 30.1 ± 1.8 a 836.7 ± 60.1 d 847.6 ± 59.9 d
HW 22.9 ± 1.9 b 3.9 ± 0.3 b 6.7 ± 0.2 b 31.6 ± 1.8 b 24.1 ± 1.7 b 960.0 ± 56.8 c 994.4 ± 43.1 c
MS 17.5 ± 2.3 c 2.9 ± 0.3 c 7.1 ± .03 b 31.7 ± 1.4 b 20.1 ± 1.7 c 1107.3 ± 60.9 b 1100.8 ± 58.7 b
HW + MS 7.7 ± 1.7 d 1.9 ± 0.3 d 7.9 ± 0.3 a 22.3 ± 1.7 c 15.1 ± 1.9 d 1291.3 ± 63.9 a 1278.0 ± 49.7 a

SP
At harvest 0.0 ± 0.0 e 0.0 ± 0.0 e 8.9 ± 0.4 a 19.3 ± 2.3 e 9.7 ± 1.4 e 1430.0 ± 58.7 a 1450.0 ± 63.6 a
5+2 9.9 ± 1.9 d 2.4 ± 0.3 d 7.7 ± 0.2 b 24.7 ± 1.4 d 17.2 ± 1.8 d 1220.8 ± 55.8 b 1163.0 ± 47.9 b
10 + 2 21.5 ± 2.6 c 3.6 ± 0.4 c 6.8 ± 0.4 c 30.1 ± 1.8 c 23.2 ± 1.7 c 1022.5 ± 52.6 c 1057.5 ± 43.6 b
15 + 2 28.2 ± 2.7 b 5.0 ± 0.5 b 6.2 ± 0.3 c 36.2 ± 1.6 b 27.9 ± 2.2 b 877.5 ± 68.1 d 898.0 ± 66.5 c
20 + 2 40.5 ± 2.6 a 6.3 ± 0.4 a 4.9 ± 0.3 d 45.1 ± 1.8 a 33.7 ± 1.7 a 693.3 ± 66.9 e 707.5 ± 42.5 d

GAE = Gallic acid equivalent.

*
Significant at P ≤ 0.05.

the corresponding Sum of Squares (SS) as performed by Kyriacou et al.
(2021). Mean comparisons were computed by the least significant dif­
ference (LSD) test at P ≤ 0.05. Pearson’s correlation analysis was carried
out by using the SPSS 16.0 software (USA).
3. Results and discussion
3.1. CI, weight loss, and overall visual quality
The CI index showed the severity of damage on sweet pepper fruit
skin that was subjected to below optimum storage temperature. The CI
exhibited as surface pitting appeared in all treatments but was signifi­
cantly higher in control fruits of sweet pepper (Fig. 1A) and relative
variance was substantially influenced by storage period (63.45 %) fol­
lowed by treatment (24.64 %) (Table 1). As the storage period increased,
CI incidence substantially increased in control fruits as compared with
combined HW + MS treatment after 20 + 2 days. The combined HW +
MS treatment was found to be 2.60-fold more effective in controlling the
incidence of CI than individual treatment of HW and MS after 20 + 2
days (Fig. 1A). HW treatment has been reported to mitigate the CI
incidence in several chilling sensitive horticultural crops such as
zucchini, and sweet pepper fruits (Endo et al., 2019; Zhang et al., 2019).
Nasef (2018) reported that HW treatment can induce chilling tolerance
by maintaining the membrane phospholipids at a higher level in cu­
cumber fruits. Our results are consistent with the findings of Sayyari
et al. (2011) and Zhang et al. (2011) who found that MS treatment
significantly alleviated the symptoms and degree of CI in pomegranate
and cherry tomatoes by maintaining higher membrane integrity. In our
case, the combined application of HW and MS possibly had a synergistic
effect in reducing the incidence of CI in sweet pepper by maintaining the
the incidence of CI (Table 3). HW treatment has been extensively re­
ported to impede weight loss in different horticultural commodities as
reported earlier in cucumber (Nasef, 2018) and zucchini fruits (Zhang
et al., 2019). Rodov et al. (1995) suggested that HW treatment can bring
the microscopic structural changes by reducing the cuticular crakes on
fruit surface and improving the cellular barrier function against water
loss thus maintains the quality for a longer period. Besides this, MS
treatment delays the rate of respiration which might be the key reason
for the reduction of weight loss as noted previously in sweet cherry
(Giménez et al., 2016). Hence, sweet pepper fruits treated with HW +
MS showed significantly reduced weight loss during the post-storage
conditioning period.
Variation in the overall visual quality was mainly influenced by
storage period and treatment which determined 69.42 % and 17.21 % of
the total variance, respectively (Table 1). A precipitous reduction in
visual quality was observed in the control group (Fig. 1C). The control
group lost the market potential after 10 + 2 days (as the score was less
than 6); while HW + MS treated fruits had markedly higher overall vi­
sual quality score until day-20 + 2 of storage (Fig. 1C). Besides this,
overall visual quality was severely affected (r = − 0.99; P ≤ 0.01) by the
occurrence of CI (Table 3). MS treatment delays the post-harvest
senescence by up-regulating the antioxidant potential and reducing
the lipid peroxidation. Hence, the storage potential of horticultural
commodities can be extended for a longer period while maintaining the
overall quality (Giménez et al., 2016). So, the combined treatment of
HW and MS extended the shelf life of sweet peppers by improving
chilling tolerance and maintaining the overall visual quality for an
extended storage period compared with the control.
3.2. Relative electrolyte leakage, MDA, H2O2, and O2– contents
•

