Food Chemistry 221 (2017) 16501657

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Melatonin treatment attenuates postharvest decay and maintains

nutritional quality of strawberry fruits (Fragaria
anannasa cv. Selva)
by enhancing GABA shunt activity
Morteza Soleimani Aghdam a,, Javad Rezapour Fard b
a
b

Young Researchers and Elite Club, Ahar Branch, Islamic Azad University, Ahar, Iran
Department of Horticultural Sciences, Faculty of Agricultural Sciences, Urmia University, Urmia, Iran

a r t i c l e

i n f o

Article history:
Received 24 August 2016
Received in revised form 16 October 2016
Accepted 26 October 2016
Available online 27 October 2016
Keywords:
c-Aminobutyric acid transaminase
Energy status
Phenylalanine ammonia lyase
Postharvest decay
Nutritional quality

a b s t r a c t
Fresh strawberry fruits as perishable commodities have a short postharvest life and are prone to postharvest fungal decay. In this study, the impact of 0, 1, 10, 100 and 1000 lmol/L melatonin on attenuating
fungal decay and maintaining nutritional quality of strawberry fruits was investigated during storage
at 4 C for 12 days. Melatonin treatment at 100 lmol/L triggered H2O2 accumulation, which result from
higher superoxide dismutase (SOD) activity, associated with lower catalase (CAT) and ascorbate peroxidase (APX) activities, leading to fruits with lower decay. Higher H2O2 accumulation was concurrent with
higher phenylalanine ammonia lyase (PAL) enzyme activity leading to higher total phenols and
anthocyanins accumulation along with higher DPPH scavenging capacity. Also, strawberry fruits treated
with melatonin exhibited higher c-aminobutyric acid transaminase (GABA-T) enzyme activity which
ensured sufficient ATP supplying leading to higher unsaturated/saturated fatty acids (unSFA/SFA) ratio.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Strawberries as important fruits produced in commercial scale
in the world and Iran with 7,739,622 and 33,752 tons of production
rates, respectively (FAOSTAT, 2013), are perishable commodities
which have a short postharvest life and are prone to postharvest
fungal decay by Botrytis cinerea and Rhizopus stolonifer leading to
losses of quality and quantity (Hashmi, East, Palmer, & Heyes,
2013). The use of synthetic fungicides for controlling postharvest
fungal decay in strawberry fruits may bring potential risks to consumers and the environment. Therefore, there is a conspicuous
need for alternative strategies for attenuation decay and extending
postharvest life of strawberry fruits (Romanazzi, Smilanick,
Feliziani, & Droby, 2016).
Since endogenous melatonin content increases in response to
abiotic and biotic stress, melatonin is considered as an endogenous
signaling molecule for attenuating abiotic and biotic stress such as
salt, drought, cold, and pathogens (Arnao & Hernndez-Ruiz, 2015;
Tan et al., 2012). Higher melatonin accumulation in horticultural
commodities not only is beneficial for human health but also can
be beneficial for commodities due to attenuating roles of mela Corresponding author.
E-mail addresses: m-aghdam@iau-ahar.ac.ir, aghdamm@ut.ac.ir (M.S. Aghdam),
jrfard2005@gmail.com (J.R. Fard).
http://dx.doi.org/10.1016/j.foodchem.2016.10.123
0308-8146/ 2016 Elsevier Ltd. All rights reserved.

tonin on biotic and abiotic stress (Tan et al., 2012). Melatonin as

a safe and beneficial indole amine molecule acts not only as
endogenous elicitor and signaling molecule for attenuation of
biotic and abiotic stress but also have a direct antioxidant activity
(Arnao & Hernndez-Ruiz, 2015). Yin et al. (2013) reported that the
melatonin treatment at 0.1 mM alleviated marssonina apple blotch
caused by fungus Diplocarpon mali, which is caused by higher H2O2
accumulation leading to enhancing pathogenesis related (PR)
proteins accumulation such as peroxidase, chitinase and b-1,3glucanase, and triggering phenylpropanoid pathway by enhancing
phenylalanine ammonia lyase (PAL) enzyme activity.
In addition to antifungal function, melatonin can be used for
enhancing postharvest sensory and nutritional quality of horticultural commodities (Cao et al., 2016; Gao et al., 2016; Liu et al.,
2016; Ma, Zhang, Zhang, & Wang, 2016; Meng et al., 2015). Meng
et al. (2015) reported that grape berries treated with melatonin
at pre-veraison exhibited higher endogenous melatonin accumulation, which not only increases berry size and weight, as a result of
higher sugars accumulation and higher endogenous hormones GA/
ABA ratio, but also increases synchronicity of berry ripening. Ma
et al. (2016) reported that the melatonin treatment delayed
cassava roots postharvest physiological deterioration (PPD), which
is achieved by lower H2O2 accumulation as a result of increasing
antioxidant enzymes superoxide dismutase (SOD), catalase (CAT)
and glutathione reductase (GR) activity and causes higher

