Postharvest Biology and Technology 101 (2015) 88–95

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Postharvest Biology and Technology

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

The role of gibberellins in the mitigation of chilling injury in cherry

tomato (Solanum lycopersicum L.) fruit
Yang Ding a , Jiping Sheng b , Shuying Li a , Ying Nie a , Jinhong Zhao a , Zhen Zhu a ,
Zhidong Wang a , Xuanming Tang a, *
a
Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Opening Laboratory of Agricultural Products Processing and
Quality Control, Ministry of Agriculture, Beijing 100193, China
b
School of Agricultural Economics and Rural Development, Renmin University of China, Beijing 100872, China

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

Article history: Tomato fruit is cold-sensitive and susceptible to chilling injury during cold storage. Bioactive gibberellins
Received 6 June 2014 (GAs), which play an important role in regulating various physiological processes, have been reported to
Received in revised form 4 November 2014 be involved in stress responses of plant. In this study, we investigated the effect of GA on chilling injury
Accepted 13 December 2014
of harvested cherry tomato fruit. We found that low temperature significantly inhibited increase of
endogenous levels of gibberellic acid (GA3) in fruit. This effect was associated with lower expression of
Keywords: key GA metabolic genes, GA3ox1,GA20ox1 and GA2ox1. GA3 treatment reduced the chilling injury index,
Chilling injury
whereas treatment with paclobutrazol (PAC), a GA biosynthesis inhibitor, exacerbated chilling injury.
Gibberellin homeostasis
Antioxidant capacity
Compared with the control, GA3 treatment decreased electrolyte leakage and malondialdehyde content,
Cherry tomato fruit increased proline content, and improved antioxidant enzyme activities. Treatment with PAC caused the
opposite effects. These results suggest that endogenous GA levels were positively associated with lipid
peroxidation and oxidative stress in chilled fruit. GA appears to play an important role in cold response
regulation in fruit.
ã 2014 Elsevier B.V. All rights reserved.

1. Introduction abiotic stresses. Several naturally occurring plant hormones, such

Cold storage is one of the most effective postharvest technolo-
gies to control quality of postharvest fruit and vegetable. However,
storage of cold sensitive commodities at low temperature is
limited by the great risk of chilling injury (Bourne, 2006; Hong and
Gross, 2006). Tomato (Solanum lycopersicum L.) fruit, a good model
system, is susceptible to chilling injury at low non-freezing
temperatures, i.e. below 12
C (Zhang et al., 2010). Chilled tomato
fruit often exhibit various symptoms, such as surface lesions,
diseases caused by pathogens, and loss of the ability to obtain full
color (Wang, 1993; Zhao et al., 2009a). These detrimental changes
reduce quality and consumer acceptability leading to substantial
economic loss (Luengwilai et al., 2012). Therefore, there is a
practical and urgent need to understand the basis of this
physiological disorder.
Phytohormones play crucial roles in modulating multiple
developmental processes and cellular responses to biotic and
* Corresponding author. Tel.: +86 10 62811868.
E-mail address: tangxuanming@caas.cn (X. Tang).
as gibberellins (GAs), salicylic acid (SA), ethylene (ETH) and
brassinosteroids (BR), have been associated with the response to
cold stress in fruit. Recent studies revealed that exogenous
application of SA was able to significantly remit chilling symptoms
in harvested tomato fruit (Aghdam et al., 2012). Treatment with
ETH or BR, at an effective concentration, could also alleviate
chilling injury of tomato fruit (Zhao et al., 2009b; Aghdam and
Mohammadkhani, 2014). It has been demonstrated that exogenous
GA3 can delay fruit ripening process, increase firmness, and
improve the cold storage life of peach fruit (Dagar et al., 2012;
Martínez-Romero et al., 2000). However, little information is
available concerning the role of GA in response to chilling stress in
postharvest tomato fruit.
Bioactive GA is a large group of naturally occurring tetracyclic
diterpenoids regulating various plant physiological processes such
as seed germination, vegetative growth, flowering induction and
fruit development (Sponsel and Hedden, 2004; Sun and Gubler,
2004). In higher plant, biosynthetic enzymes GA 3b-hydroxylases
(GA3oxs) and GA 20-oxidases (GA20oxs), and catabolic enzymes
GA 2-oxidases (GA2oxs), are particularly crucial for control of
bioactive GA levels (Fleet and Sun, 2005; Zhu et al., 2006;