membrane integrity during cold-storage.

Weight loss has a negative impact on market value as well as con­
sumer acceptability of fresh produce (Ali et al., 2020). Weight loss of the
treated and non-treated sweet pepper fruits increased as the storage
period proceeded (Fig. 1B). Fresh weight loss was significantly increased
in non-treated fruits followed by HW, MS, and HW + MS treatments. On
day-20 + 2, HW + MS treatment showed a markedly (2.05-fold) reduced
weight loss as compared to the control fruits (Fig. 1B). In addition to
this, weight loss showed a positive correlation (r = 0.99; P ≤ 0.01) with
As far as relative electrolyte leakage and MDA are concerned, all
treatments are mainly influenced by 59.17 % and 60.31 % in total
variance respectively, due to storage period (Table 1). Overall after 20 +
2 days, non-treated fruits had significantly higher relative electrolyte
leakage and MDA (1.94-fold and 1.85-fold), as compared with HW + MS
treatment (Fig. 2A, B). On the other hand, HW + MS treatment showed
the lowest MDA content and relative electrolyte leakage in contrast with
the individual application of HW and MS on day-20 + 2 (Fig. 2A, B).

4
R.N.U. Rehman et al.
Fig. 2. Effect of alone or combined application of hot water (HW) and methyl
salicylate (MS) treatments on electrolyte leakage (A), malondialdehyde (B),
H2O2 (C) and O2– (D) contents of sweet pepper fruits. Each bar is the mean ±
•
SE (n = 3). Results were analyzed using two-way analysis of variance (treat­
ment × storage period) and different letters show significant differences ac­
cording to least significant difference test at P ≤ 0.05.
Moreover, electrolyte leakage and MDA content showed a significant
positive correlation (r = 0.99 and 0.98 respectively; P ≤ 0.01) with CI
incidence (Table 3).
MDA is produced by the oxidative reactions and enzymatic degra­
dation of poly-unsaturated fatty acids in the cellular membranes. This
end product of lipid peroxidation coupled with relative electrolyte
leakage is considered as an excellent marker of oxidative damage
induced by chilling stress (Ali et al., 2016). Lipid peroxidation is perhaps
one of the earlier phenomena in the expression of CI. The exposure and
severity of CI impose a negative impact on the structure of the cellular
membranes and thus degrades the membrane integrity due to lipid
peroxidation (Endo et al., 2019). The subsequent alternations in the
cellular membranes lead to disturbance in its functional capacity and
proteins or phospholipids molecules associated with it. Our results
5
Scientia Horticulturae 283 (2021) 110113
confirmed the findings of Zhang et al. (2016) and Zhang et al. (2019) as
they reported that the application of HW markedly reduced the MDA
content in loquat and zucchini fruits.
H2O2 content was substantially affected by the storage period (67.84
%) followed by the treatment (18.60 %) and their interaction (treatment
× storage period) (9.08 %) (Table 2). The accumulation of H2O2 content
was significantly high (2.03-fold) in the control group as compared to
HW + MS treated fruits on day-20 + 2 (Fig. 2C). The alone applications
of HW and MS also showed the suppressed accumulation of H2O2
throughout the storage period; however, the suppression was substan­
tially less in contrast to the combined HW + MS treatment (Fig. 2C). The
CI showed a significant positive correlation (r = 0.99; P ≤ 0.01) with
H2O2 and MDA (Table 3). H2O2 is a reactive constituent of metabolism
and it can damage the membranes by inducing the oxidative breakdown
of the double bond in the structural lipid molecules such as fatty acids.
The CI induces the generation and accumulation of H2O2 that cause
oxidative damage due to lipid peroxidation and yield higher MDA
(Zhang et al., 2019). The higher accumulation of H2O2 in plant cells
leads to disruption of metabolic processes, loss of cellular integrity, and
aggravation of oxidative damage (Lin et al., 2020). H2O2 plays a dual
