Plant Physiology and Biochemistry 63 (2013) 209e216

Contents lists available at SciVerse ScienceDirect

Plant Physiology and Biochemistry

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

Research article

Effects of exogenous spermine on chlorophyll fluorescence,

antioxidant system and ultrastructure of chloroplasts in Cucumis
sativus L. under salt stress
Sheng Shu 1, Ling-Yun Yuan 1, Shi-Rong Guo*, Jin Sun, Ying-Hui Yuan
College of Horticulture, Nanjing Agricultural University, Key Laboratory of Southern Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing 210095, People’s Republic
of China

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

Article history: The effects of exogenous spermine (Spm) on plant growth, chlorophyll fluorescence, ultrastructure and
Received 5 July 2012 anti-oxidative metabolism of chloroplasts were investigated in Cucumis sativus L. under NaCl stress. Salt
Accepted 16 November 2012 stress significantly reduced plant growth, chlorophylls content and Fv/Fm. These changes could be alle-
Available online 12 December 2012
viated by foliar spraying with Spm. Salt stress caused an increase in malondialdehyde (MDA) content and

superoxide anion ðO2  Þ generation rate in chloroplasts. Application of Spm significantly increased
Keywords:
activities of superoxidase dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), and ascorbate
Antioxidant system 
peroxidase (APX, EC 1.11.1.11) which decreased the levels of O2  and MDA in the salt-stressed chloro-
Photochemical efficiency
Chloroplast ultrastructure
plasts. Salt stress decreased the activities of dehydroascorbate reductase (DHAR, EC 1.8.5.1) and gluta-
Cucumber thione reductase (GR, EC 1.6.4.2) in the chloroplasts and reduced the contents of dehydroascorbate
Spm (DAsA) and glutathione (GSH), but increased monodehydroascorbate reductase (MDAR, EC 1.6.5.4)
Salinity activity. On the other hand, Spm significantly increased the activities of antioxidant enzymes and levels
of antioxidants in the salt-stressed chloroplasts. Further analysis of the ultrastructure of chloroplasts
indicated that salinity induced destruction of the chloroplast envelope and increased the number of
plastoglobuli with aberrations in thylakoid membranes. However, Spm application to salt-stressed plant
leaves counteracted the adverse effects of salinity on the structure of the photosynthetic apparatus.
These results suggest that Spm alleviates salt-induced oxidative stress through regulating antioxidant
systems in chloroplasts of cucumber seedlings, which is associated with an improvement of the
photochemical efficiency of PSII.
Ó 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction Polyamines (PAs) are ubiquitous low-molecular-weight nitrog-

Salinity is one of the most deleterious environmental factors
limiting crop growth and yield [1]. Salt stress causes an initial water
deficit and ion specific toxicity resulting from changes in Kþ/Naþ
ratios. Thus, it leads to increased Naþ and Cl levels in cells that
inhibit plant growth by disrupting physiological processes, espe-
cially photosynthesis [2]. Salt stress affects plant photosynthetic
efficiency through stomatal limitation [3] and non-stomatal limi-
tations [4] including stomatal closure, chlorophyll loss, inhibition of
Rubisco activity, and degradation of membrane proteins in the
photosynthetic apparatus.
* Corresponding author. Tel./fax: þ86 2584395267.
E-mail address: srguo@njau.edu.cn (S.-R. Guo).
1
Equal contributions to this paper.
enous compounds found in all living organisms [5]. In higher
plants, the most common PAs are spermidine (Spd), spermine
(Spm), and their diamine obligate precursor putrescine (Put). They
are formed by aliphatic hydrocarbons substituted with two or more
amino groups. Because of the polycationic nature at physiological
pH, PAs are present in the free form or as conjugates bound to
phenolic acids and other low-molecular-weight compounds or to
proteins and nucleic acids [6]. Like hormones, PAs displaying high
biological activities are involved in a wide array of fundamental
processes in plants, such as replication and gene expression,
growth and development, senescence, membrane stabilization, and
adaptation to abiotic and biotic stresses [7,8].
It has been suggested that exogenous application of PAs
can alleviate salinity-induced decrease in photosynthetic efficiency
of higher plants to some extent, but this effect strongly depends
on both PAs concentrations, or types, and stress levels [9]. The