1651

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

membrane integrity demonstrated by lower malondialdehyde

(MDA) accumulation. Gao et al. (2016) reported that the peach
fruits treated with melatonin exhibited lower O-2 and H2O2 accumulation results from higher antioxidant enzymes SOD, CAT,
ascorbate peroxidase (APX) activities, which concurrent with lower
lipoxygenase (LOX) enzyme activity, caused higher membrane
integrity demonstrated by lower MDA accumulation. Cao et al.
(2016) reported that the melatonin treatment attenuated chilling
injury in peach fruits, which was resulted from 1) higher endogenous polyamines putrescine, spermidine and spermine accumulation via enhancing arginine decarboxylase and ornithine
decarboxylase genes expression 2) higher c-aminobutyric acid
(GABA) accumulation via enhancing glutamate decarboxylase
(GAD) and polyamine oxidase genes expression 3) higher proline
accumulation via enhancing pyrroline-5-carboxylate synthetase
and ornithine aminotransferase genes expression, concurrent with
decreasing proline dehydrogenase genes expression. Liu et al.
(2016) reported that tomato fruits treated with preharvest
melatonin exhibited higher fruits weight which result from higher
sugars accumulation, and concurrent with higher organic acids
accumulation led to tomato fruits with favorable flavor. Also,
tomato fruits treated with preharvest melatonin exhibited significantly higher phytonutrient lycopene and ascorbic acid accumulation, which are beneficial for human health.
The purpose of the present study was to evaluate the impact of
postharvest melatonin treatment on GABA shunt pathway activity
in strawberry fruits and its contributions to attenuating fruits
decay and enhancing fruits nutritional quality via H2O2 signaling
and phenylpropanoid pathway activity during storage at 4 C for
12 days.
2. Materials and methods
2.1. Fruit and melatonin treatment
Strawberry fruits (Fragaria
anannasa cv. Selva), at commercial
ripeness (>75% of the surface showing red color), were harvested
from a commercial production greenhouse in Karaj (Iran) and
transported to the postharvest laboratory at University of Tehran.
Fruits were selected for uniformity of color and size and any fruit
with apparent injuries, disease or infections were removed. For
postharvest treatment, 900 fruits were selected and divided into
5 lots of 180 fruits for the following treatments in triplicate (60
fruits per replicate) by dipping of fruits at 0 (control), 1, 10, 100,
1000 lmol/L melatonin solution for 5 min at 20 C and then
removed from the melatonin solution and were allowed to airdry at room temperature for 2 h. Then 60 fruits of each replicate
were put in 3 plastic jars, 20 fruits in each plastic jars, and stored
at 4 0.5 C with 9095% RH for 12 days (Wei, Mao, & Tu, 2014).
Every 4 days during storage at 4 0.5 C followed by shelf life at
20 0.5 C for 2 days, GABA shunt pathway activity and its contribution to fruits decay attenuation and enhancing fruits nutritional
quality via triggering H2O2 accumulation and phenylpropanoid
pathway activity of strawberry fruits were evaluated.
2.2. Fruit decay
Every 4 days storage at 4 0.5 C followed by shelf life at
20 0.5 C for 2 days, the percentages of decayed strawberry fruit
as decay incidence were recorded. Decay severity was assessed
by visualizing the decay area on the surface of strawberry fruits
using a scale from 1 to 5; 0, healthy fruit; 1, 120% fruit surface
infected; 2, 2140% fruit surface infected; 3, 4160% fruit surface
infected; 4, 6180% fruit surface infected; 5, P81% fruit surface
infected and showing sporulation (Romanazzi, Nigro, Ippolito,

DiVenere, & Salerno, 2002). The infection or McKinney index,

which integrates fruits decay incidence and its severity, was
calculated using the formula:

Infection or McKinney index

X

d
f=N
D
100

where d is the category of decay intensity scored on the fruit, and f

is its frequency, N is the total number of examined fruits, and D is
the highest category of decay intensity that occurred on the severity
scale (McKinney, 1923).
2.3. Antioxidant system activity
For analysis of antioxidant enzymatic activities, 5 g of fruits tissue were homogenized with 50 mmol/L phosphate buffer (pH 7.8)
containing 0.2 mmol/L EDTA and 2% PVP. The homogenate was
centrifuged at 12,000
g for 20 min at 4 C and the supernatant
was used for antioxidant enzymes CAT, APX, and SOD activities.
CAT and APX enzymes activity measured according to Zhang,
Huber, and Rao (2013). For determination of CAT, the reaction mixture (3 mL) consisted of 12.5 mmol/L H2O2, 50 mmol/L sodium
phosphate buffer (pH 7.0) and 0.2 mL of enzyme extract. The
decomposition of H2O2 was determined by recording the absorbance at 240 nm. One unit of CAT enzymatic activity was defined
as a decrease in absorbance at 240 nm of 0.01 per min. For determination of APX, the reaction mixture (3 mL) contained
50 mmol/L sodium phosphate buffer (pH 7.0), 9 mmol/L ascorbic
acid, 12.5 mmol/L H2O2 and 0.1 mL of enzyme extract. The activity
was calculated from a change in absorbance at 290 nm per min.
One unit of APX enzymatic activity was defined as the enzyme that
oxidizes 1 lmol of ascorbate per min. SOD activity was determined
by measuring its ability to inhibit the photochemical reduction of
nitro blue tetrazolium (NBT). For determination of SOD, the reaction mixture (3 mL) contained 50 mmol/L sodium phosphate buffer
(pH 7.8), 14 mmol/L methionine, 3 mmol/L EDTA, 1 mmol/L NBT,
60 mmol/L riboflavin and 0.1 mL of enzyme extract. The formation
of blue formazan was monitored by recording the absorbance at
560 nm. One unit of SOD enzymatic activity was defined as enzyme
that caused 50% inhibition of NBT reduction (Giannopolitis & Ries,
1977). CAT, APX and SOD activity were expressed as U mg1 protein. The protein content was determined according to Bradford
(1976), with bovine serum albumin used as the standard.
The H2O2 content measured according to Patterson, MacRae,
and Ferguson (1984). One gram of fruits tissue was homogenized
with 5 ml of cold acetone. After centrifugation for 15 min at
6000
g at 4 C, the supernatant was collected. The supernatant
(1 ml) was mixed with 0.1 ml of 5% titanium sulphate and 0.2 ml
ammonia, and then centrifuged for 10 min at 6000
g at 4 C. The
pellets were dissolved in 3 ml of 10% (v/v) H2SO4 and centrifuged
for 10 min at 5000
g at 4 C. Absorbance of the supernatant was
measured at 410 nmH2O2 content was calculated using H2O2 as
a standard and then expressed as nmol g1 fresh weight (FW).
2.4. PAL enzyme activity
PAL enzyme activity measured according to Chen et al. (2008).
Fruits tissue (2 g) was ground with 6 mL 50 mmol/L TrisHCl buffer
(pH 8.8) contained 15 mmol/L b-mercaptoethanol, 5 mmol/L EDTA,
5 mmol/L ascorbic acid, and 0.15% w/v PVP. The homogenate was
centrifuged at 12,000
g for 20 min at 4 C. The supernatant was
used as a source of crude enzyme for assaying PAL activity. The
reaction mixture (3 ml) contained 16 mmol/L l-phenylalanine,
50 mmol/L TrisHCl buffer (pH 8.9), 3.6 mmol/L NaCl and 0.5 ml
of the crude enzyme extract. Incubation was performed at 37 C
for 1 h and the reaction was stopped by the addition of 500 ml of
6 M HCl. Absorbance was measured at 290 nm before and after

