Scientia Horticulturae 220 (2017) 90–101

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

Exogenous glutathione alleviates salt-induced oxidative stress in

tomato seedlings by regulating glutathione metabolism, redox status,
and the antioxidant system
Yan Zhou a,b,1 , Zelin Wen a,b,1 , Jianwei Zhang a,b , Xianjun Chen a,b , Jinxia Cui a,b , Wei Xu a,b ,
Hui-ying Liu a,b,∗
a
Department of Horticulture, Agricultural College, Shihezi University, Shihezi 832003, Xinjiang, PR China
b
Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization of Xinjiang Production and Construction
Crops, Shihezi 832003, Xinjiang, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this experiment was to investigate the effects of exogenous glutathione (GSH) on
Received 14 November 2016 tomato (Solanum lycopersicum L. cv. Zhongshu No. 4) seedlings under NaCl stress (100 mM), particularly
Received in revised form 23 January 2017 the effects on (1) redox status, (2) antioxidative metabolism, and (3) the gene expression and activ-
Accepted 10 February 2017
ity of five enzymes related to GSH synthesis and metabolism. Salt stress resulted in an oxidative burst
Available online 2 April 2017
in tomato seedling leaves, as evidenced by increases in hydrogen peroxide (H2 O2 ), malondialdehyde
(MDA), and superoxide radical (O2 • − ). Salt stress also reduced leaf GSH level and GSH redox homeostasis,
Keywords:
whereas application of GSH reversed the negative effects of salt stress. Furthermore, application of GSH
Glutathione
NaCl stress
increased the transcript levels and activities of enzymes related to GSH synthesis and metabolism, includ-
Solanum lycopersicum ing gamma-glutamylcysteine synthetase (␥-ECS), glutathione synthetase (GS), glutathione-S-transferase
Glutathione metabolism (GST), glutathione peroxidase (GPX), glutathione reductase (GR). Application of GSH also enhanced the
Redox status activities of antioxidant enzymes such as superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT)
and the enzymes involved in the ascorbate-glutathione cycle, and the contents of GSH and the ratios of
reduced and oxidized glutathione (GSH/GSSG) in the salt-stressed plants or salt-stress plants treated
with buthionine sulfoximine (BSO, inhibitor of GSH synthesis key enzyme gamma-glutamylcysteine
synthetase). Taken together, our findings demonstrate that exogenous GSH application increases resis-
tance to salt-induced oxidative stress by enhancing the antioxidant defense system, and regulating GSH
synthesis and metabolism to maintain cellular redox homeostasis.
© 2017 Published by Elsevier B.V.

1. Introduction and ion-specific toxicity, which in turn inhibits corp growth by

Salt stress is one of the most harmful environmental factors
limiting crop growth and yield. Salt stress causes water deficit
Abbreviations: GSH, reduced glutathione; GSSH, oxidized glutathione; BSO, l-
buthionine-sulfoximine; AsA, reduced ascorbate; DHA, oxidized ascorbate; DTNB,

5,5 -dithiobis-(2-nitrobenzoic acid); FW, fresh weight; SOD, superoxide dis-
mutase; POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase; MDHAR,
monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glu-
tathione reductase; GPX, glutathione peroxidase; GST, glutathione-S-transferase;
GS, glutathione synthetase; ␥-ECS, gamma-glutamylcysteine synthetase; MDA, mal-
ondialdehyde; ROS, reactive oxygen species.
∗ Corresponding author at: Department of Horticulture, Agricultural College, Shi-
hezi University, Shihezi 832003, Xinjiang, PR China.
E-mail address: hyliuok@aliyun.com (H.-y. Liu).
1
These authors contributed equally to this study.
disrupting plant physiological processes. Salinity can promote the
accumulation of reactive oxygen species (ROS) in leaves when the
antioxidant capacity to detoxify ROS is low (Khan and Panda, 2002;
Apel and Hirt, 2004). The ROS can attack biomacromolecules, caus-
ing membrane lipid peroxidation, changes in antioxidative enzyme
activity and antioxidant content, and finally cell death (Li et al.,
2013). Plants have evolved detoxification strategies to cope with
excessive ROS and maintain the ROS balance within cells (Mittler
et al., 2004). Glutathione (GSH), its redox status (GSH/GSSG), and
GSH-related enzymes, including glutathione reductase (GR), glu-
tathione peroxidase (GPX) and glutathione-S-transferase (GST), are
found to play an indispensable role in detoxification of ROS (Anjum
et al., 2012).
Reduced glutathione (g-Glu-Cys-Gly, GSH), a major low
molecular-weight free thiol tripeptide, is ubiquitous in plant and