0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2012.11.028
210 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216

maximum photochemical efficiency of PSII (Fv/Fm) is not greatly influ-
enced by 1 mM Spd application, although Spd increases net photosyn-
thetic rate (PN), stomatal conductance (Gs), intercellular CO2 concentration
(Ci), actual photochemical efficiency of PSII (VPSII), and photochemical
quenchingcoefficient(qP)ofcucumberseedlings undersalt stress [10]. Put
also alleviates the reduction of salt stress on PN, but has no effect on Gs and
transpiration rate (Tr), whereas it aggravates the reduction of salt stress on
Ci [11]. Under high light conditions, Put treatment can improve photo-
chemical capacity of Scenedesmus obliquus by altering levels of LHCII
monomers and oligomers, PSI and PSII core proteins in the thylakoid
membranes [12]. In green alga Scenedesmus exposed to high salinity, Put
effectively decreases the functional antenna size and increases the density
of active PSII reaction centers, thereby alleviating photo-oxidative damage
to the photosynthetic apparatus from increased excitation pressure [13].
Application of Put, Spd and Spm through the root is also effective in alle-
viating salinity damage to PSI and PSII activities [14].
PAs as a class of growth substances are well known for their
positive effects on the photosynthetic efficiency under various
stress conditions due to their acid neutralizing and antioxidant
properties [15,16]. Pretreatment with Spd markedly reduced lipid
peroxidation and membrane relative permeability in wheat leaves
under water stress [17]. They found that Spd also improved the
under non-saline conditions (Table 2). Salt stress significantly
decreased contents of Chl a, Chl b, Chl a/Chl b and total Chl in leaves.
However, exogenous Spm supplied to leaves alleviated the loss of
chlorophylls induced by salt stress. Spm treatment increased the
levels of Chl a, Chl b, Chl a/Chl b and total Chl by 61.0%, 31.0%, 23.1%
and 44.6% in the salt-stressed plants, respectively.
2.3. Chlorophyll fluorescence
Under the saline conditions, the values of Fv/Fm, qP, VPSII and
rETR were significantly reduced by 8.79%, 48.5%, 53.4% and 53.4% as
compared with the control, respectively (Fig. 1). Salt stress also
caused a significant decrease in NPQ value in cucumber leaves.
Application of exogenous Spm significantly increased the values of
Fv/Fm, Fv0 /Fm0 , qP, VPSII and rETR of plants with NaCl. Moreover,
Spm further increased the NPQ value in leaves of the salt-stressed
plants. There were no significant differences in these parameters
between the control plants and those sprayed with Spm.
2.4. Oxidative stress and antioxidant metabolites
2.4.1. Levels of MDA and O2  in the chloroplasts


transcription of PSII genes and translation of the corresponding

Under the non-saline conditions, exogenous Spm showed no
proteins, thus increased PSII activity. The enhanced tolerance of
significant effects on the generating rate of O2  and content of


Welsh onion to flooding induced by exogenous 2 mM Put pre-

MDA in chloroplast of cucumber seedlings (Fig. 2). MDA contents
treatment appears due to the increased activities of various anti-
and O2  production levels were significantly increased in the


oxidative systems [18]. It has been shown that lipid peroxidation

chloroplasts with the salinity treatment. However, Spm signifi-
levels were significantly reduced by PAs treatment in Physcia
cantly reduced MDA levels and O2  generation rate in the chlo-


semipinnata during UV-A stress [19].