http://dx.doi.org/10.1016/j.postharvbio.2014.12.001
0925-5214/ ã 2014 Elsevier B.V. All rights reserved.
 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95
Yamaguchi, 2008). GA homeostasis is maintained through a
negative feedback regulation of GA biosynthesis and a positive
feedback regulation of GA deactivation (Hedden and Phillips, 2000;
Hedden and Thomas, 2012). In Arabidopsis, a single GA3ox gene
(GA3ox1) and three GA20ox genes (GA20ox1, GA20ox2 and GA20ox3)
whose expression levels are all highly are elevated when
endogenous levels of bioactive GA are low, but are reduced when
the GA levels are increased by exogenous GA treatment. An
additional level of regulation that functions in GA homeostasis
involves the positive feedback regulation of GA2ox genes. GA2oxs
are transcriptionally up-regulated in response to increased GA
signaling output (Hedden and Thomas, 2012). Several studies have
been suggested that the effect of low temperature on GA
homeostasis in plant depends on the developmental stage. During
imbibition of Arabidopsis seed, cold activates GA biosynthesis by
increasing GA3ox1 gene transcripts (Yamauchi et al., 2004). At the
rosette stage, cold enhances GA2ox3 and GA2ox6 transcript levels
and leads to a decrease in bioactive GA levels (Achard and
Genschik, 2009). While previous work has indicated variation in
GA metabolism caused by low temperature at different stages of
plant growth, our understanding of cold-induced shift of GA
homeostasis in harvested fruit have been not reported.
GA promotes plant growth by stimulating degradation of a
nuclear growth repressor protein called DELLA protein (Harberd,
2003; Jiang and Fu, 2007; Wang et al., 2009). Emerging evidence
suggests that GA and its signaling components play important
roles in plant response to various biotic and abiotic stresses. An
Arabidopsis loss-of-function mutant lacking four of the five DELLA-
encoding genes had greatly improved resistance to Pseudomonas
syringae pv. tomato strain DC3000 (Pto DC3000) (Navarro et al.,
2008). Exogenous application of gibberellic acid (GA3) might act
via downregulation of GAI (a DELLA gene) to mediate the defense
response to potato purple top (PPT) phytoplasma infection in
tomato plant (Ding et al., 2013a). These results indicate that GA
may promote plant resistance to biotrophs. Alonso-Ramírez et al.
(2009) found that GA3 was able to reverse the inhibitory effect of
salt, oxidative, and heat stresses in the germination and seedling
establishment of Arabidopsis. A common biochemical change
occurring when plants are subjected to stress conditions is the
accumulation of reactive oxygen species (ROS) which creates
oxidative stress that can damage DNA, inactivate enzymes and
cause lipid peroxidation (Moldovan and Moldovan, 2004). Li et al.
(2011) indicated that GA3 decreased accumulation of ROS and lipid
peroxidation in cucumber hypocotyl and radical under suboptimal
temperature. Tabatabaei (2013) reported that GA3 could increase
germination characteristics and enhance antioxidant enzyme
activities in wheat seed under salinity stress. However, the
functional relationship between GA and ROS is poorly understood
in fruit responses to chilling stresses.
In the present study, we investigated the role that GA played in
alleviating chilling injury in cherry tomato fruit. Our results
revealed that, during cold storage, endogenous levels of GA3 were
lower in chilled fruit compared to that of fruit stored at room
temperature, and this was associated with suppressed expression
of the key GA metabolic genes. The physiology indexes of
electrolyte leakage, malondialdehyde (MDA), proline content, and
activities of antioxidative enzymes, which were considered to
reflect physiological state of plant exposure to chilling stress
(Zhao et al., 2009a), were also determined in this study.
Exogenous application of GA3 attenuated chilling injury of the
tomato fruit, reduced electrolyte leakage and MDA content,
increased proline content, and induced activities of antioxidant
enzymes. PAC treatment caused the opposite effects. These
findings may provide novel insights into the mechanisms by
which GA mediates fruit tolerance to abiotic stress in general and
cold stress in particular.
89
2. Materials and methods
2.1. Fruit and treatment
Cherry tomato (Solanum lycopersicum var. cerasiforme) fruit
were harvested at the pink stage in Shandong, China, and
transported to the laboratory immediately. Harvested fruit were
selected based on uniform size and for the absence of physical
injury or infection. The fruit were disinfected with 1% sodium
hypochlorite (v/v) for 2 min, washed and their surfaces were dried
in air. The fruit were stored at (4  1)
C or (25  1)
C (room
temperature) with 80–90% relativity humidity. For GA3 and PAC
treatments, two groups of fruit (270 fruit for each group) were
dipped into 0.2 mmol L1 GA3 solution [prepared in ethanol/
distilled water (1:1000, v/v) containing 0.1% (v/v) Tween-20] and