http://dx.doi.org/10.1016/j.scienta.2017.02.021
0304-4238/© 2017 Published by Elsevier B.V.
 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101
animal cells and crucial to a variety of life processes, including the
maintenance of protein thiol, thioldisulfide exchange, removal of
hydroperoxides and free radicals, and amino acid transport across
membranes (Mendoza-Cózatl et al., 2005; Nahar et al., 2015a). The
GSH has unique redox and nucleophilic properties and is involved
in the cellular defense against the toxic actions of xenobiotics,
oxyradicals, salinity, acidity, and metal cations. The GSH can scav-
enge ROS either directly or indirectly via the ascorbate-glutathione
cycle, thioredoxin (Trx) and glutaredoxin (Grx) systems, sulphur
metabolism, regulation of cellular homeostasis, and signal trans-
duction (through either glutathionylation of proteins or thiol bridge
redox reactions) (Mittler et al., 2004; Riccillo et al., 2000; Romero-
Puertas et al., 2004; Anjum et al., 2012). Thus, GSH is considered to
be the major intracellular redox buffer (Foyer and Noctor, 2011).
GSH can act as the first line of defense against metal toxicity by
complexing metals before induced synthesis of phytochelatin (PC)
reaches an effective level. GSH is also an immediate substrate for
synthesis of PCs (Flores-Cáceres et al., 2015). Some reports suggest
that intracellular GSH biosynthesis and accumulation can enhance
the resistance and adaptation of plants to various environmental
stresses (Foyer and Noctor, 2011; Rausch et al., 2007). Glutathione
is synthesized from l-glutamate, l-cysteine and glycine in two
ATP-dependent reactions catalyzed by gamma-glutamylcysteine
synthetase (␥-ECS) and glutathionesynthetase (GS). Glutathione is
oxidized to GSSG as part of its cellular antioxidant defense, and
then, in turn, GSSG is reduced back to GSH by the action of GR using
NADPH to maintain the highly reduced state of GSH/GSSG (Foyer
and Noctor, 2011; Seth et al., 2012). Both GPX and GST use the GSH
pool as a substrate to detoxify H2 O2 and the xenobiotics by cat-
alyzing their conjugation with GSH. Ramakrishna and Rao (2013)
reported that exogenous HBR application regulated the activities
of enzymes related to GSH synthesis and metabolism to keep H2 O2
levels under control and maintained cellular redox homeostasis,
allowing radish seedlings to cope better with Zn2+ stress.
Many studies have shown that exogenous GSH enhances the
growth and the antioxidant response of plants subjected to abi-
otic stresses such as high temperature (Nahar et al., 2015a; Zhu
et al., 2016), drought (Nahar et al., 2015b), isoproturon (Nemat Alla
and Hassan, 2014) and heavy metal toxicity (Chen et al., 2010; Cai
et al., 2011; Estrella-Gómez et al., 2012; Qiu et al., 2013). In a previ-
ous study, we observed that GSH application promoted the growth
of salt-stressed tomato plants and enhanced salt-tolerance (Zhou
et al., 2016), and also increased net photosynthetic rate by protect-
ing PSII from damage caused by excess energy, thus increasing the
photochemical efficiency of PSII and the activity of the photosyn-
thetic dark reaction (Liu et al., 2014a,b).
However, there is no information about the effects of exogenous
GSH on glutathione synthesis, glutathione metabolism, and redox
homeostasis in plants under salt stress. Furthermore, little is known
about the mechanism by which GSH acts. Thus, the objective of this
study was to test the hypothesis that exogenous GSH application
may regulate or provide an effective strategy for combating salt
stress by stimulating the antioxidant defense system, GSH synthe-
sis and metabolism, and by maintaining redox homeostasis.
2. Materials and methods
2.1. Plant materials and treatments
Hydroponic experiments were carried out in a solar green-
house at the Shihezi University Agricultural Experiment Station,
Shihezi University, China. Tomato seeds (Solanum lycopersicum L.
cv. Zhongshu No. 4) were germinated on moist filter paper in the
dark at 28 ◦ C for 2 days. Then, the germinated seeds were sown
in trays filled with a 2:1 peat:vermiculite (v/v). When the second
91
leaves were fully expanded, the seedlings were transplanted into
12 L plastic containers (five seedlings per container) containing 10 L
aerated full-strength nutrient solution.
The seedlings were pre-cultured for 7 days before treatments.
The experimental plots included five treatments: (a) Control:
no NaCl, no GSH (Roche, China), and no BSO (l-buthionine-
sulfoximine, an inhibitor of GSH synthesis key enzyme ␥-ECS)
(Sigma, USA); (b) NaCl: 100 mM NaCl; (c) NG: 100 mM NaCl + 5 mM
GSH; (d) NB: 100 mM NaCl + 1 mM BSO; and (e) NBG: 100 mM
NaCl + 1 mM BSO + 5 mM GSH. The NaCl was added to the nutrient
solution. For the NG and NB treatment, 150 mL GSH and 150 mL
BSO were sprayed onto the seedling leaves at 10:00 a.m. every day
for 15 days, respectively. For the NBG treatment, the entire foliar
regions of salt-stressed plants were pretreated with 150 mL BSO for
1 h and subsequently treated with 150 mL GSH. The concentrations
of GSH, and BSO were selected according to the results of prelim-
inary experiments (Supplementary material). The containers were
arranged in a randomized complete block with three replicates per
treatment. There were three containers per treatment, giving a total
of 15 containers. The light period in the greenhouses was about
14 h. The air temperatures were 24–30 ◦ C during the day time and
17–20 ◦ C at night. The nutrient solutions were replaced every 3
days. Leaf samples were analyzed at day 5, 10, and 15 after the
treatments.
2.2. Measurement of leaf Na+ and Cl− content
For the determination of Na+ and Cl− contents, plant samples
(0.2 g) were digested in Triacid mixture, a mixture of HNO3 and
H2 O2 in the ratio of 2:1. The content of ions was extracted in dis-
tilled water by boiling for 30 min twice. The filtered extract was
used to measure specific ions. The Na+ content was analyzed using
an inductively coupled plasma optical emission spectrometer (ICP-
OES, Thermo Scientific ICAP 6000 Series, Boston, USA), Cl− content
was determined by titration against 0.02 N silver nitrate solution
using 5% K2 CrO4 as an indicator as described by Nazar et al. (2011).
2.3. Lipid peroxidation
Lipid peroxidation was estimated by measuring leaf malon-
dialdehyde (MDA) content. Leaf MDA was determined using the
method of Health and Packer (1968) with thiobarbituric acid (TBA)
as the reactive material. The MDA contents were calculated based
on absorption at 532 and 600 nm, with an extinction coefficient of
155 mM−1 cm−1 .
2.4. H2 O2 content and O2 •− formation rate
Leaf H2 O2 was assayed according to the method described by
Yu et al. (2003). H2 O2 was extracted by homogenizing 0.5 g leaf
tissue with 3 mL of phosphate buffer (50 m M, pH 6.5) at 4 ◦ C. The
homogenate was centrifuged at 6000 × g for 25 min. A supernatant
of 3 mL was mixed with 1 mL of 0.1% TiCl4 in 20% H2 SO4 (v/v), and
the mixture was then centrifuged at 6000 × g for 15 min at room
temperature. The optical absorption of the supernatant was mea-
sured spectrophotometrically at 410 nm to determine the H2 O2
content (expressed as ␮mol g−1 fresh weight).
The O2 − formation rate in the leaves was determined fol-
lowing Elstner and Heupel (1976) with some modifications. Fresh
leaves (0.3 g) were homogenized in 3 cm3 of 65 mM potassium