roplasts of salt-stressed plants.
Cucumber is an economically important horticultural crop and
highly sensitive to salinity, especially at the seedling stage [9]. In
2.4.2. Activities of superoxidase dismutase (SOD) and peroxidase
this study, we examined the effects of exogenous Spm on chlo-
(POD) in the chloroplasts
rophyll fluorescence, ultrastructure, and antioxidant system of the
There were no large differences in two enzyme activities of the
chloroplasts in salt stressed cucumber seedlings. The objective
chloroplasts between the control plants and those sprayed with
was to elucidate the mechanism of PAs how protects photo-
Spm (Fig. 3). The SOD and POD activities in chloroplasts of the
chemical efficiency from salt-induced damages on cucumber
cucumber seedlings leaves were significantly decreased during the
seedlings.
exposure to salt stress. Exogenous Spm markedly increased their
activities in the chloroplasts of salt-stressed plants, and the activity
2. Results
levels were significantly higher than the control.
2.1. Plant growth
2.4.3. Activities and levels of antioxidant
As compared with the control, salt stress significantly decreased
Salt stress caused severe inhibition of plant height, stem diameter,
activities of APX, DHAR and GR, and contents of DAsA, AsA, GSSG
fresh weight and dry weight of cucumber seedlings (Table 1). The
and GSH in the chloroplasts of the cucumber seedlings leaves, but
values of those parameters were reduced by 61.1%, 34.6%, 71.5% and
MDAR activity increased markedly (Figs. 4 and 5). Application of
72.2%, respectively, as compared with control plants. The reduction of
exogenous Spm significantly increased the activities of APX, DHAR,
the above parameters was partially alleviated by application of
GR and MDAR in the chloroplasts of the salt-stressed plants and the
exogenous Spm. Under non-saline conditions, exogenous Spm exer-
activities of these enzymes were significantly higher than the
ted no effect on these parameters of cucumber seedlings.
control. In addition, Spm alleviated salinity-induced decrease in the
contents of DAsA, AsA, GSSG and GSH in the chloroplasts. There
2.2. Chlorophylls contents
were no differences in metabolism of ascorbate-glutathione
There was no difference in the photosynthetic pigment content
of cucumber seedlings between PAs treatments and the control
Table 2
Table 1 Effects of application of exogenous spermine on content of chlorophylls in cucumber
Effects of application of exogenous spermine on growth parameters of cucumber seedlings exposed to 75 mM NaCl stress for 7 days.
seedlings exposed to 75 mM NaCl stress for 7 days.
Treatments Chlorophyll Chlorophyll b Chlorophyll a/ Total
Treatments Plant Stem Fresh weight Dry weight a content content Chlorophyll b Chlorophyll
height (cm) diameter (mm) (g plant1) (g plant1) (mg cm2) (mg cm2) (mg cm2)
Cont 24.5  0.15a 6.19  0.10a 43.2  3.40a 3.63  0.40a Cont 35.1  2.70a 9.23  0.19a 3.60  0.21a 44.8  4.05a
Spm 25.4  0.98a 6.45  0.23a 44.3  1.50a 3.73  0.24a Spm 35.6  1.50a 9.74  0.88a 3.65  0.31a 45.3  1.10a
NaCl 10.1  0.19c 4.27  0.16c 12.3  0.93c 1.01  0.12c NaCl 17.2  1.34c 6.84  0.52b 2.51  0.23b 24.0  2.15c
NaCl þ Spm 15.4  0.61b 4.79  0.02b 27.1  1.59b 1.92  0.13b NaCl þ Spm 27.7  3.60b 8.96  1.40a 3.09  0.26a 34.7  2.98b

Each value is the mean  standard error of 15 independent experiments. Different Each value is the mean  standard error of 15 independent experiments. Different
letters indicate significant differences between treatments (P < 0.05). letters indicate significant differences between treatments (P < 0.05).
 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216 211

Fig. 1. Effects of exogenous spermine on chlorophyll fluorescence parameters in leaves of cucumber seedlings under salt stress. The data were taken on the third leaves, numbered
basipetally, after 7 days of the final concentration salt treatment. Each histogram represents a mean  standard error of three independent experiments (n ¼ 3). Different letters
indicate significant differences between treatments (P < 0.05).

pathway of the chloroplasts between the control and the control
plus Spm treatment.
2.5. Ultrastructure of the chloroplasts
There were no significant differences between the control and
the control plus Spm in ultrastructure of chloroplasts and thyla-
koids (Fig. 6). Salt stress induced alterations in the structure of
chloroplasts and thylakoids as compared to the control. The chlo-
roplasts in salt-stressed plants were separated from the plasma
membrane, in contrast to those in control plants where chloro-
plasts were in closely contact with the membrane. The shape of
chloroplasts changed slightly from elliptical to nearly round. The
envelope membrane in the NaCl treated samples appeared to be
ruptured. Chloroplast membranes were notably disorganized in the
NaCl-treated leaves and the chloroplasts showed disarranged


Fig. 2. Effects of exogenous spermine on MDA content and O2  generation rate in chloroplast of cucumber seedlings under salt stress. The data were taken on the third leaves,
numbered basipetally, after 7 days of the final concentration salt treatment. Each histogram represents a mean  standard error of three independent experiments (n ¼ 3). Different
letters indicate significant differences between treatments (P < 0.05).
212 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216

Fig. 3. Effects of exogenous spermine on the activities of SOD and POD in chloroplast of cucumber seedlings under salt stress. The data were taken on the third leaves, numbered
basipetally, after 7 days of the final concentration salt treatment. Each histogram represents a mean  standard error of three independent experiments (n ¼ 3). Different letters
indicate significant differences between treatments (P < 0.05).