1652

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

incubation. One unit of PAL activity was defined as enzyme that

caused an increase in absorbance of 0.01at 290 nm per hour. PAL
activity was expressed as U mg1 protein.
2.5. Total phenol and anthocyanin contents and DPPH scavenging
activity
Total phenols content was determined according to the
FolinCiocalteu method (Chen et al., 2008). One gram of fruits tissue was homogenized with 8 mL of 1% HClmethanol and
extracted for 24 h in the dark. Then the homogenate was centrifuged at 12,000
g for 20 min at 4 C. The supernatant reacted
with FolinCiocalteu reagent and 150 g L1 Na2CO3. The total phenols content was expressed as mg of gallic acid equivalent (GAE)
per 100 g of FW. Total anthocyanins content was determined by
using pH differential method (Cheng & Breen, 1991). One gram of
fruits tissue was homogenized with 10 ml of 1% HClmethanol
and held at 0 C for 10 min. Then the homogenate was centrifuged
at 12,000
g for 15 min at 4 C and then the supernatant was used.
Absorption was measured at 530 and 700 nm in buffers at pH 1.0
and 4.5, using A = [(A530  A700) pH1.0  (A530  A700) pH4.5] with
a molar extinction coefficient of 22,400 L cm1 mg1 and molecular weight of 433.2 g mol1 of pelargonidin-3-glucoside for strawberry fruits. Total anthocyanins content was expressed as mg of
pelargonidin-3-glucoside equivalent (P3GE) per 100 g of FW.
Free radical DPPH scavenging activity was measured according
to Nakajima, Tanaka, Seo, Yamazaki, and Saito (2004). One gram of
fruits tissue was homogenized with 8 mL of 1% HClmethanol and
extracted for 24 h in the dark. Then the homogenate was centrifuged at 12,000
g for 20 min at 4 C. Fifty mL of the supernatant
were added to 1.0 mL of 6
105 mol/L DPPH in methanol. The
mixture was shaken and left at room temperature for 30 min;
the absorbance was measured at 515 nm. The percent of reduction
of DPPH was calculated according to the following equation.

% inhibition of DPPH Abs control  Abs sample=Abs control

100

sium phosphate (pH 5.8), 0.1 mmol/L PLP and 20 mmol/L glutamate in a final volume of 200 ll. Blanks were without glutamate.
After the incubation at 30 C for 1 h, 0.6 M perchloric acid was
added to stop the reaction, and neutralized immediately with
3 M KOH. For compute of GAD activity, the GABA content in the
neutralized samples was measured by GABase assay. GAD activity
expressed as nmol GABA mg1 protein s1.
GABA-T was assayed using pyruvate as an amino acceptor and
based on alanine production, according to Ansari, Lee, and Chen
(2005). GABA-T activity was determined by incubating 25 lL of
enzyme extract in the assay mix with 50 mmol/L TrisHCl (pH
8.0), 1.5 mmol/L DTT, 0.75 mmol/L EDTA, 0.1 mmol/L PLP, 10%
(v/v) glycerol, 16 mmol/L GABA and 4 mmol/L of pyruvate in a final
volume of 500 ll. Blanks were without substrates, GABA and pyruvate. After the incubation at 30 C for 1 h, 4 mmol/L sulfosalicylic
acid was added to stop the reaction. For computed of GABA-T activity the alanine synthesized was measured by alanine dehydrogenase (ADH) assay. The ADH assay was performed in the assay
mix with 150 mmol/L TrisHCl (pH 9.0), 1.0 mmol/L NAD+ and
0.02 units Bacillus subtilis ADH (Sigma). The absorbance at
340 nm was read using the 96-well plate reader, and alanine content was determined by comparison with a standard curve of alanine. GABA-T activity expressed as nmol alanine mg1 protein s1.
2.7. Assays of ATP/ADP/AMP content
ATP, ADP, and AMP contents in strawberry fruits were measured according to Yi et al. (2008). Fruits tissue (2 g) was homogenized with 6 ml of 0.6 M perchloric acid. After centrifugation at
16,000
g for 15 min at 4 C, the supernatant (3 ml) was quickly
neutralized to pH 6.56.8 using 1 M KOH, diluted to 4 ml and
passed through a 0.45 lm filter. ATP, ADP and AMP were analyzed
by HPLC with a 4.6 mm
250 mm C18 column and an UV detector
at 254 nm. Mobile phase A consisted of 0.06 M K2HPO4 and 0.04 M
KH2PO4 dissolved in deionized water and adjusted to pH 7.0 with
0.1 M KOH. Mobile phase B was acetonitrile. ATP, ADP, and AMP
contents were computed based external standard and expressed
as lg g1 of FW. Adenylate energy charge (EC) was calculated by
[ATP + 1/2 ADP]/[ATP + ADP + AMP] (Yi et al., 2008).

2.6. GABA shunt pathway activity

2.8. Fatty acid quantification
GABA content in strawberry fruits was measured enzymatically
using
a
Pseudomonas
fluorescens
GABase
(Sigma)
(Deewatthanawong, Rowell, & Watkins, 2010). Fruits tissue
(0.5 g) was homogenized in 1 mL of methanol for 10 min at room
temperature. After vacuum dried, dissolved in 1 mL 70 mmol/L lanthanum chloride followed by 15 min of shaking, and centrifugation
at 13,000
g for 15 min. Then, 800 lL supernatant was mixed with
160 lL 1 M KOH. After shaken for 5 min, and centrifugation at
13,000
g for 10 min, the supernatant was used for GABA determination. The GABase assay mixture contained 75 mmol/L potassium
pyrophosphate (pH 8.6), 3.3 mmol/L b-mercaptoethanol, 10 mmol/
L 2-oxoglutarate, 1.25 mmol/L NADP+, 0.016 unit GABase. The
absorbance at 340 nm was read before and 10 min after adding
a-ketoglutarate using the 96-well plate reader, and GABA was
determined by comparison with a standard curve of GABA and
expressed as lmol g1 FW.
For GAD and GABA transaminase (GABA-T) extraction, 2 g of
fruits tissue was homogenized with 6 mL of extraction buffer containing 0.1 M Tris-HCl (pH 9.1), 10% (v/v) glycerol, 1 mmol/L DTT,
5 mmol/L EDTA, 0.5 mmol/L PLP and 1 mmol/L PMSF. After centrifugation at 20,000
g for 30 min at 4 C, the supernatant was
used for GAD and GABA-T assay. GAD activity was assayed based
on GABA production, according to Bartyzel, Pelczar, and
Paszkowski (2003). GAD activity was determined by incubating
20 lL of enzyme extract in the assay mix with 150 mmol/L potas-

Fatty acids in strawberry fruits were separated and quantified

according to Zhang and Tian (2010), by gas chromatography
equipped with flame ionization detector (FID). Fruits tissue (5 g)
was homogenized in 10 ml chloroform: methanol:0.1 N HCl
(200:100:1), then 10 ml of 0.1 N HCl was added before centrifugation at 4000
g for 10 min. The organic phase was collected and
taken to dryness. Methylation of fatty acids was carried out by adding 1 ml boron trifluoride/methanol at boiling temperature for
10 min. Methylated fatty acids were extracted with hexane, taken
to dryness and redissolved in 200 ll chloroform before injection.
Identification and quantification of fatty acids were performed by
comparing retention times and peak areas with authentic
standards. The unSFA/SFA ratio was calculated by the formula:
(18:1 + 18:2 + 18:3)/(16:0 + 18:0) where 16:0 = Palmitic acid;
18:0 = Stearic acid; 18:1 = Oleic acid; 18:2 = Linoleic acid;
18:3 = Linolenic acid.
2.9. Statistical analysis
The experiment was arranged as split plots for time on the basis
of completely randomized design with three replications. Analysis
of variance (ANOVA) was carried out with SPSS software. Differences between means were assessed by Tukey test with differences
being considered significant at P < 0.05.