phosphate buffer (pH 7.8) and centrifuged at 5000 × g for 10 min.
The supernatant was mixed with the extraction buffer and 10 mM
hydroxylamine hydrochloride (at the ratio of 10.7:9.1:1) and incu-
bated at 25 ◦ C for 20 min. The solution was then mixed with 17 mM
sulfanilamide and 7 mM naphthylamine (at the ratio of 1:1) and
incubated again at 25 ◦ C for 20 min. The absorbance was measured
92 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101
at 530 nm. Absorption was measured at 530 nm. The O2 − formation
rate was calculated from a standard curve prepared with NaNO2 .
2.5. Histochemical staining
Histochemical staining of H2 O2 and O2 − was conducted accord-
ing to the method described by Thordal-Christonson et al. (1997).
2.6. Determination of redox state
Tomato leaves (0.3 g fresh weight) were homogenized in 3 mL
ice-cold acidic extraction buffer (6% metaphosphoric acid contain-
ing 1 mM EDTA) using a mortar and pestle. The homogenates were
centrifuged at 12,000 × g for 15 min at 4 ◦ C. The supernatant was
then neutralized with 0.5 M K-phosphate buffer (pH 7.5). The total
glutathione (GSH + GSSG) content in the leaves was determined
according to the procedure by Yu et al. (2002). Glutathione was oxi-
dized by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and reduced by
NADPH in the presence of glutathione reductase. Total glutathione
content was calculated by measuring the generation of 2-nitro-5-
thiobenzoic acid (NTB) from DTNB. This was indicated by changes
in absorption at 412 nm. The GSSG content was determined after
removing GSH by 2-vinylpyridine derivatization. A standard curve
was prepared with GSH. The GSH content was determined by sub-
tracting the GSSG content from the total glutathione content.
The total ascorbate content (AsA plus DHA) was determined
following the procedure by Costa et al. (2002) with slight mod-
ifications. Leaves (0.3 g) were homogenized in ice-cold 5% (w/v)
TCA and centrifuged at 12,000 × g for 10 min. Half of each sample
was assayed for total ascorbate, and the other half was used for
AsA analysis. Absorbance was recorded at 525 nm. The DHA con-
tent was calculated as the difference between the total ascorbate
concentration and the AsA content.
2.7. Extraction and assay of antioxidant enzymes
Antioxidant enzymes were extracted from leaves using the
method of Grace and Logan (1996). Samples (0.3 g) of the leaves
were ground with a pre-cooled mortar and pestle in 1.6 mL ice-cold
50 mM potassium phosphate extraction buffer (pH 7.5), 0.1 mM
ethylenediaminetetraacetic acid (EDTA), 0.3% (w/v) Triton X100,
and 4% (w/v) polyvinylpolypyrrolidone (PVPP). The extracts were
then centrifuged at 13,000 × g for 10 min at 4 ◦ C. The supernatant
(hereafter referred to as enzyme extract) was immediately ana-
lyzed for the following antioxidant enzymes.
Superoxidase dismutase (SOD; EC: 1.15.1.1) activity was
assayed using a xanthine-xanthine oxidase system (El-Shabrawi et
al., 2010). A reaction mixture of 1 mL was prepared by mixing the
enzyme extract (0.10 mL) with 50 mM potassium phosphate buffer
(pH 7.8), 2.24 mM nitroblue tetrazoliam chloride (NBT), catalase
(0.1 units), xanthine oxidase (0.1 units), and xanthine (2.36 mM).
Catalase was added to avoid H2 O2 -mediated inactivation of CuZn-
SOD. The change in absorbance was read at 560 nm. The SOD
activity was expressed as the amount of enzyme needed to inhibit
NTB reduction by 50%.
Peroxidase (POD; EC: 1.11.1.7) activity was determined using
the method by Shannon et al. (1966). The reaction mixture con-
tained 2.9 mL of 0.1 M phosphate buffer (pH 7.0), 0.04 mL of 0.1 M
H2 O2 , 0.04 mL of 0.2% o-dianisidine, and 0.02 mL of enzyme extract.
The change in absorbance was read at 470 nm for 4 min. One
enzyme unit is defined as the change in 1 unit of absorbance min−1 .
Catalase (CAT; EC: 1.11.1.6) activity was assayed according to
Hasanuzzaman et al. (2011a). A reaction mixture of 2 mL was pre-
pared by mixing the enzyme extract (0.1 mL) with distilled water,
50 mM potassium phosphate buffer (pH 7.0), and H2 O2 (15 mM).
The reaction was initiated with the enzyme extract. The decrease
in the absorbance (due to decomposition of H2 O2 ) at 240 nm was
recorded for 1 min. The activity was calculated using an extinction
coefficient of 39.4 M−1 cm−1 .
Ascorbate peroxidase (APX; EC: 1.11.1.11) activity was mea-
sured according to Nakano and Asada (1981). A reaction mixture
of 2 mL was prepared by mixing the enzyme extract (0.1 mL) with
50 mM potassium phosphate buffer (pH 7.0), 0.5 mM AsA, 0.1 mM
H2 O2 , and 0.1 mM EDTA. The reaction was started by adding H2 O2 .
The absorbance was measured at 290 nm for 1 min. The APX activity
was determined using an extinction coefficient of 2.8 mM−1 cm−1 .
Monodehydroascorbate reductase (MDHAR; EC: 1.6.5.4) activity
was assayed according to Hossain et al. (1984). A reaction mixture
of 2 mL was prepared by mixing the enzyme extract (0.1 mL) with
50 mM Tris–HCl buffer (pH 7.5), 0.2 mM NADPH, 2.5 mM AsA, and
0.5 units of ascorbate oxidase (AO). The reaction was started by
adding AO. The absorbance was measured at 340 nm. The activity
was calculated from the change in absorbance after 1 min (extinc-
tion coefficient was 6.2 mM−1 cm−1 ).
Dehydroascorbate reductase (DHAR; EC: 1.8.5.1) activity was
measured according to Nakano and Asada (1981). The reaction
mixture was prepared by mixing the enzyme extract (0.1 mL)
into a solution containing 50 mM potassium phosphate buffer (pH
7.0), 2.5 mM GSH, 0.1 mM EDTA, and dehydroascorbic acid solu-
tion (0.001 g dehydroascorbic acid dissolved in 4.2 mL distilled
water). The activity was calculated from the change in absorbance
at 265 nm after 1 min (extinction coefficient was 14 mM−1 cm−1 ).
2.8. Extraction and analysis of key enzymes in glutathione
synthesis and metabolism
Using a pre-cooled mortar and pestle, 0.3 g of leaf tissue was
homogenized in equal volumes of 50 mM ice-cold K-phosphate
buffer (pH 7.0) containing 100 mM KCl, 1% (w/v) ascorbate, and 10%
(w/v) glycerol. The homogenates were centrifuged at 12,000 × g
for 10 min and the supernatants (i.e., enzyme extracts) were used
to determine enzyme activity. All procedures were performed
between 0 and 4 ◦ C.
Activity of gamma-glutamylcysteine synthetase (␥-ECS;
EC:6.3.2.2) was determined using the method by Rüegsegger and
Brunold (1992). A reaction mixture of 500 ␮L was prepared by
mixing the enzyme extract (150 ␮L) with 100 mM HEPES (pH 8.0),
50 mM MgCl2 , 20 mM glutamate, 1 mM cysteine, 5 mM ATP, 5 mM
phosphoenolpyruvate, 5 mM DTT, and 10 units mL−1 pyruvate
kinase. The reaction mixture was incubated for 45 min at 37 ◦ C and
the reaction was stopped by addition of 100 ␮L of 50% TCA. The
mixture was centrifuged and the supernatant was used to estimate
the phosphate content by the phosphomolybdate method.
The activity of glutathione synthetase (GS; EC: 6.3.2.3) was
determined according to the method of Cobbett et al. (1998). The
reaction mixture contained 100 mM Tris–HCl (pH 8.0), 50 mM KCl,
20 mM MgCl2 , 1 mM ␥-EC, 2 mM glycine, 5 mM ATP, 5 mM phos-
phoenolpyruvate, 5 mM DTT, and 10 units mL−1 pyruvate kinase.
The reaction was started by adding 140 ␮L of extract to the reac-
tion mixture. The mixture was incubated for 45 min at 37 ◦ C. The
reaction was then stopped by adding 100 ␮L of 50% TCA. The mix-
ture was centrifuged and the supernatant was used to estimate the