thylakoids with numerous osmiophilic plastoglobuli. Furthermore,
the internal lamellae of stromal thylakoids (SL) were damaged with
disintegration of the stacked granal thylakoid (GL) system.
However, exogenous Spm alleviated the structural changes of
chloroplasts (or thylakoid membranes) induced by salt stress. Spm
maintained a well-preserved internal lamellar system in the chlo-
roplasts of salt-stressed leaves and the chloroplasts contained less
osmiophilic plastoglobuli.
3. Discussion
PAs have been extensively described in ecological, physiological,
cellular, and molecular biological aspects and have been shown to
increase crop yield and stress tolerance [20]. In this study, we found
that application of exogenous Spm led to an improvement of the
growth of salt-stressed cucumber seedlings, because Spm treated
plants exhibited higher biomass after being exposed to saline
stresses than non-Spm treated plants. PAs treatment resulted in the
improvement of growth in many plants exposed to various envi-
ronmental stressors that are often associated with increased
chlorophylls and photosynthetic efficiency [11,18]. In this work,
Spm prevented chlorophyll degradation in the salt-stressed
cucumber leaves. This enhanced effect by Spm was similar to
those of Unal et al. [19] who showed that PAs increased the Chl
content in P. semipinnata under UV-A stress. These results indicated
that Spm may protect light harvesting complex (LCH) and photo-
system II from salinity-induced damage.
Chlorophyll fluorescence is a subtle reflection of primary reac-
tions of photosynthesis. The intricate relationships between fluo-
rescence kinetics and photosynthesis are the keys to our
understanding of photosynthetic biophysical processes [21]. The
values of Fv/Fm, qP and FPSII were significantly reduced under salt
stress (Fig. 2) suggesting that salinity induced an inhibition of PSII
electron transport. This inhibition of electron transfer may be from
the primary acceptor plastoquinone (QA) to the secondary acceptor
plastoquinone (QB) at the acceptor side of PSII. These results are in
agreement with those of Mehta et al. [22] who reported that salt
stress led to the decrease in the Fv/Fm, mainly due to inhibition of
electron transport at the acceptor side of the PSII reaction center.
On the other hand, Fv/Fm and FPSII were increased in the salt-

Fig. 4. Effects of application of exogenous spermine on activities of APX, DHAR, MDAR and GR in the chloroplast of cucumber seedlings exposed to 75 mM NaCl stress for 7 days.
Each histogram represents a mean  standard error of three independent experiments (n ¼ 3). Different letters indicate significant differences between treatments (P < 0.05).
 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216
Fig. 5. Effects of application of exogenous spermine on contents of DAsA, AsA, GSSG and GSH in the chloroplast of cucumber seedlings exposed to 75 mM NaCl stress for 7 days. Each
histogram represents a mean  standard error of three independent experiments (n ¼ 3). Different letters indicate significant differences between treatments (P < 0.05).
stressed plants when cucumber seedlings were sprayed with Spm.
The value of NPQ is indicative of a high photo-protective capacity of
a plant to protect itself against damage by excess energy. In our
study, we also observed that exogenous Spm treatment signifi-
cantly increased the NPQ of the salt-stressed plants. These results
indicated that Spm helped the protection of PSII against over
excitation through regulating of photo-protective mechanism,
under salt stress, that could have caused damage perhaps from the
loss of integrity in the thylakoid membranes.
Chloroplasts are the major source of reactive oxygen species
(ROS) such as superoxide radical ðO2  Þ, hydroxyl ions (OH$), and

H2O2 that may oxidize proteins, lipids, and nucleic acids which
results in abnormalities at the level of cell, when the plants are
exposed to environmental stresses [23]. ROS can be generated by
the direct transfer of the excitation energy from Chl to produce
singlet oxygen or by oxygen reduction in the Mehler reaction in the
chloroplasts [24] which leads to the production of the membrane
lipid peroxidation [25]. PAs are also well known for their positive
effects on the photosynthetic efficiency under stress conditions due
to their acid neutralizing and antioxidant properties, as well as
their membrane and cell wall stabilizing abilities [15,16]. Our
previous study showed that application of exogenous Put induced
the changes of endogenous PAs in the chloroplasts and thylakoids
that, to some extent, might be involved in the reduction of
membrane lipid peroxidation under salinity [26]. In the present
study, exogenous application of Spm significantly increased the
activities of ROS scavenging enzymes of SOD, POD, APX and GR in
chloroplasts of the salt-stressed plants. Moreover, exogenous Spm
induced synthesis of antioxidant metabolites that provided addi-
tional power of resistance to neutralize the toxic effects of salt
stress generated ROS [18]. We observed that Spm increased the
contents of DAsA, AsA, GSSG and GSH in the chloroplasts
which enhanced the salinity tolerance of the photosynthetic
apparatus. The contents of antioxidant metabolites and activities of
enzymes in the salt-stressed chloroplasts were enhanced by Spm
213
application, which was consistent with Spm reducing the gener-
ating rate of O2  and content of MDA in chloroplast of cucumber