1653

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

3. Results and discussion

3.1. Fruit decay
As depicted in Table 1, melatonin treatment significantly attenuated postharvest fungal decay of strawberry fruits during storage
at 4 C for 12 days. In comparison with the control, the melatonin
treatment at 100 lmol/L significantly decreased decay incidence,
decay severity and McKinney index (P < 0.01; Table 1).
3.2. Antioxidant system activity
In control fruits and fruits treated with melatonin H2O2 content
increased within the first 8 days of storage and then began to
decrease, whereas fruits treated with melatonin exhibited significantly higher H2O2 content during storage (P < 0.01; Table 2). The
SOD activity in control fruits and fruits treated with melatonin
decreased during storage, whereas fruits treated with melatonin
exhibited significantly higher SOD activity during storage
(P < 0.05; Table 2). The CAT and APX activities in control fruits
and fruits treated with melatonin increased the during storage,
whereas fruits treated with melatonin exhibited significantly
lower CAT and APX activities during storage (P < 0.01; Table 2).
Our results suggest that the melatonin treatment promoted H2O2
accumulation in strawberry fruits via increasing SOD enzyme
activity concurrent with decreasing APX and CAT enzymes activities, so higher H2O2 accumulation might act as a signaling molecule for the fortification of the defense system in strawberry fruits.
According to the Seifi et al. (2013) suggested scenario, the reinforcement of strawberry fruits defense system via higher H2O2
accumulation may be results from synchronous triggering of the
cytosolic glutamine synthetase/glutamate synthase (GS/GOGAT)
cycle and the GABA shunt, which enhanced resistance of ABAdeficient sitiens tomato mutant to Botrytis cinerea. In spite of direct
antifungal function, higher H2O2 accumulation, in epidermis cell of
sitiens tomato in response to infection of necrotroph pathogens
Botrytis cinerea, so-called oxidative burst, which results from
NADPH oxidase and apoplastic peroxidase activation, increased
PAL enzyme activity which lead to cell wall fortification by deposition of phenols and lignin to cell wall and arrest penetrating pathogen to epidermis. Also, synchronous with phenylpropanoid
pathway activity, the NH+4 production by PAL enzymatic activity
in epidermis cell was reassimilated by GS/GOGAT cycle in mesophyll cell for glutamate production. Then, glutamate consumed
by GABA shunt pathway for providing carbon skeleton in succinate
form for tricarboxylic acid (TCA) cycles (Asselbergh et al., 2007;
Seifi et al., 2013). Under pathogen infection which leads to
oxidative stress, succinyl-CoA synthetase and a-ketoglutarate

dehydrogenase as key TCA cycle enzymes are deactivated, resulting in hastening cell death and senescence due to lower energy
and carbon skeleton production (Fait, Fromm, Walter, Galili, &
Fernie, 2008). Since responding to pathogen infection needs higher
requirements of energy and carbon skeleton, GABA shunt pathway,
by anaplerotic route, could provide NADH and succinate for mitochondrial electron transport chain and TCA cycle for ensure sufficient ATP (Seifi et al., 2013). In addition to ensuring sufficient
energy and NADH production, higher GABA shunt activity replenished TCA cycle in response to pathogens infection with oxidative
fact, leading to higher carbon skeleton in phosphoenolpyruvate
(PEP) form which in combination with Er4P can trigger phenylpropanoid pathway activity and enhance pathogen resistance
(Seifi et al., 2013). Higher ATP and carbon skeleton not only are
required for higher PAL enzyme activity, as a key enzyme in the
phenylpropanoid pathway, but also are crucial for maintaining
higher unsaturated/saturated fatty acids (unSFA/SFA) ratio, higher
antioxidant system activity and higher PRs proteins accumulation,
all of them are pivotal for attenuating decay and maintaining nutritional quality (Aghdam, Naderi, Malekzadeh, & Jannatizadeh, 2016;
Aghdam et al., 2016; Seifi et al., 2013).

3.3. PAL enzyme activity

As depicted in Fig. 2A, PAL enzyme activity in control fruits and
fruits treated with melatonin increased within the first 8 days of
storage and then began to decrease, whereas fruits treated with
melatonin exhibited significantly higher PAL enzyme activity during storage, which was concurrent with higher H2O2 accumulation
(P < 0.01). Kogovsek et al. (2016) reported that the potato leaves in
response to infection with potato virus Y (PVY) exhibited higher
GABA shunt, TCA cycle and phenylpropanoid pathway activities.
This can demonstrate the contribution of GABA shunt in providing
sufficient energy and carbon skeleton for responding to viral
pathogen with higher energy and carbon skeleton requirement.
Lower decay in strawberry fruits treated with melatonin may be
the result of higher PAL enzyme activity, which may have been
achieved by H2O2 signaling. Higher H2O2 accumulation and oxidative burst in strawberry fruits in response to melatonin treatment
is responsible for initial triggering of PAL enzyme activity for cell
wall fortification. However, according to the scenario suggested
by Seifi et al. (2013), higher PAL enzyme activity in strawberry
fruits during 12 days storage at 4 C, which leads to total phenols
and anthocyanins accumulation, may be due to higher GABA shunt
pathway activity, responsible for supplying skeleton carbon in
PEP form, associated with Er4P which may act as starter of
phenylpropanoid pathway.

Table 1
Postharvest decay indicators of strawberry fruits treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to 12 days.
Time (day)

4
8
12
Significant
Time
Treatment
T
T
CV

Treatment

Postharvest decay indicators

Melatonin (lM)

Decay incidence (%)

Decay severity (15)

McKinney index (%)

0
100
0
100
0
100
df
2
1
2

14.8 0.18 e
7.2 0.22 f
58 0.25 b
16.2 1.41 d
97.5 0.87 a
44.4 0.55 c

1 0.00 e
1 0.00 e
2.8 0.08 b
1.4 0.08 d
3.9 0.06 a
2 0.03 c

7.9 0.07 d
1.4 0.08 e
33.8 0.12 b
18.6 0.12 e
71.2 0.37 a
25.8 0.21 c

2.44

3.68

1.86

Mean values SE (n = 3). Different letters indicate significant differences at P = 0.05.

and : significant at the 5% and 1% level of probability, respectively.