phosphate content using the phosphomolybdate method.
Glutathione reductase (GR; EC: 1.6.4.2) activity was measured
by the method of Cakmak et al. (1993). A reaction mixture of 1 mL
was prepared by mixing the enzyme extract (0.1 mL) with 0.1 M Na-
phosphate buffer (pH 7.8), 1 mM EDTA, 1 mM GSSG, and 0.2 mM
NADPH. The reaction was initiated with GSSG. The decrease in
absorbance at 340 nm due to NADPH oxidation was recorded for
1 min. The activity was calculated using the extinction coefficient
of 6.2 mM−1 cm−1 .
 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101
Glutathione peroxidase (GPX; EC: 1.11.1.9) activity was mea-
sured as described by Elia et al. (2003) using H2 O2 as a substrate.
The reaction mixture consisted of 20 ␮L enzyme mixture combined
with 100 mM Na-phosphate buffer (pH 7.5), 1 mM EDTA, 1 mM
NaN3 , 0.12 mM NADPH, 2 mM GSH, 1 unit GR, and 0.6 mM H2 O2 .
The reaction was started by adding H2 O2 . The oxidation of NADPH
was recorded at 340 nm for 1 min and the activity was calculated
using an extinction coefficient of 6.62 mM−1 cm−1 .
Glutathione-S-transferase (GST; EC: 2.5.1.18) activity was deter-
mined spectrophotometrically using the method described by
Booth et al. (1961) with modifications. A reaction mixture of
0.7 mL was prepared by mixing the enzyme extract (0.1 mL)
with 100 mM Tris–HCl buffer (pH 6.5), 1 mM 1-chloro-2,4-
dinitrobenzene (CDNB), and 1.5 mM reduced glutathione (GSH).
The enzyme reaction was initiated by the addition of CDNB.
The increase in absorbance was measured at 340 nm for 1 min.
The activity was calculated using an extinction coefficient of
9.6 mM−1 cm−1 .
2.9. Total RNA extraction and GPX, GST, GR, -ECS and GS
transcript levels analysis
Total RNA was extracted using Trizol according to the supplier’s
recommendation (Sangon, China). After extraction, total RNA was
dissolved in diethyl pyrocarbonate-treated water. The cDNA tem-
plate for real-time PCR was synthesized using a RevertAidTM first
strand cDNA Synthesis Kit (Fermentas) from 2 ␮g RNA purified
using RNeasy Mini Kit (Qiagen). Quantitative real-time PCR was
performed using the iCycler iQ Multicolor Real-time PCR Detection
System (Bio-Rad, Hercules, CA, USA). Each reaction (20 ␮L) con-
sisted of 2 ␮L of diluted cDNA, 10 ␮L iQ SYBR Green Supermix, and
0.4 ␮L of forward and reserve primers. The PCR conditions consisted
of denaturation at 95 ◦ C for 30 s, followed by 40 cycles of 95 ◦ C for
30 s, denaturation at 95 ◦ C for 5 s, annealing at 56 ◦ C for 10 s and
extension at 72 ◦ C for 15 s. Tomato actin gene was used as an inter-
nal control. Relevant gene expression was calculated according to
Livak and Schmittgen (2001). The following primers were designed
for gene specific transcript amplification:
Tomato actin gene was used as the control (SLU60478),
forward (fw)-5 -TGGTCGGAATGGGAAAG-3 , and reverse (rv)-5 -
CTCAGTCAGGAGAACAGGGT-3 ;
GPX (XM006347213.2), fw-5 -CCGGAACAAATGAGGAGATT-3 , rv-
5 -TCCACAAGGAATTTGGTGAA-3 ;
GST (EF409975.1), fw-5 -GGCTCGTTTTGTTGATGGCAA-3 , rv-5 -
CCACCGATTCAACTCCCTC T-3 ;
GR (NM001321393.1), fw-5 -TTGGTGGAACGTGTGTTCTT-3 , rv-5 -
TCTCATTCACTTCCCATCCA-3 ;
-ECS (AF017983.1), fw-5 -CCTCAGCACACCAAAATCCT-3 , rv-5 -
GCTTTGTGCCTTGGCTCTAGT-3 ;
GS (AF017984.1), fw-5 -AGTGGAAAGCTAGGCTGCTG-3 , rv-5 -
TCATCCAAGCTCCACAACCC-3 .
2.10. Statistical analysis
The data were analyzed by one-way analysis of variance
(ANOVA) and post hoc multiple comparisons using SPSS version
13.0 (SPSS Inc., Chicago, IL, USA). Differences were considered to be
significant when P < 0.05. The values in the figures and tables are
means ± SE (n = 3).
93
3. Results
3.1. Effect of GSH on the accumulation of ions and oxidative stress
Plants treated with 100 mM NaCl showed increased accumu-
lation of Na+ and Cl− in the leaves of tomato seedlings (Fig. 1).
However, supplementation of GSH to salt-stressed seedlings signif-
icantly decreased the Na+ and Cl− uptake and accumulation. Leaf
Na+ and Cl− contents were reduced by 5–38% and 17–32%, when
compared to the salt-stressed plants, respectively. The NB treat-
ment compared with the NaCl treatment significantly increased
Na+ by 11% and 10% on 10 and 15 d and Cl− by 7.9% on 10 d. During
the whole sampling period, application of GSH also exerted positive
effects on lowering Na+ and Cl− accumulation in plants under NB
treatment (P < 0.05), decreasing the values by 3–36% and 10–28%,
when compared with the NB treatment, respectively (Fig. 1).
Plants with higher Na+ and Cl− content showed higher oxidative
stress. Exposure to NaCl stress caused an increase in ROS generation
and lipid peroxidation in the leaves of tomato seedlings. The NaCl
stress significantly increased MDA and H2 O2 in leaves on all sample
dates (Fig. 2A and B). The NG treatment compared with the NaCl
treatment decreased MDA by 28–80% and H2 O2 by 4–13% on all
sample dates (P < 0.05). In contrast, NB significantly increased MDA
by 7 and 11% on 5 and 15 d, and H2 O2 by 7–11% on all three sample
dates compared with the NaCl treatment. Among all treatments,
NB had the greatest MDA content on 5 and 15 d and the greatest
H2 O2 content on all sample dates. The NBG treatment significantly
decreased the MDA and H2 O2 contents in tomato leaves compared
with the NB treatment. Leaf MDA and H2 O2 contents were 38–177%
and 5–7% less in NBG than in NB on all sample dates (P < 0.05).
Salt stress also significantly increased the O2 •− formation rate on
all sample dates (P < 0.05) (Fig. 2C). Compared with the NaCl treat-
ment, NG reduced the O2 •− formation rate by 27–58% (P < 0.05). In
contrast, NB increased O2 •− formation rates by 9–24%. Seedlings in
NB had the highest O2 •− formation rates. The O2 •− formation rates
were 44–82% less in NBG than in NB (P < 0.05).
3.2. In situ detection of leaf O2 •− and H2 O2
Similar to the above measured results of H2 O2 content and O2 •−
formation rate (Fig. 2B and C), the accumulation of O2 •− (dark
spots) and H2 O2 (brown spots) was observed in tomato leaves in
the NaCl treatment on all sample dates compared with the control
(Fig. 3A and B). In addition, O2 •− and H2 O2 were both distributed
throughout the entire leaf. Compared with the NaCl treatment,
NG diminished salt-induced ROS accumulation in tomato leaves,
whereas BSO application had an opposite effect. More O2 •− (dark
spots) and H2 O2 (brown spots) were observed in NB than in the
NBG on all sample dates (Fig. 3A and B).
3.3. GSH, AsA level and redox status in tomato leaves
Compared with the control, the NaCl treatment significantly
reduced the contents of GSH and AsA, and the ratios of GSH/GSSG
and AsA/DHA in tomato leaves throughout the experiment
(P < 0.05) (Table 1). The NG treatment compared with the NaCl
treatment significantly increased leaf GSH and AsA levels, and the
ratios of GSH/GSSG and AsA/DHA (P < 0.05). The greatest effects
were observed at the time point of 5 d, when GSH was signifi-
cantly higher in NG than in the control (P < 0.05). However, the
content of AsA, and the ratios of GSH/GSSH and AsA/DHA in NG
were less than those in the control during the entire experiment
(P < 0.05). In contrast, NB significantly decreased GSH and AsA by
5–20% and 11–17%, respectively, compared with the NaCl treat-
ment (P < 0.05). The GSH/GSSG ratio and the AsA/DHA ratio were
58–112% and 11–116% less in the NB treatment than in the NaCl
94 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101