0.3 mmol L1 PAC solution [prepared in ethanol/distilled water
(1:1000, v/v) containing 0.1% (v/v) Tween-20] for 15 min,
respectively. The fruit immersed in ethanol/distilled water
(1:1000, v/v) with 0.1% (v/v) Tween-20 for 15 min were used as
the control. Ten fruit were removed at 0, 3, 7, 14, 21 and 28 d from
room temperature or cold storage for GA3 content and gene
expression. Samples of 30 fruit from Control, GA3 and PAC
treatment were respectively removed at 14, 21 and 28 d from
cold storage and held at room temperature for 3 d for chilling
injury evaluation. In addition, 30 fruit from each treatment were
sampled before treatment (time 0) and at 3, 7, 14, 21, 28 d of cold
storage and held at room temperature for 3 d for GA3, electrolyte
leakage, MDA, proline and enzymatic analyses. The mesocarp from
the fruit equator area was cut into small pieces, frozen in liquid
nitrogen, and stored at 80
C for gene assays. Each treatment was
replicated three times.
2.2. GA3 content
GA3 content was measured using liquid chromatography-
tandem mass spectrometry (LC–MS/MS) according to the method
of Shi et al. (2012), with minor modifications.
GA3 isolation and purification: A fully homogenized sample
(10 g) was weighed in a 50 mL plastic centrifuge tube. With the
addition of 10 mL of 0.1% (v/v) acetic acid in acetonitrile, the tube
was vigorously shaken for 1 min. Afterward, 4 g of anhydrous
magnesium sulfate was added, and the solution was immediately
shaken for another 1 min, then homogenized, and centrifuged at
12,000  g for 5 min. A total of 1 mL of the clarified supernatant was
transferred into a clean plastic centrifuge tube containing 50 mg of
C18 sorbents. The mixture was then shaken for 30 s, centrifuged at
12,000  g for 5 min, and finally, filtered through a 0.22 mm
membrane prior to LC–MS/MS analysis.
LC–MS/MS conditions: Chromatographic analyses were con-
ducted using an Agilent series 1200 high performance liquid
chromatography (HPLC) system (Agilent, Santa Clara, CA, USA)
equipped with a binary pump, a column oven, and an auto
sampler. A Waters SunFire C18 reversed-phase column
(2.1 150 mm, with a 5.0 mm particle size) was used and the
column temperature was maintained at 30
C. The injection
volume was 5 mL. The mobile phases consisted of 0.5% (v/v) acetic
acid (A) and methanol (B). The column was developed with
stepwise linear gradient elution according to the following
timetable: 0–2 min, 80% B; 3–6 min, 10% B; 9–10 min, 80% B;
and 13.1 min, 80% B at a flow rate of 3.3 mL s1.
Mass spectrometric detection was carried out using an API
5000 tandem quadrupole mass spectrometer (Applied Biosystems,
Foster City, CA, USA) in multiple-reaction monitoring mode.
The transition from [M—H] to [M—H—(COO)3—C5H10] (m/z
345 ! 143) was chosen as the quantitation transition in the
present study. Electrospray ionization (ESI) parameters were used
90 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95
as follows: curtain gas (CUR), 0.2 MPa; atomization air pressure
(GS1), 0.1 MPa; auxiliary gas (GS2), 0.3 MPa; ion spray voltage (IS),
4.5 kV; ion source temperature (TEM), 500
C; collision cell exit
potential (CXP), 17 V; entrance potential (EP), 10 V; collision energy
(CE), 50 V; and declustering potential (DP), 70 V. Data acquisition
was performed under time-segmented conditions based on the
chromatographic separation of the target compounds to maximize
the sensitivity of detection. Segment 1–17 min was detected in the
negative-ion mode. All system control, data acquisition, and data
analysis were performed with the AB Sciex Analyst 1.4.2 software
(Applied Bioscience, Foster City, CA, USA).
Quantification was based on peak area ratios of the analyte to
the corresponding internal standard. A standard curve was
established by using a linear least-squares regression analysis. A
good linear correlation was observed between peak area and GA3
concentrations (y = 6.27  103 x–777, R2 = 0.9957). GA3 content was
expressed as mg kg1 fresh weight.
2.3. RNA isolation and reverse transcription
Total RNA was isolated from approximately 2 g of frozen fruit
tissue using an RNA prep Pure Plant Kit (Tiangen, Beijing, China)
according to the manufacturer’s instructions. The RNA concen-
trations were determined photometrically with a SMA 4000
UV–vis spectrophotometer (Merinton, Beijing, China) and were
normalized to 200 mg L1. One microgram of total RNA was used in
each cDNA synthesis reaction with the QuantScript RT Kit
(Tiangen) using oligo (dT) primers.
2.4. Quantitative real-time polymerase chain reaction (PCR)
expression analysis
Relative expression levels of GA3ox1 (AB010991), GA20ox1
(AF049898) and GA2ox1 (EF441351) in fruit stored at cold
temperature and room temperature were detected via quantitative
real-time PCR (qPCR) using SuperReal PreMix Plus (SYBR Green)
(Tiangen) as described by Ding et al. (2013b). b-actin (SGN-
U144149) was used as an internal control. qPCR reactions were