1654

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

Table 2
Antioxidant system activity of strawberry fruits treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to 12 days.
Time (day)

0
4
8
12
Significant
Time
Treatment
T
T
CV

Treatment

Antioxidant System Activity

Melatonin (lM)

APX (U mg1 protein)

CAT (U mg1 protein)

SOD (U mg1 protein)

H2O2 (nmol g1 FW)

0
100
0
100
0
100
df
2
1
2

12.21 1.02
24.12 1.15d
18.33 0.88d
45.24 1.73b
33.68 0.92c
78.64 1.76a
44.42 2.40b

27.21 1.02
42.32 1.86c
38.21 0.58c
84.62 1.45a
69.25 1.45b
71.51 0.58b
65.25 1.37b

186.21 0.84
146.32 0.88b
168.42 2.03a
95.24 1.20d
124.12 1.53c
65.74 1.15f
87.49 1.20e

38.29 0.58
46.12 2.31f
61.32 1.15e
102.20 1.76c
125.25 1.54a
70.65 2.21d
114.48 1.34b

3.76

1.65

2.84

6.55

Mean values SE (n = 3). Different letters indicate significant differences at P = 0.05.

and : significant at the 5% and 1% level of probability, respectively.

3.4. GABA shunt pathway activity

During storage at 4 C for 4 days, strawberry fruits exhibited
higher GABA content in response to melatonin treatment
(P < 0.01; Fig. 1A), which resulted from higher GAD enzyme
activities (P < 0.01; Fig. 1B) and higher GAD/GABA-T enzymatic
activities ratio (P < 0.05; Fig. 1D). Since low temperature led to
cytosol acidification, biosynthesis of GABA by GAD enzyme activity
in cytosol via consumption of H+ prevents of cytosol acidification
(Fait et al., 2008). So, higher GABA accumulation in strawberry
fruits treated with melatonin during storage at 4 C for 4 days
can be a defense response of fruits to low temperature (4 C).
But, during storage at 4 C for 8 and 12 days, strawberry fruits
exhibited lower GABA content in response to melatonin treatment
(P < 0.01; Fig. 1A), which is due to higher GABA-T enzyme activity
(P < 0.01; Fig. 1C) and lower GAD/GABA-T enzymatic activity ratio
(P < 0.05; Fig. 1D). These results indicated that melatonin treatment in strawberry fruits boosted GABA shunt pathway activity
during storage at 4 C. Through increasing GABA-T activity, GABA
shunt exert not only as provider of C and N, of NADH as reducing
molecule, and of ATP, but also exert as H2O2 scavenger. Aghdam,
Naderi, Jannatizadeh et al. (2016) and Aghdam et al. (2016)
reported that the GABA and salicylic acid (SA) treatments alleviated chilling injury in anthurium cut flowers which was associated
with consumption of GABA via higher GABA-T activities for providing higher ATP, higher phenols and higher unSFA concurrent with
lower H2O2 in flowers during cold storage which resulted in flowers with lower spathe browning. Since extra H2O2 accumulation in
the strawberry fruits treated with melatonin can be damaging for
fruits, by utilization of GABA via GABA shunt pathway extra H2O2
could be scavenged in strawberry fruits, which keep fruits safe
from the damaging impact of extra H2O2.
3.5. Energy status
During storage of strawberry fruits at 4 C, ATP and ADP contents decrease and in AMP content increase, which led to decrease
in energy charge (Table 3). Strawberry fruits displayed higher ATP
content (P < 0.01) and lower AMP content (P < 0.01) in response to
melatonin treatment, which led to maintaining higher energy
charge (P < 0.05) in fruits during storage at 4 C (Table 3). The function of GABA shunt as provider ATP supported by the higher ATP
content and energy charge in strawberry fruits in response to
melatonin treatment during storage at 4 C.
ATP can be used by pathogens infected fruits and vegetables for
the accumulation of phytoalexins and PRs, which have antimicrobial functions and for phenylpropanoid pathway activity, which

is crucial for cell wall fortification (Yi et al., 2008). Bolton,

Kolmer, Xu, and Garvin (2008) reported that high resistance of
wheat to leaf rust caused by fungus Puccinia triticina results from
Lr34 gene which have higher energy requirement, and is achieved
by higher energy procreate metabolic pathways such as TCA cycle,
glycolysis pathway, oxidative pentose phosphate pathway, fatty
acids b-oxidation, pyruvate dehydrogenase (PDH) bypass and
GABA shunt pathway. Under resistance of wheat to Puccinia
triticina conferred by Lr34 gene, with higher energy requirement,
higher pyruvate produced by glycolysis pathway which cannot
be entirely converted to acetyl CoA by PDH, may be utilized by
GABA shunt for providing energy via production of succinate for
TCA cycle. Also, GABA shunt pathway has antioxidant function
and protects ROS sensitive enzymes in TCA cycle from oxidative
damage, which ensures providing sufficient energy and NADH by
TCA cycle (Bolton et al., 2008). Recently, Sheng et al. (2017)
reported that the lower postharvest decay in citrus fruits in
response to exogenous GABA treatment was associated with higher
ATP content, which probably results from higher GABA shunt pathway activity, demonstrated by lower GAD and higher GABA-T
genes expression. Based on these results, we suggest that the lower
decay in strawberry fruits treated with melatonin may result from
higher energy status, which is achieved by higher GABA shunt
pathway activity.
3.6. Fatty acids status
In strawberry fruits, during storage at 4 C, the linoleic and
linolenic acid contents decreased, while palmitic, stearic and oleic
acids contents increased. As a consequence, the unSFA/SFA ratio
decreased (Table 4). Melatonin treatments delayed the increases
in palmitic acid (P < 0.01), stearic acid (P < 0.05) and oleic acid
(P < 0.01) contents and delayed the decreases in linoleic
(P < 0.01) and linolenic acids (P < 0.01) contents. Thus, the melatonin treated strawberry fruits maintained higher unSFA/SFA ratio
than control fruits during storage at 4 C (P < 0.05; Table 4). Since
ATP plays a crucial role in biosynthesis of fatty acids and their
unsaturation, having higher ATP status can lead to higher membranes unSFA/SFA (Yi et al., 2008). Higher unSFA/SFA ratio in
strawberry fruits treated with melatonin may results from higher
ATP content, which is achieved by higher GABA shunt pathway
activity. In response to pathogen infection, lipase enzyme activity
increased leading to releasing free fatty acid from the membrane.
These free fatty acids directly have pivotal roles in response to
pathogen infection as free fatty acids or indirectly via biosynthesis
of oxylipins (Walley, Kliebenstein, Bostock, & Dehesh, 2013).
Yaeno, Matsuda, and Iba (2004) reported that the chloroplastic