role in plant cells i.e. it also plays an important role in the stress toler­
ance signaling cascade to tap the potential of the antioxidant defense
system (Huan et al., 2017). The consistent accumulation of H2O2 may be
intimately related to the redox balance inside the cell as it is closely
associated with modulation of gene expression and thus strengthen the
defense responses in the plant cell (Endo et al., 2019). Alone or com­
bined treatment of HW and MS, possibly inhibited H2O2 production due
to suppressed CI and higher enzymes (APX and CAT) activity as these
antioxidant enzymes decompose H2O2 into water and oxygen.
As expected, O2– content significantly increased in HW, MS as well
•
as combined HW + MS treated fruits and control group largely influ­
enced by the storage period (66.69 %) followed by treatment (21.52 %)
(Table 2). However, the production of O2– radical was more obvious in
•
the control group. Alone HW and MS treatments also showed higher O2–
•
but significantly lower than the control fruits. In general, the control
group had 2.13-fold higher production of O2– compared with HW + MS
•
treatment on day-20 + 2 (Fig. 2D). Generation of O2– is considered as a
•
marker of oxidative stress and its production may be reduced with
suitable physical and chemical treatments. Wang et al. (2015) reported
the reduced O2– content in response to exogenous application of sali­
•
cylic acid in apricot fruits. Authors also found that O2– production
•
instigated the activity of SOD. SOD enzyme converts free radical (O2– )
•
into H2O2 that is further catalyzed by the activities of CAT or APX (Ali
et al., 2019). Similar results have been reported by Huan et al. (2017) as
they found that pre-treatment with HW significantly reduced the over­
production of O2– in peach fruits during refrigerated storage. Similarly,
•
the exogenous application of salicylic acid significantly reduced the
production of O2– in loquat fruits (Cai et al., 2006). Hence the syner­
•
gistic impact of HW + MS treatment impeded chilling stress by reducing
the production of O2– and H2O2 and inducing higher activity of anti­
•
oxidative enzymes.
3.3. Ascorbic acid content, TPC, and DPPH-RS activity
The variation in the ascorbic acid content was significantly affected
by the storage period (59.11 %) followed by treatments (25.68 %) and
their interaction (8.51 %) (Table 1). In general, untreated control sweet
peppers exhibited a sharp decrease in ascorbic acid content from day-
0 to day-20 + 2 (Fig. 3A). However, the loss of ascorbic acid content
in sweet peppers was markedly less in HW + MS treatment followed by
alone application of MS and HW. After 20 + 2 days of storage, HW + MS
treatment maintained 2.49-fold higher content of ascorbic acid,
compared to control (Fig. 3A). In addition to this, ascorbic acid showed
substantial negative association with CI (r = − 0.97; P ≤ 0.05), elec­
trolyte leakage (r = − 0.98; P ≤ 0.01), and MDA content (r = − 0.94; P ≤
0.05) (Table 3). Ascorbic acid plays a significant part in quenching ROS
R.N.U. Rehman et al. Scientia Horticulturae 283 (2021) 110113

Table 2
Analysis of variance, percentage of total variance, means ± standard error (SE) and least significant difference (LSD) for factors (treatment, storage period and their
interaction) for the H2O2 and O−2 , antioxidant activity (AoA), ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in sweet
•

pepper fruits treated with hot water (HW), methyl salicylate (MS) or combine (HW + MS) and stored for 5 + 2, 10 + 2, 15 + 2 and 20 + 2 days.
H2O2 O–•
2 AoA APX CAT POD SOD
Source of Variance
Percentage of total variance

Treatment (T) 18.60* 21.52* 25.87* 23.13* 27.67* 23.70* 33.44*

Storage period (SP) 67.84* 66.69* 59.02* 60.19* 56.57* 62.39* 49.14*
T × SP 9.08* 7.27* 8.40* 9.07* 9.88* 6.62* 10.24*
Error 4.54 4.49 6.71 7.59 5.88 7.28 7.17

Means ± SE
Factors
(μmol Kg− 1) (nmol min− 1
Kg− 1) (%) (U mg− 1
protein) (U mg− 1
protein) (U mg− 1
protein) (U mg− 1
protein)