performed with an ABI 7500 real-time PCR system (Applied
Biosystems). Each reaction mixture, in a volume of 20 mL,
contained 2 mL of above prepared cDNA, 5 mmol L1 of forward
and reverse primers, 10 mL of 2  SuperReal PreMix Plus (with SYBR
Green I), and 0.4 mL of 50  ROX Reference Dye. The qPCR cycling
program consisted of an initial denaturation step at 95
C for
15 min followed by 40 cycles of denaturation at 95
C for 30 s,
annealing at 60
C for 1 min, and extension at 72
C for 30 s.
Quantitation of the target gene transcript levels was carried out
using the comparative Ct method (DDCt method) (Schmittgen and
Livak, 2008).
2.5. Chilling injury index
Chilling injury index of chilled fruit was evaluated at room
temperature for 3 d after 14, 21 and 28 d in cold storage period to
allow development of chilling injury symptoms. Symptoms were
manifested as surface pitting according to the method of Ding et al.
(2002), where 0 = no pitting; 1 = pitting covering <25% of the fruit
surface; 2 = pitting covering 25%–50% of the surface; 3 = pitting
covering 50%–75% of the surface; and 4 = pitting covering >75% of
the surface. The average extent of cold damage was expressed as
the chilling injury index, which was calculated using the
P
following formula: chilling injury index (%) = [(chilling injury
level)  (number of fruit at the chilling injury level)]/[4  (total
number of fruit)]  100.
2.6. Electrolyte leakage
Electrolyte leakage was measured after 0, 3, 7, 14, 21 and 28 d of
cold storage at room temperature for 3 d using the methods of
Luo et al. (2012) and Luengwilai et al. (2012) with modifications. A
3 mm-thick mesocarp tissue was excised from equator part of five
fruit. The disks were briefly rinsed with deionized water and
blotted dry on a slightly moistened Whatman filter paper. Then the
disks were put into 30 mL of 0.3 mol L1 mannitol for 3 h in a 50 mL
plastic centrifuge tube under constant shaking. The conductivity of
the bathing solution was measured with MP513 conductivity
meter (Sanxin, Shanghai, China). The disks and bathing solutions
were frozen at 20
C for 24 h, warmed to room temperature, and
boiled in water for 30 min, and frozen, thawed and boiled once
again before the total conductivity of the solution was measured at
room temperature after 1 h of shaking. The electrolyte leakage was
expressed as a percentage of the conductivity of total tissue
electrolyte.
2.7. MDA content
MDA content was measured after 0, 3, 7, 14, 21 and 28 d of cold
storage at room temperature for 3 d using the thiobarbituric acid
(TBA) method described by Ding et al. (2007) with modifications.
MDA formation was determined by measuring the absorbance of
the reaction product of MDA with 3 mL of 0.6 mL L1 TBA and 1 mL
of the supernatant at 532 nm, corrected for nonspecific turbidity by
subtracting the absorbance at 600 nm and interference generated
by TBA-sucrose complexes at 450 nm. MDA content was expressed
as mmol kg1 fresh weight.
2.8. Proline content
Proline content was measured after 0, 3, 7, 14, 21 and 28 d of
cold storage at room temperature for 3 d using the acid ninhydrin
method described by Shan et al. (2007). Proline in tissues was
extracted using 3% (v/v) sulfosalicylic acid at 100
C for 10 min with
constant shaking. After cooling to room temperature, the extract
was mixed with an equal volume of glacial acetic acid and acid
ninhydrin reagent, and boiled for another 30 min. After cooling to
room temperature, the reaction mix was partitioned against
toluene, and the absorbance of the organic phase was recorded at
520 nm. The resulting values were compared with a standard curve
constructed using known amounts of proline and expressed as
mg kg1 fresh weight.
2.9. Antioxidant enzyme assays
Enzyme extracts for antioxidant enzyme activity assays were
prepared according to the method of Zhao et al. (2011). The
enzyme extraction procedure was conducted at 4
C. Three grams
of fresh tissue was extracted with 6 mL of 50 mmol L1 sodium
phosphate buffer (pH 7.0) containing 1 mmol L1 ethylene diamine
tetraacetic acid (EDTA) and 5% (w/v) polyvinyl polypyrrolidone
(PVPP). After centrifugation of the homogenate at 12,000  g for
30 min at 4
C, the supernatant was used for the enzymatic assays.
Activity of the enzymes was determined after 0, 3, 7, 14, 21 and
28 d of cold storage at room temperature for 3 d by a TU-1901
spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.,
Beijing, China). Superoxide dismutase (SOD, EC 1.15.1.1) activity
was determined according to the method of Dhindsa et al. (1981).
One unit SOD activity was defined as the amount of enzyme that
caused 50% inhibition of nitroblue tetrazolium (NBT). The specific
SOD activity was expressed as mol kg1 protein. Catalase (CAT, EC
1.11.1.6) activity was assayed using the modified method of Aebi
(1984). One unit of CAT activity was defined as the amount of
 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95 91