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

Fig. 1. GAD and GABA-T enzymes activity and GABA content of strawberry fruits
treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to
12 days. Data shown are mean values of n = 3 and the error bars represent standard
errors of the means. Tukey test at P = 0.05 level.

membrane linolenic acid (18:3) results in H2O2 accumulation by

activating NADPH oxidase, so oxidative burst results from higher
linolenic acid enhanced pathogen resistance in Arabidopsis. Higher
pathogen susceptibility of Arabidopsis fad7 fad8 mutants resulted
from lower linolenic acid which in turn decreased H2O2 accumulation and hampered oxidative burst (Yaeno et al., 2004). Higher
linolenic acid in strawberry fruits treated with melatonin was concurrent with lower oleic acid (18:1). Kachroo and Kachroo (2009)
suggested that the lower oleic acid under pathogen infection is

1655

Fig. 2. PAL enzyme activity, total phenols, anthocyanins contents and DPPH
scavenging capacity of strawberry fruits treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to 12 days. Data shown are mean values of n = 3
and the error bars represent standard errors of the means. Tukey test at P = 0.05
level.

crucial for boosting the resistance of plants to pathogen infection.

Arabidopsis sis2 mutant which results from a mutation in SSI2,
encodes stearoyl-ACP desaturase 2 (SACPD2), which catalyzes the
desaturation of stearic acid (18:0) to oleic acid (18:1), have higher
stearic acid (18:0) and lower oleic acid (18:1) contents. Lower oleic
acid enhanced resistance to pathogens by triggering higher SA
accumulation via EDS1 and SID2 and higher nitric oxide accumulation via NOA1 and NIA1 (Mandal et al., 2012; Venugopal et al.,
2009). Also, unSFA can be converted to phyto-oxylipins, which
are crucial for the pathogens infection resistance. Phyto-oxylipins
which results from unsaturated fatty acid by oxidation via

1656

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

Table 3
Energy status of strawberry fruits treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to 12 days.
Time (day)

0
4
8
12
Significant
Time
Treatment
T
T
CV

Treatment

Energy status

Melatonin (lM)

ATP (lg g1 FW)

ADP (lg g1 FW)

AMP (lg g1 FW)

AEC

0
100
0
100
0
100
df
2
1
2

28.11 0.32
24.92 0.31
27.99 0.27
21.21 0.51
23.70 0.29
13.40 0.61
20.86 0.42

14.97 0.54
11.91 0.21 b
14.32 0.26 a
8.71 0.41 d
10.59 0.16 c
6.12 0.44 e
8.54 0.22 d

5.04 0.34
5.88 0.39 cd
5.24 0.04 d
8.66 0.38 b
6.61 0.18 c
12.04 0.37 a
8.48 0.28 b

0.73 0.01
0.72 0.01
0.74 0.02
0.66 0.01
0.71 0.01
0.52 0.01
0.66 0.02

ns
3.501

4.131

1.731

b
a
c
b
d
c

3.541

a
a
b
a
c
b

Mean values SE (n = 3). Different letters indicate significant differences at P = 0.05.

and : significant at the 5% and 1% level of probability, respectively.

Table 4
Fatty acids status of strawberry fruits treated with postharvest 100 lM melatonin and stored at 4 0.5 C for up to 12 days.
Time (day)

0
4
8
12
Significant
Time
Treatment
T
T
CV

Treatment

Fatty acids status

Melatonin (lM)

C16:0 (%)

C18:0 (%)

C18:1 (%)

C18:2 (%)

unSFA/SFA

0
100
0
100
0
100
df
2
1
2

5.24 0.42
9.07 0.62 e
7.86 0.32 e
19.34 0.58 c
15.67 0.52 d
35.98 0.47 a
23.72 1.25 b

3.25 0.52
4.70 0.29 e
3.47 0.27 e
10.08 0.32 c
7.73 0.14 d
21.79 0.35 a
15.29 0.64 b

28.57 0.49
24.77 0.28 b
27.67 0.68 a
17.34 0.57 d
20.18 0.49 c
8.93 0.37 f
15.51 0.46 e

38.85 0.48
35.22 0.58
38.37 0.29
24.89 0.32
30.59 0.85
10.17 0.51
18.84 0.17

2.76

3.08

4.65

9.64

4.89

4.66

C18:3 (%)
b
a
d
c
f
e

25.34 0.52
22.17 0.54 b
24.35 0.61 a
15.50 0.24 d
19.21 0.48 c
8.85 0.31 e
16.19 0.50d

10.92 0.58
5.98 0.27 b
8.00 0.03 a
1.96 0.01 d
2.98 0.04 c
0.48 0.02 e
1.30 0.05 de

Mean values SE (n = 3). Different letters indicate significant differences at P = 0.05.

and : significant at the 5% and 1% level of probability, respectively.

cytochrome P450 and LOX and or direct oxidation by ROS, participate in resistance to pathogens infection through eliciting systemic
resistance or directly inhibiting the growth of the pathogens
(Kachroo & Kachroo, 2009). Madi, Wang, Kobiler, Lichter, and
Prusky (2003) reported that the treatment of avocado fruits with
ethylene, CO2 and low temperature (4 C), also inoculation of avocado fruits with Colletotrichum gloeosporioides increased FAD9 gene
expression which resulted in the accumulation of linoleic acid
(18:2) leading to higher resistance to Colletotrichum gloeosporioides. Higher resistance to the pathogen in avocado fruits with
higher linoleic acid is due to incorporation of linoleic acid within
oxylipins with direct antifungal activity (Madi et al., 2003). Based
on these results, we suggest that the lower decay in strawberry
fruits treated with melatonin may be due to higher unSFA/SFA,
which results from higher ATP provided by GABA shunt pathway
activity.
3.7. Total phenols and anthocyanins accumulation and DPPH
scavenging capacity
As depicted in Fig. 2B and C, total phenols and anthocyanins
content in control strawberry fruits increased within the first
8 days of storage and then began to decrease, whereas strawberry
fruits treated with melatonin exhibited significantly higher total
phenols and anthocyanins content during storage at 4 C
(P < 0.01). Also, DPPH scavenging capacity in control strawberry
fruits increased within the first 8 days of storage and then began
to decrease, whereas strawberry fruits treated with melatonin
exhibited significantly higher DPPH scavenging capacity during