T
Control 42.57 ± 2.4 a 27.45 ± 2.2 a 49.5 ± 3.7 d 654.6 ± 29.3 c 88.2 ± 6.3 d 768.2 ± 41.1 c 529.3 ± 30.2 d
HW 37.51 ± 2.4 b 21.54 ± 1.7 b 57.1 ± 3.1 c 716.1 ± 25.2 b 103.1 ± 5.7 c 886.1 ± 38.2 b 617.7 ± 27.4 c
MS 30.68 ± 2.4 c 17.54 ± 1.7 c 64.6 ± 3.4 b 752.2 ± 32.1 b 121.0 ± 6.3 b 952.3 ± 44.5 b 671.3 ± 28.2 b
HW + MS 23.65 ± 2.6 d 10.82 ± 2.1 d 74.4 ± 3.5 a 868.7 ± 37.1 a 144.1 ± 8.1 a 1083.4 ± 51.4 a 786.7 ± 35.1 a

SP
At harvest 14.09 ± 2.6 e 5.62 ± 1.5 e 83.7 ± 3.8 a 948.6 ± 31.2 a 163.1 ± 6.4 a 1233.6 ± 47.7 a 815.4 ± 29.2 a
5+2 24.21 ± 2.4 d 12.21 ± 1.9 d 69.9 ± 2.5 b 810.4 ± 28.3 b 127.1 ± 5.6 b 984.8 ± 41.1 b 719.6 ± 27.6 b
10 + 2 35.25 ± 2.3 c 16.37 ± 1.7 c 61.8 ± 3.5 c 738.5 ± 30.4 c 110.6 ± 7.4 c 910.1 ± 37.7 b 664.6 ± 28.8 b
15 + 2 40.81 ± 2.4 b 27.00 ± 2.2 b 50.5 ± 3.8 d 656.8 ± 33.9 d 93.7 ± 6.9 d 790.8 ± 47.8 c 558.3 ± 33.6 c
20 + 2 53.66 ± 2.6 a 35.48 ± 2.2 a 41.1 ± 3.5 e 585.1 ± 30.7 e 76.1 ± 6.5 e 693.1 ± 44.6 d 498.3 ± 31.9 c
*
Significant at P ≤ 0.05.

Table 3
Correlation coefficients among different attributes of sweet pepper fruits.
CI WL AA TPC AoA OVQ MDA EL H2O2 O2–• APX CAT POD

WL 0.99**
AA − 0.97* − 0.39**
TPC − 0.99** − 0.69** 0.77*
AoA − 0.98* − 0.39** 0.99** 0.98*
OVQ − 0.99** − 1.00** 0.98* 0.49** 0.59ns
MDA 0.98** 0.56ns − 0.94* − 0.77* − 0.74* − 0.66*
EL 0.99** 0.28ns − 0.98** − 0.69** − 0.67* − 0.48* 0.99**
H2O2 0.99** 0.17ns − 0.76* − 0.98* − 0.96* − 0.77* 0.99** 0.99**
O−2 • 0.96* 0.55* − 0.69* − 0.97* − 0.99** − 0.88* 0.93* 0.96* 0.95*
APX − 0.98* − 0.69ns 0.49** 0.59ns 0.99** 0.59ns − 0.94* − 0.98* − 0.96* − 0.29ns
CAT − 0.94* − 0.26ns 0.29ns 0.44ns 0.98* 0.65ns − 0.90* − 0.94* − 0.92* − 0.19ns 0.97*
POD − 0.97** − 0.46ns 0.56* 0.65ns 0.96* 0.85* − 0.98* − 0.98* − 0.98* − 0.46ns 0.25ns 0.95*
SOD − 0.99** − 0.27ns 0.25ns 0.37ns 0.95* 0.57* − 0.99** − 0.99** 1.00** − 0.94* 0.45ns 0.91* 0.98*

CI = chilling injury; WL = weight loss; AA = ascorbic acid; TPC = total phenolic contents; AoA = antioxidant activity; OVQ = overall visual quality; MDA =
malondialdehyde; EL = electrolyte leakage; APX = ascorbate peroxidase; CAT = catalase; POD = peroxidase; SOD = superoxide dismutase. ns = non-significant. *, **
Significant at P ≤ 0.05, and 0.01, respectively.