Fig. 1. The Na+ content (A) and Cl− content (B) in leaves of salt-stressed tomato seedlings as affected by exogenous GSH and BSO. Values are means ± SD (n = 3). Values with
a different letter within a sampling date are significantly different (P < 0.05).

Table 1
The GSH content (nmol g−1 FW), GSH/GSSG ratio, AsA content (nmol g−1 FW), and AsA/DHA ratio in leaves of salt-stressed tomato seedlings as affected by exogenous GSH
and BSO. Values are means ± SD (n = 3). Values with a different letter within a sampling date are significantly different (P < 0.05).

Treatment Time GSH GSH/GSSG AsA AsA/DHA

CK 5d 82.99 ± 0.24b 25.96 ± 0.06a 10.74 ± 0.18a 19.13 ± 0.06a

NaCl 68.20 ± 0.40d 12.98 ± 0.05c 7.69 ± 0.03d 15.58 ± 0.06d
NG 119.83 ± 0.48a 23.03 ± 0.23b 9.48 ± 0.01b 18.43 ± 0.28b
NB 64.97 ± 0.53e 6.13 ± 0.22e 6.90 ± 0.03e 14.04 ± 0.05c
NBG 74.98 ± 0.52c 8.99 ± 0.35d 8.49 ± 0.02c 16.07 ± 0.08e

CK 10 d 80.47 ± 0.45a 20.27 ± 0.34a 10.25 ± 0.05a 21.65 ± 0.10a

NaCl 60.23 ± 0.38d 12.96 ± 0.35c 8.23 ± 0.07d 14.57 ± 0.02c
NG 75.29 ± 0.28b 16.92 ± 0.59b 9.24 ± 0.06b 18.03 ± 0.01b
NB 50.02 ± 0.03e 8.19 ± 0.60e 7.25 ± 0.04e 9.82 ± 0.04e
NBG 66.10 ± 0.29c 10.95 ± 0.09d 8.69 ± 0.03c 13.71 ± 0.16d