storage at 4 C (P < 0.01; Fig. 2D). Higher PAL enzyme activity not
only leads to cell wall fortification by deposition of phenols and
lignin to cell wall and arresting penetrations of pathogen to cells
(Seifi et al., 2013), but also leads to phenols and anthocyanins accumulation which are crucial for fruits and vegetables nutritional
quality and helps in maintaining human health by their ROS scavenging capacity (Zhang et al., 2016). Higher PAL enzyme activity in
strawberry fruits in response to melatonin treatment not only
results from H2O2 accumulation but also may be due to higher
GABA shunt pathway activity which is responsible for supplying
skeleton carbon and PEP associated with Er4P which act as feeder
of phenylpropanoid pathway. Zhang et al. (2016) reported that the
higher anthocyanins accumulation in cabbage seedling in response
to melatonin treatment not only is the result of higher expression
of anthocyanin biosynthesis structural genes (PAL, C4H, CHS, CHI,
F3H, F30 H, DFR, LDOX, UFGT, and GST) but also of higher expression
of anthocyanins biosynthesis regulatory genes such as transcription factors MYB, bHLH, and WD40 (MBW), which are responsible
for regulation of anthocyanin biosynthetic structural genes. Also,
higher anthocyanin accumulation in cabbage seedling treated with
melatonin results from higher PAL enzyme activity. Lower ROS
accumulation in cabbage seedling treated with melatonin results
from higher antioxidant enzymes CAT, SOD and APX activity,
which was associated with higher membrane integrity, demonstrated by lower MDA accumulation. Since higher endogenous
ATP content in litchi fruit treated with ATP was associated with
phenols accumulation, Yi et al. (2010) suggested that endogenous
ATP is required for phenols accumulation with antioxidant activity.
Based on these results, we suggest that the higher phenols and

M.S. Aghdam, J.R. Fard / Food Chemistry 221 (2017) 16501657

anthocyanins accumulation in strawberry fruits treated with melatonin which was associated with higher DPPH scavenging capacity
can be attributed to higher GABA-T enzyme activity which led to
providence of higher ATP concurrent with supplying skeleton
carbon, PEP, for feeding phenylpropanoid pathway.
4. Conclusion
In conclusion, the present study sheds lights on the beneficial
impacts of melatonin treatment on attenuating postharvest decay
in strawberry fruits during storage at 4 C for 12 days. The attenuating postharvest decay in strawberry fruits by melatonin treatment may be due to the higher GABA-T enzyme activity which
results to providing sufficient ATP in strawberry fruits. Also, higher
SOD enzyme activity concurrent with lower CAT and APX enzymes
activity in strawberry fruits caused H2O2 accumulation in response
to melatonin treatment. Higher H2O2 accumulation triggered
phenylpropanoid pathway activity, which is crucial not only for
cell wall fortification but also for fruits nutritional quality. Also,
strawberry fruits in response to melatonin treatment displayed
higher unSFA/SFA ratio, which results from higher ATP supplying
by GABA shunt pathway activity.
Conflict of interest
The authors declare that they have no conflict of interest.
References
Aghdam, M. S., Naderi, R., Jannatizadeh, A., Babalar, M., Sarcheshmeh, M. A., &
Faradonbe, M. Z. (2016). Impact of exogenous GABA treatments on endogenous
GABA metabolism in anthurium cut flowers in response to postharvest chilling
temperature. Plant Physiology and Biochemistry, 106, 1115.
Aghdam, M. S., Naderi, R., Malekzadeh, P., & Jannatizadeh, A. (2016). Contribution of
GABA shunt to chilling tolerance in anthurium cut flowers in response to
postharvest salicylic acid treatment. Scientia Horticulturae, 205, 9096.
Ansari, M. I., Lee, R. H., & Chen, S. C. G. (2005). A novel senescence-associated gene
encoding c-aminobutyric acid (GABA): Pyruvate transaminase is upregulated
during rice leaf senescence. Physiologia Plantarum, 123, 18.
Arnao, M. B., & Hernndez-Ruiz, J. (2015). Functions of melatonin in plants: a
review. Journal of Pineal Research, 59, 133150.
Asselbergh, B., Curvers, K., Frana, S. C., Audenaert, K., Vuylsteke, M., Van
Breusegem, F., & Hfte, M. (2007). Resistance to Botrytis cinerea in sitiens, an
abscisic acid-deficient tomato mutant, involves timely production of hydrogen
peroxide and cell wall modifications in the epidermis. Plant Physiology, 144,
18631877.
Bartyzel, I., Pelczar, K., & Paszkowski, A. (2003). Functioning of the c-aminobutyrate
pathway in wheat seedlings affected by osmotic stress. Biologia Plantarum, 47,
221225.
Bolton, M. D., Kolmer, J. A., Xu, W. W., & Garvin, D. F. (2008). Lr34-mediated leaf rust
resistance in wheat: Transcript profiling reveals a high energetic demand
supported by transient recruitment of multiple metabolic pathways. Molecular
Plant-Microbe Interactions, 21, 15151527.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72(1), 248254.
Cao, S., Song, C., Shao, J., Bian, K., Chen, W., & Yang, Z. (2016). Exogenous melatonin
treatment increases chilling tolerance and induces defence response in
harvested peach fruit during cold storage. Journal of Agriculture and Food
Chemistry, 64(25), 52155222.
Chen, J. Y., He, L. H., Jiang, Y. M., Wang, Y., Joyce, D. C., Ji, Z. L., & Lu, W. J. (2008). Role
of phenylalanine ammonia-lyase in heat pretreatment-induced chilling
tolerance in banana fruit. Physiologia Plantarum, 132(3), 318328.
Cheng, G. W., & Breen, P. J. (1991). Activity of phenylalanine ammonia-lyase (PAL)
and concentrations of anthocyanins and phenolics in developing strawberry
fruit. Journal of the American Society for Horticultural Science, 116(5), 865869.
Deewatthanawong, R., Rowell, P., & Watkins, C. B. (2010). C-Aminobutyric acid
(GABA) metabolism in CO2 treated tomatoes. Postharvest Biology and Technology,
57(2), 97105.
FAOSTAT (2013). Statistical databases on global food production and trade. Rome:
Food and Agriculture Organization.
Fait, A., Fromm, H., Walter, D., Galili, G., & Fernie, A. R. (2008). Highway or byway:
The metabolic role of the GABA shunt in plants. Trends in Plant Science, 13(1),
1419.
Gao, H., Zhang, Z. K., Chai, H. K., Cheng, N., Yang, Y., Wang, D. N., & Cao, W. (2016).
Melatonin treatment delays postharvest senescence and regulates reactive
oxygen species metabolism in peach fruit. Postharvest Biology and Technology,
118, 103110.