in the plant cells. As an antioxidant, ascorbic acid works for the detox­
ification of free radicals. The relative change in ascorbic acid content can
also be considered important for the assessment of the quality of horti­
cultural produce. Our results were consistent with the findings of Zhang
et al. (2019) who noted that the amount of ascorbic acid decreased in
zucchini fruits during cold storage because it neutralizes free radicals in
the plant tissues. Likewise, Huang et al. (2008) reported that the
pre-storage treatment of navel orange fruits with salicylic acid showed
higher ascorbic acid content. The combined application of salicylic acid
and CaCl2 also enhanced the ascorbic acid content in sweet peppers due
to the reduced activities of ascorbic acid oxidase (Rao et al., 2011). In
addition to this, combine treatment of HW and glycine betaine main­
tained higher levels of ascorbic acid in loquat fruits during cold storage
(Zhang et al., 2016). However, reduced loss of ascorbic acid in sweet
peppers treated with HW + MS may be due to the low permeability of
oxygen into the cell thus diminishing the oxidation of ascorbic acid
(Sayyari et al., 2011).
Variance of TPC and DPPH scavenging activity was mainly deter­
mined by storage period (61.22 % and 59.02 % respectively) and
treatment (24.08 % and 25.87 % respectively) (Tables 1 and 2).
Nevertheless, the declining rate of DPPH scavenging activity and TPC
was considerably lower in HW + MS treatment followed by MS and HW
alone, compared to control. Although the alone application of MS and
HW retained relatively higher DPPH scavenging activity, and TPC but
the impact was significantly better in the combined HW + MS treatment.
In contrast, untreated fruits showed a sharp decline in DPPH scavenging
activity, and TPC as the storage period proceeded. In general, after 20 +
2 days, HW + MS treated fruits showed 2.32-fold and 2.31-fold higher
scavenging activity of DPPH radical, and TPC respectively, compared to
untreated control (Fig. 3B and C). The DPPH scavenging activity and
TPC also showed a significant negative correlation (r = − 0.99; r =
− 0.98; P ≤ 0.01) with CI incidence (Table 3).
TPC of sweet peppers decreased in the control group at a higher rate
as compared with alone HW, MS, and the combined HW + MS treat­
ment. The alone MS and HW also showed lower TPC in sweet peppers,
however, it was noticeably higher as compared with the control fruits.
HW treatment reduces the loss of phenolic compounds by maintaining
the integrity of the vacuolar membranes (Nasef, 2018). The scientific
literature available for the elucidation of physiological cross-talk be­
tween MS and TPC accumulation is limited (Sayyari et al., 2011).
Nevertheless, it has been reported that the exogenous application of MS
may trigger the activities of phenylalanine ammonia-lyase, one of the