CK 15 d 82.10 ± 0.38a 21.09 ± 0.18a 9.77 ± 0.09a 19.53 ± 0.03a

NaCl 57.06 ± 0.80d 11.11 ± 0.47c 7.28 ± 0.06d 13.11 ± 0.16c
NG 72.03 ± 0.23b 17.05 ± 0.45b 9.37 ± 0.04b 17.10 ± 0.08b
NB 47.97 ± 0.32e 6.20 ± 0.22e 6.21 ± 0.07e 6.06 ± 0.05e
NBG 60.18 ± 0.26c 9.12 ± 0.20d 7.89 ± 0.07c 10.05 ± 0.07d

treatment, respectively. Compared with NB, NBG increased GSH
by 15–32%, AsA by 20–27%, the GSH/GSSG ratio by 34–47%, and
the AsA/DHA ratio by 14–66% during the whole sampling period
(P < 0.05).
3.4. Activities of antioxidant enzymes in tomato leaves
increased by GSH application. The activities of all those enzymes
were higher in the NBG treatment than in the corresponding NB
treatment (P < 0.05).
3.5. Activities of key enzymes related to glutathione synthesis and
metabolism

Salt stress significantly decreased the activities of SOD, POD,
CAT, APX, DHAR, and MDHAR in tomato leaves on all three sam-
pling dates (P < 0.05) (Fig. 4). Compared with NaCl treatment, NG
significantly increased the activities of SOD, POD, CAT, APX, DHAR,
and MDHAR (P < 0.05). In contrast, NB reduced the activities of
SOD, POD, CAT, APX DHAR, and MDHAR (P < 0.05). The activities
of SOD, POD, CAT, APX, DHAR, and MDHAR in NB were significantly
The above results indicate that exogenous GSH increases the leaf
GSH content and maintains the cells in a reduced state in tomato
plants under salt stress with BSO or without BSO (Table 1).
To further investigate the protective mechanism of GSH against
salt stress, we analyzed the temporal changes in the activities of
enzymes related to GSH biosynthesis and metabolism. Salt stress
significantly reduced the activities of GPX, GST, GR, ␥-ECS, and GS
 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101 95

Fig. 2. Leaf MDA content (A), H2 O2 content (B) and O2 • − formation rate (C) in salt-stressed tomato seedlings as affected by exogenous GSH and BSO. Error bars represent SD
(n = 3). Bars with a different letter within a sampling date are significantly different (P < 0.05).

(P < 0.05) (Fig. 5). Compared with the NaCl treatment, NG signifi- 4. Discussion
cantly increased the activities of GPX by 19–55%, GST by 36–43%,
GR by 14%, ␥-ECS by 7–28%, and GS by 21–25%, respectively. In con- The ROS and MDA levels are well-known indexes for determin-
trast, NB significantly reduced the activities of GPX by 92–118%, ing the degree of oxidative stress. It has been proposed that plants
GST by 8–9%, GR by 26–28%, ␥-ECS by 4–33%, and GS by 8–12%. The increase stress tolerance partly by controlling MDA content, H2 O2
activities of all of the above enzymes were significantly higher in content, and O2 •− formation rate (Chattopadhayay et al., 2002; Bor
NBG than in NB (P < 0.05). et al., 2003). Increases in MDA, H2 O2 content, and O2 •− formation
rate have been reported in many plant species under salt stress
(Sheokand et al., 2008; Sumithra et al., 2006; Seckin et al., 2009).
In the present study, we also demonstrated that excess Na+ and
3.6. Analysis of GPX, GST, GR, -ECS and GS transcript levels by
Cl− accumulation in leaves of plants under NaCl treatment resulted
real-time RT-PCR
in the increase of MDA and H2 O2 content, and O2 •− formation
rate, reflecting NaCl stress induced oxidative stress (Figs. 1 and 2).
Fig. 5 shows the relative transcript levels of five key enzyme
This agrees with the reports by Leyva et al. (2011) and Queirós
genes related to GSH biosynthesis and metabolism. Compared with
et al. (2011). Application of BSO aggravates the oxidative stress
the control, salt stress significantly reduced the transcript levels
induced by NaCl stress, implicating enhanced oxidative degrada-
of GPX, GST, GR, -ECS, and GS genes (P < 0.05). The NG treatment
tion of lipids. To protect themselves against oxidative stress, plants
significantly increased the transcript levels of GPX, GST, GR, -ECS,
have evolved enzymatic and non-enzymatic ROS scavenging sys-
and GS on all three sampling dates compared with NaCl treatment
tems. These systems play a crucial role in protecting the structure
(P < 0.05). The highest expression levels of GPX, GST, GR, -ECS, and
and function of membrane systems and in maintaining the cellular
GS in tomato seedlings in NG were 6.10, 8.74, 6.83, 13.49 and 9.29
redox state (Baisak et al., 1994). In plants, SOD constitutes the first
times higher than those in the corresponding NaCl treatment. In
line of defense to funnel O2 •− into H2 O2 and O2 . Subsequently, H2 O2
contrast, NB significantly down-regulated GPX, GST, GR, -ECS, and
is eliminated by conversion to water by the action of CAT, POD, the
GS expression (P < 0.05). The NBG treatment significantly increased
ascorbate-glutathione cycle, the thioredoxin (Trx) system, and the
GPX, GST, GR, -ECS, and GS expression in the tomato leaves com-
glutaredoxin (Grx) system (Hasanuzzaman et al., 2012; Garg and
pared with NB treatment (P < 0.05). The highest expression levels
Manchanda, 2009; Mittler et al., 2004). In the ascorbate-glutathione
of GPX, GST, GR, -ECS, and GS in the NBG treatment were 3.26,
cycle, APX plays the most important role in removing H2 O2 . The
5.58, 4.20, 2.92 and 6.92 times higher, respectively, than those in
MDHAR, DHAR and GR are mainly responsible for providing sub-
the corresponding NB treatment, respectively.
96 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101

Fig. 3. Staining of O2 • − (A) and H2 O2 (B) in leaves of salt-stressed tomato seedlings as affected by exogenous GSH and BSO.