1657

Giannopolitis, C. N., & Ries, S. K. (1977). Superoxide dismutases: I occurrence in

higher plants. Plant Physiology, 59(2), 309314.
Hashmi, M. S., East, A. R., Palmer, J. S., & Heyes, J. A. (2013). Hypobaric treatment
stimulates defence-related enzymes in strawberry. Postharvest Biology and
Technology, 85, 7782.
Kachroo, A., & Kachroo, P. (2009). Fatty Acid-derived signals in plant defense. Annual
Review of Phytopathology, 47, 153176.
Kogovsek, P., Pompe-Novak, M., Petek, M., Fragner, L., Weckwerth, W., & Gruden, K.
(2016). Primary metabolism, phenylpropanoids and antioxidant pathways are
regulated in potato as a response to Potato Virus Y infection. PLoS One, 11(1),
e0146135.
Liu, J., Zhang, R., Sun, Y., Liu, Z., Jin, W., & Sun, Y. (2016). The beneficial effects of
exogenous melatonin on tomato fruit properties. Scientia Horticulturae, 207,
1420.
Ma, Q., Zhang, T., Zhang, P., & Wang, Z. Y. (2016). Melatonin attenuates postharvest
physiological deterioration of cassava storage roots. Journal of Pineal Research,
60(4), 424434.
Madi, L., Wang, X., Kobiler, I., Lichter, A., & Prusky, D. (2003). Stress on avocado fruits
regulates D9-stearoyl ACP desaturase expression, fatty acid composition,
antifungal diene level and resistance to Colletotrichum gloeosporioides attack.
Physiological and Molecular Plant Pathology, 62(5), 277283.
Mandal, M. K., Chandra-Shekara, A. C., Jeong, R. D., Yu, K., Zhu, S., Chanda, B., ...
Kachroo, P. (2012). Oleic acid-dependent modulation of NITRIC OXIDE
ASSOCIATED1 protein levels regulates nitric oxide-mediated defense signaling
in Arabidopsis. Plant Cell, 24(4), 16541674.
McKinney, H. (1923). Influence of soil temperature and moisture on infection of
wheat seedlings by Helminthosporium sativum. Journal of Agricultural Research,
26(5), 195217.
Meng, J. F., Xu, T. F., Song, C. Z., Yu, Y., Hu, F., Zhang, L., ... Xi, Z. M. (2015). Melatonin
treatment of pre-veraison grape berries to increase size and synchronicity of
berries and modify wine aroma components. Food Chemistry, 185, 127134.
Nakajima, J.-I., Tanaka, I., Seo, S., Yamazaki, M., & Saito, K. (2004). LC/PDA/ESI-MS
profiling and radical scavenging activity of anthocyanins in various berries.
Biomed Research International, 2004(5), 241247.
Patterson, B. D., MacRae, E. A., & Ferguson, I. B. (1984). Estimation of hydrogen
peroxide in plant extracts using titanium(IV). Analytical Biochemistry, 139(2),
487492.
Romanazzi, G., Nigro, F., Ippolito, A., DiVenere, D., & Salerno, M. (2002). Effects of
pre- and postharvest chitosan treatments to control storage grey mold of table
grapes. Journal of Food Science, 67(5), 18621867.
Romanazzi, G., Smilanick, J. L., Feliziani, E., & Droby, S. (2016). Integrated
management of postharvest gray mold on fruit crops. Postharvest Biology and
Technology, 113, 6976.
Seifi, H. S., Curvers, K., De Vleesschauwer, D., Delaere, I., Aziz, A., & Hofte, M. (2013).
Concurrent overactivation of the cytosolic glutamine synthetase and the GABA
shunt in the ABA-deficient sitiens mutant of tomato leads to resistance against
Botrytis cinerea. New Phytology, 199(2), 490504.
Sheng, L., Shen, D., Luo, Y., Sun, X., Wang, J., Luo, T., ... Cheng, Y. (2017). Exogenous caminobutyric acid treatment affects citrate and amino acid accumulation to
improve fruit quality and storage performance of postharvest citrus fruit. Food
Chemistry, 216, 138145.
Tan, D. X., Hardeland, R., Manchester, L. C., Korkmaz, A., Ma, S., Rosales-Corral, S., &
Reiter, R. J. (2012). Functional roles of melatonin in plants, and perspectives in
nutritional and agricultural science. Journal of Experimental Botany, 63(2),
577597.
Venugopal, S. C., Jeong, R. D., Mandal, M. K., Zhu, S., Chandra-Shekara, A. C., Xia, Y., ...
Kachroo, P. (2009). Enhanced disease susceptibility 1 and salicylic acid act
redundantly to regulate resistance gene-mediated signaling. PLoS Genetics, 5(7),
e1000545.
Walley, J. W., Kliebenstein, D. J., Bostock, R. M., & Dehesh, K. (2013). Fatty acids and
early detection of pathogens. Current Opinion in Plant Biology, 16(4), 520526.
Wei, Y., Mao, S., & Tu, K. (2014). Effect of preharvest spraying Cryptococcus laurentii
on postharvest decay and quality of strawberry. Biological Control, 73, 6874.
Yaeno, T., Matsuda, O., & Iba, K. (2004). Role of chloroplast trienoic fatty acids in
plant disease defense responses. The Plant Journal, 40(6), 931941.
Yi, C., Jiang, Y., Shi, J., Qu, H., Xue, S., Duan, X., ... Prasad, N. K. (2010). ATP-regulation
of antioxidant properties and phenolics in litchi fruit during browning and
pathogen infection process. Food Chemistry, 118(1), 4247.
Yi, C., Qu, H. X., Jiang, Y. M., Shi, J., Duan, X. W., Joyce, D. C., & Li, Y. B. (2008). ATPinduced changes in energy status and membrane integrity of harvested litchi
fruit and its relation to pathogen resistance. Journal of Phytopathology, 156(6),
365371.
Yin, L., Wang, P., Li, M., Ke, X., Li, C., Liang, D., ... Ma, F. (2013). Exogenous melatonin
improves Malus resistance to Marssonina apple blotch. Journal of Pineal
Research, 54(4), 426434.
Zhang, C., & Tian, S. (2010). Peach fruit acquired tolerance to low temperature stress
by accumulation of linolenic acid and N-acylphosphatidylethanolamine in
plasma membrane. Food Chemistry, 120(3), 864872.
Zhang, N., Sun, Q., Li, H., Li, X., Cao, Y., Zhang, H., ... Guo, Y. D. (2016). Melatonin
improved anthocyanin accumulation by regulating gene expressions and
resulted in high reactive oxygen species scavenging capacity in cabbage.
Frontiers in Plant Science, 7, 197.
Zhang, Z., Huber, D. J., & Rao, J. (2013). Antioxidant systems of ripening avocado
(Persea americana Mill.) fruit following treatment at the preclimacteric stage
with aqueous 1-methylcyclopropene. Postharvest Biology and Technology, 76,
5864.