6
R.N.U. Rehman et al.
Fig. 3. Effect of alone or combined application of hot water (HW) and methyl
salicylate (MS) treatments on ascorbic acid (A), total phenolic content (B), and
DPPH scavenging activity (C) of sweet pepper fruits. Each bar is the mean ± SE
(n = 3). Results were analyzed using two-way analysis of variance (treatment ×
storage period) and different letters show significant differences according to
least significant difference test at P ≤ 0.05.
major enzymes of the phenylpropanoid pathway involved in the
biosynthesis of various phenolic compounds (Rehman et al., 2017,
2018). Thus, despite decreasing content of TPC along with the pro­
gressed storage period, the consistent biosynthesis maintains its content
high as compared to the control group. Giménez et al. (2016) reported
the MS-induced accumulation of TPC in sweet cherry. In addition to
TPC, DPPH scavenging activity was also higher in HW + MS treatment.
This may be due to the higher levels of membrane integrity and
consistent accumulation of hydrophilic antioxidant compounds such as
TPC and ascorbic acid in the vacuole of pepper fruits in response to HW
and pre-treatment with salicylic acid (Wang et al., 2015). Our results
corroborate the findings of Sayyari et al. (2011) and Wang et al. (2015)
who observed that the exogenous application of salicylic acid can
enhance the accumulation of hydrophilic antioxidants such as phenolic
acids thus improving the health-related compounds in the stored hor­
ticultural commodities.
3.4. Activities of POD, APX, SOD, and CAT
The activities of POD, CAT, APX, and SOD constantly declined from
day-5 + 2 to day-20 + 2 (Fig. 4A–D). The fruits of the control group
exhibited the lowest activities of the said enzymes. However, their ac­
tivities were significantly higher in HW + MS treated sweet peppers
followed by MS and HW treated fruits. Variation in APX, CAT, POD, and
7
Scientia Horticulturae 283 (2021) 110113
Fig. 4. Effect of alone or combined application of hot water (HW) and methyl
salicylate (MS) treatments on ascorbate peroxidase (A), catalase (B), peroxidase
(C), and superoxide dismutase (D) enzymes activities of sweet pepper fruits.
Each bar is the mean ± SE (n = 3). Results were analyzed using two-way
analysis of variance (treatment × storage period) and different letters show
significant differences according to least significant difference test at P ≤ 0.05.
SOD activities had been greatly affected by the storage period that
accounted for 60.19 %, 56.57 %, 62.39 % and 49.14 % of total variance,
respectively (Table 2). Overall, on day-20 + 2, activities of APX, CAT,
POD, and SOD were 1.55-fold, 3.01-fold, 1.67-fold, and 1.92-fold higher
in HW + MS treated fruits than in the control group, respectively
(Fig. 4A–D). In addition, CI incidence showed significant negative cor­
relation with APX (r = − 0.98; P ≤ 0.05), CAT (r = − 0.94; P ≤ 0.05), POD
(r = − 0.97; P ≤ 0.01) and SOD (− 0.99; P ≤ 0.01) enzymes activities
(Table 3).
The antioxidant enzyme system played an imperative role in the
scavenging of ROS (O2– and H2O2) in sweet pepper fruits treated with
•
HW + MS. Similar to our work, numerous other researchers reported
that HW treatment up-regulates the activities of APX, CAT, POD, and
SOD enzyme as found in cucumber (Nasef, 2018), sweet pepper (Endo
et al., 2019), peach (Huan et al., 2017), and zucchini (Zhang et al.,
R.N.U. Rehman et al.
2019). Huan et al. (2017) suggested that HW treatment can improve the
activities of antioxidant enzymes by transcriptional up-regulation of the
related genes as observed in peach fruits. Our results of CAT activity
were not in line with the findings of Sánchez-Bal et al. (2012) who
showed a consistent decrease in CAT activity in sweet pepper stored
under chilling temperature. They found that the CAT enzyme is located
inside the peroxisome and during chilling stress; the peroxisome disin­
tegrates, and thus down-regulates the abundance of CAT protein
(Sánchez-Bal et al., 2012). In our case, the combined application of HW
and MS probably reduced the decrease of CAT activity by up-regulation
of CAT associated genes as reported by Huan et al. (2017). Similarly,
higher activity of APX may be due to reduced senescence that conserved
higher APX activity and ameliorated CI associated stress by quenching of
ROS (Sato et al., 2001). In addition to this, higher activity of SOD and
POD enzymes was also closely associated with the reduction of CI owing
to reduced H2O2 and O2– related deleterious effects as noted in grapes
•
(Khan et al., 2015). The enhanced activities of antioxidant enzymes can
improve the shelf life as well as conserve the nutraceutical compounds of
fruits and vegetables (Ling et al., 2018).
4. Conclusion
In conclusion, HW treatment of sweet peppers conserved membrane
integrity by reducing the de-compartmentalization of cell membranes
and maintained higher content of TPC and other antioxidants. Similarly,
the exogenous application of MS enhanced CI tolerance in sweet peppers