strates for APX through the formation of AsA and GSH. Here, we
showed that NaCl stress reduced the activities of SOD, CAT, POD,
and four key enzymes (i.e., APX, DHAR, MDHAR and GR) involved in
the AsA-GSH cycle. The great accumulation of H2 O2 by salt stress is
accompanied with great drop in SOD activity, indicating a decline in
H2 O2 detoxification, rather than great production, due to the inhib-
ited activities of POD, CAT and APX. Therefore, the present results
point to a deficiency in H2 O2 detoxification in response to salt stress
Moreover, the application of GSH significantly reduced the accu-
mulation of Na+ and Cl− , and lipid peroxidation and ROS levels
in salt-stressed plants or salt and BSO combined stressed plants
(NB treatment) (Fig. 2A–C). Furthermore, exogenous GSH applica-
tion also elevated the internal content of GSH and the activities
of SOD, CAT, POD, and four key enzymes involved in the AsA-GSH
 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101 97

Fig. 4. Activities of APX (A), SOD (B), POD (C), CAT (D), DHAR (E), and MDAHR (F) in leaves of salt-stressed tomato seedlings as affected by exogenous GSH and BSO. Error
bars represent SD (n = 3). Bars with a different letter within a sampling date are significantly different (P < 0.05).

cycle in salt-stressed plants or NB treated plants, respectively. An
exogenous GSH-induced improvement in cell membrane proper-
ties (Alla and Hassan, 2014) or higher endogenous GSH level is
observed to reduce oxidative stress under various abiotic stresses
(Hasanuzzaman et al., 2014; Wu et al., 2011). These results give
strong evidence that the higher antioxidant defense capacity is one
of the important mechanisms for exogenous GSH to alleviate oxida-
tive stress induced by accumulation of Na+ and Cl− in the leaves of
tomato.
Mounting evidence shows that the intracellular redox state is
an important factor in the ability of plants to resist the effects of
biotic and abiotic stress (Dietz, 2008). Maintaining a high reducing
power level is essential for plants to quench excess ROS. Because
GSH is a major cellular antioxidant, GSH content and GSH/GSSG
ratio are regarded as determinants of cellular redox state and might
indirectly influence many fundamental cellular processes. The high
GSH content and GSH/GSSG ratio in plants could be conducive to
maintaining an appropriate redox environment and reducing the
oxidative stress caused by biotic and abiotic stress. In the present
study, a disturbance of the GSH redox balance was evidenced by
a significant decrease in the GSH/GSSG and AsA/DHA ratios of
salt-stressed tomato seedlings (Table 1), whereas exogenous GSH
application induced the accumulation of antioxidant metabolites
which provided additional resistance to the toxic effects of salt
stress-induced ROS. We observed that exogenous GSH increased
the contents of GSH and AsA, and the ratios of GSH/GSSG and
AsA/DHA in the salt-stressed tomato seedlings. Furthermore, when
GSH accumulation was inhibited in salt-stressed plants with BSO
treatment, GSH application had similar effects on NB treated plants.
Specifically, the lower H2 O2 and MDA accumulation and O2 •−
formation rates in NB-treated plants with GSH application were
observed as compared with NB treated plants. These parameters
were correlated with the higher GSH and AsA contents, and ratios
of GSH/GSSG and AsA/DHA. These changes could enable plants to
maintain the redox state in their leaves and keep the sulfhydryl
groups of soluble and membrane proteins in the reduced state,
resulting in the alleviation of salt stress. Thus, exogenous GSH
alleviated salt-induced membrane injury is closely related with
the higher reducing power levels, thereby protecting plants from
oxidative stress.
The amount of GSH in a given organism is the result of the
combined action of biosynthesis, consumption, and degradation
98 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101

Fig. 5. Activities (left) and transcript levels (right) of GPX (A), GST (B), GR (C), ␥-ECS (D) and GS (E) in leaves of salt-stressed tomato seedlings as affected by exogenous GSH
and BSO. Error bars represent SD (n = 3). Bars with a different letter within a sampling date are significantly different (P < 0.05).