enzyme that caused a 0.01 decrease of absorbance per minute. The
specific CAT activity was expressed as mol s1 kg1 protein.
Peroxidase (POD, EC 1.11.1.7) activity was assayed according to
the method by Hammerschmidt et al. (1982). One unit of enzyme
activity was defined as the amount that caused an increase of 10%
in absorbance per minute. The specific POD activity was expressed
as mol s1 kg1 protein.
2.10. Protein concentration
Protein concentration in the enzyme extracts was assayed
according to the method of Bradford (1976) with bovine serum
albumin as standard.
2.11. Statistical analysis
occurred at 14 d and 21 d, which were 89% and 92% lower than
those in fruit stored at room temperature, respectively (P < 0.05).
Relative transcript levels of GA metabolism genes, GA3ox1,
GA20ox1 and GA2ox1, were evaluated by qPCR (Fig. 1B–D). In
chilled fruit, the expression levels of GA3ox1 were declined, and the
most notable changes were observed at 14 d and 21 d, when 70%
and 88% decrease occurred relative to fruit stored at room
temperature, respectively (P < 0.05; Fig. 1B). GA20ox1 was
expressed at levels 91%, 85%, 49% and 48% lower than those in
fruit stored at room temperature at 3, 7, 14 and 21 d, respectively
(P < 0.05; Fig. 1C). The fluctuation of endogenous GA3 content
(Fig. 1A) and GA3ox1 gene expression (Fig. 1B), respectively was
greatest at 14 d and 21 d. In addition, transcript levels of GA2ox1
were consistently lower compared with fruit stored at room
temperature (P < 0.05; Fig. 1D).