by improving the enzymatic antioxidant system and thus reduced the
lipid peroxidation and other oxidative stress indicators. HW treatment
probably enhanced the impact of MS application thus, the integrated
impact of HW + MS was more appropriate for mitigation of CI and
maintenance of overall quality for the extended storage period. Thus,
pre-storage combined treatment of sweet peppers with HW + MS could
be considered potentially appropriate for CI amelioration and quality
conservation of sweet pepper fruits.
Authors contribution
Rana Naveed Ur Rehman and Mahmood Ul Hasan designed the
research and performed the experiments; Raheel Anwar and Sajid Ali
analyzed data; Rana Naveed Ur Rehman and Muhammad Wasim Haider
wrote the manuscript; Aman Ullah Malik provided ideas; Ahmad Sattar
Khan revised the manuscript; all authors read and approved the final
manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors are highly grateful to Higher Education Commission,
Pakistan for funding the project (#2315) entitled, ‘Postharvest Shelf life
Management of Sweet Pepper (Capsicum annuum L.)’. We are also very
thankful to Irfan Nawaz from MA Agri Farms, Faisalabad for providing
us sweet pepper fruits.
References
Ali, S., Khan, A.S., Malik, A.U., Shahid, M., 2016. Effect of controlled atmosphere storage
on pericarp browning, bioactive compounds and antioxidant enzymes of litchi fruits.
Food Chem. 206, 18–29. https://doi.org/10.1016/j.foodchem.2016.03.021.
Ali, S., Khan, A.S., Nawaz, A., Anjum, M.A., Naz, S., Ejaz, S., Hussain, S., 2019. Aloe vera
gel coating delays postharvest browning and maintains quality of harvested litchi
fruit. Postharvest Biol. Technol. 157, 110960. https://doi.org/10.1016/j.
postharvbio.2019.110960.
Ali, S., Khan, A.S., Anjum, M.A., Nawaz, A., Naz, S., Ejaz, S., Hussain, S., 2020. Effect of
postharvest oxalic acid application on enzymatic browning and quality of lotus
8
Scientia Horticulturae 283 (2021) 110113
(Nelumbo nucifera Gaertn.) root slices. Food Chem. 312, 126051 https://doi.org/
10.1016/j.foodchem.2019.126051.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilising the principle of protein-dye binding. Anal. Biochem.
72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.
Cai, C., Li, X., Chen, K.S., 2006. Acetylsalicylic acid alleviates chilling injury of
postharvest loquat (Eriobotrya japonica Lindl.) fruit. Eur. Food Res. Technol. 223,
533–539. https://doi.org/10.1007/s00217-005-0233-5.
Ding, F., Wang, R., 2018. Amelioration of postharvest chilling stress by trehalose in
pepper. Sci. Hortic. 232, 52–56. https://doi.org/10.1016/j.scienta.2017.12.053.
Endo, H., Miyazaki, K., Ose, K., Imahori, Y., 2019. Hot water treatment to alleviate
chilling injury and enhance ascorbate-glutathione cycle in sweet pepper fruit during
postharvest cold storage. Sci. Hortic. 257, 108715. https://doi.org/10.1016/j.
scienta.2019.108715.
Fung, R.W.M., Wang, C.Y., Smith, D.L., Gross, K.C., Tian, M., 2004. MeSA and MeJA
increase steady-state transcript levels of alternative oxidase and resistance against
chilling injury in sweet peppers (Capsicum annuum L.). Plant Sci. 166, 711–719.
https://doi.org/10.1016/j.plantsci.2003.11.009.
Giménez, M.J., Valverde, J.M., Valero, D., Zapata, P.J., Castillo, S., Serrano, M., 2016.
Postharvest methyl salicylate treatments delay ripening and maintain quality
attributes and antioxidant compounds of ‘Early Lory’ sweet cherry. Postharvest Biol.
Technol. 117, 102–109. https://doi.org/10.1016/j.postharvbio.2016.02.006.
Hasan, M.U., Rehman, R.N.U., Malik, A.U., Haider, M.W., Ahmed, Z., Khan, A.S.,
Anwar, R., 2019. Pre-storage application of L-arginine alleviates chilling injury and
maintains postharvest quality of cucumber (Cucumis sativus). J. Hortic. Sci. Technol.
2 (4), 102–108.
Huan, C., Han, S., Jiang, L., An, X., Yu, M., Ma, R., Yu, Z., 2017. Postharvest hot air and
hot water treatments affect the antioxidant system in peach fruit during refrigerated
storage. Postharvest Biol. Technol. 126, 1–14. https://doi.org/10.1016/j.
postharvbio.2016.11.018.
Huang, R.H., Liu, J.H., Lu, Y.M., Xia, R.X., 2008. Effect of salicylic acid on the
antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck)
at different storage temperatures. Postharvest Biol. Technol. 47, 168–175. https://
doi.org/10.1016/j.postharvbio.2007.06.018.
Khan, M.I.R., Fatma, M., Per, T.S., Anjum, N.A., Khan, N.A., 2015. Salicylic acid-induced
abiotic stress tolerance and underlying mechanisms in plants. Front. Plant Sci. 6,
462. https://doi.org/10.3389/fpls.2015.00462.
Korkmaz, A., Korkmaz, Y., Demirkıran, A.R., 2010. Enhancing chilling stress tolerance of
pepper seedlings by exogenous application of 5-aminolevulinic acid. Environ. Exp.
Bot. 67, 495–501. https://doi.org/10.1016/j.envexpbot.2009.07.009.
Kyriacou, M.C., Antoniou, C., Rouphael, Y., Graziani, G., Kyratzis, A., 2021. Mapping the
primary and secondary metabolomes of Carob (Ceratonia siliqua L.) fruit and its