(Tripathi et al., 2012). GSH is synthesized in two sequential reac-
tions catalyzed by ␥-ECS and GS (Foyer and Noctor, 2011; Yadav,
2012). Arabidopsis cad2-1 mutant accumulates lower levels of GSH
than the wild type due to the lack of ␥-ECS (Cobbett et al., 1998).
Some studies have found that an increase in the activities of ␥-ECS
and GS can lead to GSH accumulation, and hence enhance resistance
of plants to heavy metal, ozone, and oxidative stress. However,
there is also evidence that increases in the activities of the two
enzymes do not account for GSH accumulation (Noctor et al., 1998;
Di Baccio et al., 2005). The cellular GSH pool is determined not only
by the rates of GSH synthesis but also by GSH regeneration (Foyer
and Noctor, 2011). Our results showed that salt stress reduced the
content of GSH and the activities of ␥-ECS and GS (Fig. 5D and
E). This suggests that NaCl stress reduces the rate of GSH synthe-
sis, making it impossible to rapidly replenish the depleted GSH
pool and maintain the GSH/GSSG ratio. Salt stress also affected the
recovery of GSH from GSSG in seedlings with reduced GR activity
(Fig. 5C). Reduced GR activity may therefore lead to the accumu-
lation of GSSG, which would help explain the lower GSH/GSSG
ratios of seedlings under salt stress conditions (Table 1). However,
GSH application to salt stressed seedlings switched the GSH pools
towards reduced forms, thereby improving the GSH/GSSG ratio.
The observed higher levels of GSH could result from the increased
GSH synthesis catalyzed by ␥-ECS and GS and from the increased
GSH regeneration catalyzed by GR. To further investigate the role
of GSH, we used BSO, an inhibitor of ␥-ECS, which is believed to
be the rate-limiting enzyme in the synthesis of GSH. The depletion
of GSH by pretreatment with BSO is found to augment the oxida-
tive damage induced by Cd in tomato (Ammar et al., 2008) and A.
thaliana (Wójcik and Tukiendorf, 2011), primarily due to impaired
GSH-dependent redox homeostasis. In our study, application of BSO
reduced not only the activities of ␥-ECS, GS and GR and but also cell
GSH concentrations and GSH/GSSG ratio in salt-stressed seedlings
during the entire sampling period. Previous studies in our labo-
ratory have shown that BSO significantly depleted GSH pools and
augments the oxidative damage in tomato seedlings after salt stress
 Y. Zhou et al. / Scientia Horticulturae 220 (2017) 90–101
for 5 d (Liu et al., 2014a). However, application of GSH reversed the
effects of BSO on salt-stressed plants, as evidenced by significant
increases in the activities of ␥-ECS, GS and GR. These results indicate
that exogenous GSH maintains GSH redox homeostasis and helps
to meet the increasing demand for GSH by regulating GSH synthe-
sis and regeneration, thus increasing resistance to the damaging
oxidative effects of salt stress.
Enzymes involved in the GSH metabolism may have vital roles in
plant tolerance to environmental stress. GPX and GST are important
such enzymes involved in GSH-metabolism and play significant
roles in the cellular ROS detoxification (Anjum et al., 2012). In
plants, GST is a multigene family of isoenzymes that catalyze the
conjugation of GSH with toxic compounds and may relieve the
oxidative stress by scavenging various ROS (Zhang et al., 2013;
Haluskova et al., 2009). GPX is another GSH dependent enzyme
that serves as an intrinsic defense tool, catalyzing the detoxifi-
cation of H2 O2 as well as organic and lipid hydroperoxides (Gill
and Tuteja, 2010; Passaia and Margis-Pinheiro, 2015). The GPX also
plays an important role in modulating the GSH/GSSG ratio to main-
tain the higher reducing power and to deliver information about
ROS to the redox signal network (Noctor et al., 2002; Sarvajeet
and Narendra, 2010). Roxas et al. (1997) indicate the overexpres-
sion and improvement in the activity of GST and GPX in transgenic
tobacco increase GSH-dependent peroxide scavenging and alter-
ations in GSH metabolism that lead to less oxidative damage. In the
present study, the reduced of GST and GPX activity and higher H2 O2
and MDA levels were observed in salt-stressed seedlings (Figs. 1A,
B and 5A, B). Application of GSH affects GPx- and GST-catalyzed
protective mechanisms that utilize glutathione in salt-stressed
seedlings (Fig. 5A and B), There was an up-regulation of GST and
GPX activity in response to salt-stress with GSH treatment, which
is consistent with an increase in ROS detoxification. Furthermore,
in our study, BSO application to salt-stressed seedlings further
aggravated the oxidative stress induced by salt stress and signifi-
cantly decreased GST and GPX activities on all three sample periods,
whereas exogenous GSH application stimulated the detoxification
of oxidative degradation of lipids in salt-stressed plants with BSO
treatment by increasing GST and GPX activity. These results fur-
ther confirm that exogenous GSH-induced GSH-related antioxidant
defense system might be the main mechanism of ROS detoxification
in salt-stressed tomato seedlings.
There is no information about the effect of exogenous GSH on the
expression of genes involved in GSH biosynthesis and metabolism.
B. Juncea overexpressing GS or -ECS has been reported to exhibit
its enhanced tolerance to a variety of heavy metals including As,
Cd, Zn and Pb due to higher capacity of GSH synthesis as well as PC
synthesis (Reisinger et al., 2008). The transcription of -ECS, GS and
GR is significantly induced after ochratoxin A treatment, consistent
with the increased GSH content in these tissues (Wang et al., 2014).
Our RT-PCR results showed that the transcript levels of the GPX, GST,
GR, -ECS, and GS genes in salt-stressed plants were all elevated by
GSH application, but were reduced by BSO application when com-
pared to the salt-stressed plants during the entire sampling period.
Similar to the gene expression, the activities of GPX, GST, GR, ␥-
ECS, and GS and the contents of GSH in the tomato leaves of GSH-
and BSO-treated under salt stress were increased and decreased,
respectively. Again, the BSO-induced decreases in the transcript
levels and activities of these enzymes were recovered by the sub-
sequent application of GSH during the entire sampling period (Fig. 5
and Table 1). This indicated that exogenous GSH may increase the
activation of existing enzyme pools, thus using metabolic means
to increase tolerance salt stress. Increasing antioxidant levels in
tomato cells through modification of gene expression and adaptive
metabolism protects plants from salt-caused oxidative damage. It is
possible that both the anti-oxidative capacity and the GSH-induced
response in tomato contribute to the protection against salt stress.
99
In addition, under salt stress, the GSH-induced increase in the tran-
scripts of GPX, GST, GR, -ECS, and GS genes was not accompanied by
enhanced enzymes activity. We observed that the transcript levels
of GPX, GST, GR, -ECS, and GS genes in leaves of tomato seedlings
in NG treatment were higher than CK treatment. However, activ-
ities of GPX, GST, GR, ␥-ECS, and GS in leaves of tomato seedlings
in NG treatment were lower than CK treatment. The discrepancies
between changes in expression and activity of enzymes related to
GSH synthesis and metabolism under NG treatment might be due
to the presence of multiple allo- or iso-zymes or post-translational
modification. The reasons remain to be further studied.
In conclusion, our results clearly demonstrate that exogenous
application of GSH increase the transcript levels and activities of
enzymes related to GSH synthesis and metabolism, including ␥-
ECS, GS, GST, GPX and GR, and the contents of intracellular GSH and
AsA and the ratios of GSH/GSSH and AsA/DHA in the salt-stressed
plants or salt-stress plants treated with BSO. Application of GSH also
enhanced the activities of SOD, CAT, POD and enzymes related to the
ascorbate-glutathione cycle including APX, DHAR, MDHAR and GR,
and decreased the contents of H2 O2 and O2 •− , lipid peroxidation.
Therefore, application of exogenous GSH to salt-stressed tomato
can be a successful strategy for enhancing ROS detoxification and
regulating GSH synthesis and metabolism. These changes keep ROS
levels low and maintain the cellular redox homeostasis.
Authors’ contributions
The work was carried out in collaboration between all the
authors. Huiying Liu and Yan Zhou defined the research theme.
Huiying Liu and Yan Zhou designed the experiment, determined
the methods, carried out the laboratory experiments, analyzed the
data, interpreted the results, and wrote the paper. Zelin Wen co-
designed experiments, carried out the laboratory experiments, and
discussed the analyses and interpretation. Jianwei Zhang, Xianjun
Chen, Jinxia Cui and Wei Xu worked together on total RNA extrac-
tion and gene expression analysis. Huiying Liu conceived of and
coordinated the study. All authors have contributed to, seen, and
approved the final manuscript.
Conflict of interest
None.
Acknowledgments
This work was funded by National Natural Science Foundation of
China, China (No. 31360478 and No. 31160391), and International
Cooperation Project of Xinjiang Production and Construction Corps,
China (No. 2014BC002).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.
02.021.
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