Three samples were assayed for each treatment, and three
technical replicates were run for each sample. Data were analyzed
statistically with Independent-Samples T test and one-way
analysis of variance (ANOVA) using SPSS (version 19.0) statistical
analysis software (IBM SPSS, Inc., Chicago, IL, USA). A difference
was considered to be statistically significant when P < 0.05.
3. Results
3.1. Effects of low temperature on GA homeostasis in tomato fruit
Endogenous GA3 levels in cherry tomato fruit were measured at
0, 3, 7, 14, 21 and 28 d after room temperature and cold storage. The
GA3 content of fruit stored at room temperature increased
significantly and reached a maximum at 21 d (P < 0.05).
Cold treatment retarded the increase in GA3 content, which
showed a slight increase in the first 3 d. Over the entire
experimental time course, the GA3 content in fruit at cold storage
was consistently lower compared with that in fruit stored at room
temperature (Fig. 1A). Substantial reductions in GA3 content
3.2. Effects of GA3 and PAC treatments on chilling injury index of
tomato fruit
Chilling injury symptoms in cherry tomato fruit appeared in all
treatments after 14 d of cold storage plus 3 d at room temperature,
and continued to progress over time. Chilling injury index in fruit
treated with GA3 was 12% less than that in control fruit at 28 d. In
contrast, PAC treatment caused severe chilling injury, with an
index 17% higher than that in control fruit (P < 0.05; Fig. 2).
3.3. Effects of GA3 and PAC treatments on GA3 content of tomato fruit
As shown in Fig. 3, when exogenous GA3 was applied, tissue GA3
levels at 3 d and 7 d were 2.24- and 1.67-fold higher, respectively,
than those in control fruit (P < 0.05). By contrast, GA3 levels in PAC-
treated fruit were inhibited at 3, 7 and 28 d, which were 62%, 31%
and 32% lower than those in control fruit, respectively (P < 0.05).
Compared with control fruit, GA3 treatment increased endogenous
GA3 levels while alleviating chilling injury, whereas PAC treatment
reduced endogenous GA3 levels while aggravating chilling injury.