postharvest antioxidant potential at critical stages of ripening. Antioxidants 10, 57.
https://doi.org/10.3390/antiox10010057.
Lin, X., Wang, L., Hou, Y., Zheng, Y., Jin, P., 2020. A combination of melatonin and
ethanol treatment improves postharvest quality in bitter melon fruit. Foods 9, 1376.
https://doi.org/10.3390/foods9101376.
Ling, C., Xu, J., Shao, S., Wang, L., Jin, P., Zheng, Y., 2018. Effect of ultrasonic treatment
combined with peracetic acid treatment reduces decay and maintains quality in
loquat fruit. J. Food Quality 7564056, 8. https://doi.org/10.1155/2018/7564056.
Liu, L., Wei, Y., Shi, F., Liu, C., Liu, X., Ji, S., 2015. Intermittent warming improves
postharvest quality of bell peppers and reduces chilling injury. Postharvest Biol.
Technol. 101, 18–25. https://doi.org/10.1016/j.postharvbio.2014.11.006.
Møller, I.M., 2001. Plant mitochondria and oxidative stress: electron transport, NADPH
turnover, and metabolism of reactive oxygen species. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 52, 561–591. https://doi.org/10.1146/annurev.arplant.52.1.561.
Nakano, J., Asada, K., 1987. Purification of ascorbate peroxidase in spinach chloroplasts:
its inactivation in ascorbate depleted medium and reactivation by
monodehydroascorbate radical. Plant Cell Physiol. 28, 131–140. https://doi.org/
10.1016/S0168-9452(97)00063-0.
Nasef, I.N., 2018. Short hot water as safe treatment induces chilling tolerance and
antioxidant enzymes, prevents decay and maintains quality of cold-stored
cucumbers. Postharvest Biol. Technol. 138, 1–10. https://doi.org/10.1016/j.
postharvbio.2017.12.005.
Rao, T.V.R., Gol, N.B., Shah, K.K., 2011. Effect of postharvest treatments and storage
temperatures on the quality and shelf life of sweet pepper (Capsicum annum L.). Sci.
Hortic. 132, 18–26. https://doi.org/10.1016/J.SCIENTA.2011.09.032.
Rehman, R.N.U., You, Y., Zhang, L., Goudia, B.D., Khan, A.R., Li, P., Ma, F., 2017. High
temperature induced anthocyanin inhibition and active degradation in Malus
profusion. Front. Plant Sci. 8, 1401. https://doi.org/10.3389/fpls.2017.0140.
Rehman, R.N.U., You, Y., Ali, S., Wang, Y., Zhang, L., Li, P., Ma, F., 2018. Phenolic
compounds as biochemical markers of senescence in woody ornamental flowers of
Malus crabapple. Hortic. Environ. Biotechnol. 59 (1), 1–10. https://doi.org/
10.1007/s13580-018-0001-7.
Rodov, V., Ben-Yehoshua, S., Albagli, R., Fang, D.Q., 1995. Reducing chilling injury and
decay of stored citrus fruit by hot water dips. Postharvest Biol. Technol. 5, 119–127.
https://doi.org/10.1016/0925-5214(94)00011-G.
Sánchez-Bal, P., Egea, I., Sánchez-Ballesta, M.T., Martinez-Madrid, C., Fernandez-
Garcia, N., Romojaro, F., Olmos, E., Estrella, E., Bolarín, M.C., Borja Flores, F., 2012.
Understanding the mechanisms of chilling injury in bell pepper fruits using the
proteomic approach. J. Proteom. 75, 5463–5478. https://doi.org/10.1016/j.
jprot.2012.06.029.
Sato, Y., Murakami, T., Funatsuki, H., Matsuba, S., Sayuyama, H., Tanida, M., 2001. Heat
shock-mediated APX gene expression and protection against chilling injury in rice
seedlings. J. Exp. Bot. 52, 145–151. https://doi.org/10.1093/jexbot/52.354.145.
R.N.U. Rehman et al.
Sayyari, M., Babalar, M., Kalantari, S., Martínez-Romero, D., Guillén, F., Serrano, M.,
Valero, D., 2011. Vapour treatments with methyl salicylate or methyl jasmonate
alleviated chilling injury and enhanced antioxidant potential during postharvest
storage of pomegranates. Food Chem. 124, 964–970. https://doi.org/10.1016/j.
foodchem.2010.07.036.
Vicente, Ariel R., Pineda, Carlos, Lemoine, Laura, Civello, Pedro M., Martinez, Gustavo
A., Chaves, Alicia R., 2005. UV-C treatments reduce decay, retain quality and
alleviate chilling injury in pepper. Postharvest Biol. Technol. 35, 69–78. https://doi.
org/10.1016/j.postharvbio.2004.06.001.
Wang, Z., Ma, L., Zhang, X., Xu, L., Cao, J., Jiang, W., 2015. The effect of exogenous
salicylic acid on antioxidant activity, bioactive compounds and antioxidant system in
apricot fruit. Sci. Hortic. 181, 113–120. https://doi.org/10.1016/j.
scienta.2014.10.055.
Scientia Horticulturae 283 (2021) 110113
Zhang, X., Shen, L., Li, F., Meng, D., Sheng, J., 2011. Methyl salicylate-induced arginine
catabolism is associated with up-regulation of polyamine and nitric oxide levels and
improves chilling tolerance in cherry tomato fruit. J. Agric. Food Chem. 59,
9351–9357. https://doi.org/10.1021/jf201812r.
Zhang, Y., Jin, P., Huang, Y., Shan, T., Wang, L., Li, Y., Zheng, Y., 2016. Effect of hot
water combined with glycine betaine alleviates chilling injury in cold-stored loquat
fruit. Postharvest Biol. Technol. 118, 141–147. https://doi.org/10.1016/j.
postharvbio.2016.04.010.
Zhang, M., Liu, W., Li, C., Shao, T., Jiang, X., Zhao, H., Ai, W., 2019. Postharvest hot
water dipping and hot water forced convection treatments alleviate chilling injury
for zucchini fruit during cold storage. Sci. Hortic. 249, 219–227. https://doi.org/
10.1016/j.scienta.2019.01.058.