Fig. 1. GA3 content (expressed as mg kg1 fresh weight) and expression levels of key GA metabolism genes in chilled tomato fruit.
(A) GA3 content in fruit stored at (4  1)
C (Cold) versus fruit stored at (25  1)
C (room temperature). (B) Expression levels of GA3ox1. (C) Expression levels of GA20ox1. (D)
Expression levels of GA2ox1. Rectangles in Panel A and Panel B highlight the correlation between GA3 content and expression levels of GA3ox1 at 14 d and 21 d. Vertical bars
represent the standard errors of the means of triplicate assays. Data with different lowercase letters in each column are significantly different at P < 0.05.
92 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95

Fig. 2. Effects of GA3 and PAC treatments on chilling injury index of tomato fruit.
Chilling symptoms of cherry tomato fruit appeared in all treatments after 14 d of
(4  1)
C storage plus 3 d at (25  1)
C. The fruit samples were taken after cold
storage for 14, 21 and 28 d plus 3 d at room temperature. Vertical bars represent the
standard errors of the means of triplicate assays. Data with different lowercase
letters in each column are significantly different at P < 0.05.

These results indicated that GA might participate in the chilling

tolerance of tomato fruit.

3.4. Effects of GA3 and PAC treatments on electrolyte leakage, MDA

content, and proline content of tomato fruit

Electrolyte leakage and MDA content were lower in GA3-treated

fruit than those in control or PAC-treated fruit during cold storage
(Fig. 4A and B). Electrolyte leakage in fruit treated with PAC was not
significantly increased compared with control fruit (P > 0.05),
while in PAC-treated fruit it was higher than that in GA3-treated
fruit from 3 d through 28 d (P < 0.05; Fig. 4A). Compared with
control fruit, MDA content in GA3-treated fruit was maintained at
lower levels after 7 d (P < 0.05). By contrast, in PAC-treated fruit,
MDA content was increased at 14 d and 21 d (P < 0.05; Fig. 4B). GA3
treatment notably increased the free proline content, especially at
14 d, which was 1.37-fold higher than that in control fruit
(P < 0.05). Whereas proline content was significantly reduced by
Fig. 4. Effects of GA3 and PAC treatments on electrolyte leakage (A), MDA content
(expressed as mmol kg1 fresh weight) (B), and proline content (expressed as
mg kg1 fresh weight) (C) of tomato fruit.
Cherry tomato fruit were sampled before treatment (time 0) and at 3, 7, 14, 21 and
28 d at (4  1)
C plus 3 d at (25  1)
C. Vertical bars represent the standard errors of
the means of triplicate assays.

PAC treatment from 14 d through 28 d compared with control fruit

(P < 0.05; Fig. 4C).

3.5. Effects of GA3 and PAC treatments on antioxidant enzyme

activities of tomato fruit

As shown in Fig. 5, GA3 maintained relatively higher activities of

Fig. 3. Effects of GA3 and PAC treatments on GA3 content (expressed as mg kg1
fresh weight) of tomato fruit.
Cherry tomato fruit were sampled before treatment (time 0) and at 3, 7, 14, 21 and
28 d at (4  1)
C plus 3 d at (25  1)
C. Vertical bars represent the standard errors of
the means of triplicate assays.
SOD in the treated fruit after 7 d than those in control and PAC-
treated fruit (P < 0.05). SOD activities in PAC-treated fruit were 18%,
21% and 12% lower than those in control fruit at 14, 21 and 28 d,
respectively (P < 0.05; Fig. 5A). Although CAT activity gradually
decreased in all fruit during cold storage, it was at a higher level in
GA3-treated fruit compared with control and PAC-treated fruit at 3,
7 and 14 d (P < 0.05; Fig. 5B). POD activity in control fruit increased
to a peak at 3 d, and constantly decreased afterwards. A similar
trend was found in GA3-treated fruit with a higher activity than
those of control and PAC-treated fruit at 3 d (P < 0.05; Fig. 5C).
 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95
Fig. 5. Effects of GA3 and PAC treatments on SOD activity (expressed as mol kg1
protein) (A), CAT activity (expressed as mol s1 kg1 protein) (B) and POD activity
(expressed as mol s1 kg1 protein) (C) of tomato fruit.
Cherry tomato fruit were sampled before treatment (time 0) and at 3, 7, 14, 21 and
28 d at (4  1)
C plus 3 d at (25  1)
C. Vertical bars represent the standard errors of
the means of triplicate assays.
4. Discussion
Experimental observations have indicated that low tempera-
ture causes disorder of endogenous bioactive GA levels in plant
(Yamauchi et al., 2004; Achard and Genschik, 2009). However, the
underlying mechanisms of cold-induced disruption of GA homeo-
stasis in fruit remained unknown. Findings from the present study
indicated that, cold storage notably retarded the increase of GA3
levels in postharvest cherry tomato fruit (Fig. 1A). Sustained lower
GA3 content was associated with suppressed expression of key GA
metabolic genes (Fig. 1B–D). Although the expression levels of
GA3ox1 and GA20ox1 increased from 14 d to 21 d, they were still
much lower than those in fruit stored at room temperature. This
might indicate an insufficient negative feedback regulation of the
GA biosynthesis genes. The correlation between GA3ox1 gene
expression (Fig. 1B) and bioactive GA levels (Fig. 1A) indicated that
the disruption of GA homeostasis might be largely due to lower
expression of GA3ox1 gene. On the other hand, low temperature
also repressed the expression of GA2ox1 gene (Fig. 1D). The
93
elevated transcript levels of GA2ox1 at 14 d and 21 d might partly
result in sustained low levels of GA3 in chilled fruit.
From our experiments, GA3 treatment alleviated chilling injury
in cherry tomato fruit (Fig. 2). Application of exogenous GA3
rapidly contributed to the elevated GA3 levels in the treated fruit at
early stages of cold storage (Fig. 3). After a rebalance of endogenous
GA3 in the treated fruit, tissue GA3 content reduced to levels
statistically indistinguishable from those in control fruit. However,
the subsequent effect of GA3 treatment maintained lower chilling
injury index from 14 d through 28 d (Fig. 2). On the other hand, PAC
treatment reduced GA3 levels at 3, 7 and 28 d and aggravated
chilling injury in fruit (Figs. 2 and 3). Membrane leakage and lipid
peroxidation are often evaluated in studies of fruit mechanisms
under chilling stress (Zhang et al., 2010). Our present study showed
that levels of electrolyte leakage in GA3-treated fruit were
remarkably lower than those of control and PAC-treated fruit
during 28 d of cold storage. However, PAC did not significantly alter
the electrolyte leakage compared to the control (Fig. 4A). Lurie
et al. (1995) suggested that the potassium ion leakage of red bell
pepper fruit was not significantly affected by PAC treatment after
28 d at 2
C. It would seem to indicate that the effect of PAC on
electrolyte leakage depends on the initial fruit maturity. Since MDA
content is a direct measure of lipid peroxidation, it is utilized as a
marker to assess the degree of cell damage (Xu et al., 2013).
Compared with the control, MDA content in GA3-treated fruit was
markedly decreased, while in PAC-treated fruit MDA content was
increased (Fig. 4B). Dai et al. (2012) indicated that GA3 treatment
alleviated cold-induced tissue damage in zoysiagrass by reducing
MDA content, whereas PAC treatment aggravated chilling injury by
increasing MDA content. Alonso-Ramírez et al. (2009) also
reported that MDA content in Arabidopsis seed previously treated
with GA3 was significantly reduced under high-temperature
exposure. The remarkable decreases in lipid peroxidation and
electrolyte leakage caused by GA, as indicators of reduction of
membrane damage, increased membranes stability and cold
tolerance of fruit. Proline has been suggested to play multiple
roles in plant stress tolerance. Positive correlations between
accumulation of endogenous proline and improved cold tolerance
have been found mostly in chilling-sensitive plant (Chen and Li,
2002). As shown in Fig. 4C, GA3 treatment increased proline
content in chilled fruit at 14 d. These results are in agreement with
increased tolerance to cold stress in GA3-treated fruit. It is
worthwhile to note that, GA is implicated in plant responses to
biotic and abiotic stress by modulating SA levels, a hormone that
been involved in responses to diverse stressful conditions. In many
plant species, exogenous GA3 treatment is able to stimulate SA
biosynthesis and defense signaling pathway (Alonso-Ramírez
et al., 2009; Lee and Park, 2010; Ding et al., 2013a). Aghdam
et al. (2012) also suggested that SA could enhance cold tolerance,
alleviate chilling injury, reduce electrolyte leakage, MDA content
and increase proline content in postharvest tomato fruit. Based on
our present findings, we hypothesized that the subsequent effect of
GA3 treatment might activate SA production and signaling, and
thus maintain fruit tolerance to chilling stress. It would be
interesting to investigate the temporal pattern of SA signaling
pathway in response to GA3 treatment in fruit during cold storage.
Various abiotic stresses lead to the overproduction of ROS in
plant (Gill and Tuteja, 2010). The coordinated action of antioxi-
dant enzymes such as SOD, CAT and POD, which are important for
scavenging ROS to protect cell membranes, help to reduce
oxidative damage and chilling injury (Egea et al., 2010). As chilling
stress increases ROS accumulation in plant, the ability to scavenge
ROS during and after chilling determines the resistance and
adaptation to low temperature (Zhao et al., 2011). Fath et al.
(2001) indicated that GA3 application increased ROS levels in
aleurone cells. Whereas, Rosenwasser et al. (2010) pointed out
94 Y. Ding et al. / Postharvest Biology and Technology 101 (2015) 88–95
that application of GA3 inhibited ROS increase in chloroplasts
during dark-induced senescence of Pelargonium cuttings. Howev-
er, the mode of action of GA3 in reducing ROS is still not clear. As
shown in Fig 5A, although GA3 and PAC treatment had no
significant effect on SOD activity during first 7 d of cold storage,
there was relatively higher activity of SOD in GA3-treated fruit
compared with the control from 14 d through 28 d. PAC treatment
resulted in an opposite effect. Saeed et al. (2014) considered that
exogenous GA3 application enhanced the SOD activity in gladiolus
cut flower during storage. Hajihashemi and Ehsanpour (2014) also
suggested that PAC treatment decreased SOD activity in Stevia
rebaudiana Bertoni in vitro under polyethylene glycol (PEG)-
induced drought stress. CAT activity was increased in GA3-treated
fruit compared with control and PAC-treated fruit from 3 d
through 14 d (Fig. 5B). Pretreatment with GA3 induced CAT activity
in cucumber hypocotyl under suboptimal temperature (Li et al.,
2011). In GA-primed wheat seeds, CAT activity was also enhanced
under salinity stress (Tabatabaei, 2013). Under nickel toxicity, POD
activity was increased in response to exogenous GA3 in Triticum
aestivum L. (Siddiqui et al., 2011). We also found that GA3
treatment improved activity of POD in fruit at 3 d of cold storage
(Fig. 5C). Thus, elevated activities of CAT and POD by exogenous
application of GA3 at early stages of cold storage might partly
result in an increase in the capacity of detoxification mechanism
and also in an enhancement in the capacity of tolerance to chilling
stress in fruit. If it could be learned how antioxidant capacity is
regulated by GA and how GA is linked with SA signaling, deeper
understanding of the chilling stress-fruit interaction could be
reached, and it might be possible to devise approaches for effective
chilling injury management.
In conclusion, we have demonstrated that cold storage caused
lower GA3 levels in tomato fruit and this was associated with lower
transcript levels of three GA metabolic enzymes. Exogenous GA3
treatment improved fruit tolerance during cold storage. The
elevated GA3 content was positively associated with lipid
peroxidation and oxidative stress in chilled fruit. The findings
from the current study open new opportunities for in-depth
studies of the functional role of the GA signaling network in fruit
during cold storage.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 31401551) and the Agricultural Science
and Technology Innovation Program (ASTIP) from the Chinese
Central Government.
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