seedlings. These results showed that Spm alleviated salt-induced
membrane injury of the chloroplasts through increasing the ROS
scavenging ability, which indicated that Spm may protect photo-
system II from oxidative stress.
Maintaining structural integrity and orderliness of chloroplast is
necessary in the conversion of light energy for photosynthesis. In
higher plants, the photosynthetic machinery is mainly localized in
thylakoid membranes of the chloroplasts, including photosystem I
and II. It was reported that many stressors led to the decrease in the
photochemical efficiency and electron transport activity that might
be associated with the changes of the structure of photosynthetic
apparatus [4]. PAs as organic cations are involved in stabilizing the
structure and function of photosynthetic apparatus in response to
unfavorable environmental factors [13]. Tiburcio et al. [27] reported
that Put dramatically enhanced lipid accumulation in the chloro-
plasts and prevented the membrane degradation in the granal and
stromal thylakoids under NaCl stress. However, Pjon et al. [28] re-
ported that Spd treatment with chloroplast for about 10 s displayed
the envelope and the typical dense network of the thylakoid
lamellae interspersed with numerous areas of stacked grana. The
discrepancy was dependent on different stress levels and types of
polyamines. In the present study, we observed that exogenous Spm
decreased the number of plastoglobules in the thylakoid membrane
which was consistent with the decline in ROS accumulation and
MDA content in the chloroplasts. These results demonstrated that
Spm was involved in protecting photosynthetic apparatus by
enhancing the capacity of antioxidant metabolites and antioxidant
enzymes activities, thus inducing a normal stacking order in the
adjacent grana thylakoids.
In conclusion, our results suggested that exogenous Spm could
significantly alleviate salt-induced oxidative damage on cucumber
seedlings most likely through regulation of antioxidant enzymes
and non-enzymatic systems in the chloroplast. This was associated
214 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216

Fig. 6. The effects of salt stress with and without exogenous spermine on the ultrastructure of chloroplasts and thylakoid membranes in leaves of cucumber plants. (a) Cont; 0 mM
NaClþ0 mM spermine, (b) Spm; 0 mM NaClþ0.3 mM spermine, (c) NaCl; 75 mM NaClþ0 mM spermine, (d) NaCl þ Spm; 75 mM NaClþ0.3 mM spermine. The third fully expanded
leaves, numbered basipetally, were sampled for ultramicroscopic observation on day 7 after the onset of the final concentration salt treatment. SL, stroma lamella; GL, grana
lamellae; SG, starch grain; P, plastoglobule. Scale bars for chloroplasts and thylakoids are 1 mm and 500 nm, respectively.

with an improvement in the photochemical efficiency of PSII of the
salt stressed plants.
4. Material and methods
4.1. Plant material and growth conditions
The seeds of cucumber (Cucumis sativus L., cv. Jingyou No. 4)
were germinated for 24 h at 29  C in Petri plates lined with two
layers of filter paper moistened with sterile distilled water. The
germinated seeds were sown in washed quartz sand. The seedlings
were grown in an artificial climate chamber where the air
temperature was kept at 28  1  C/19  1  C (day/night), 14 h/10
(day/night) photoperiod was imposed with a day light intensity of
800 mmol m2 s1, and the relative humidity was maintained at
50%e60 %. After two true leaves emerged, the seedlings were
transplanted to plastic containers (the length, width and depth
are 55, 40 and 10 cm, respectively), each containing 12 plants and
20 L of full-strength Hoagland solution (pH 6.5  0.1, EC 2.0e
2.2 dS m1). Nutrient solution was aerated using an air pump
 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216
that was switched on and off at an interval of 30 min to maintain
the dissolved oxygen at 8.0  0.2 mg L1.
4.2. Salt and Spm treatments
After 2 days of pre-culture under control conditions, NaCl and
polyamines treatments were commenced by adding NaCl to the
nutrient solution and by foliar spraying with 0.3 mM Spm,
respectively. NaCl concentrations were increased by 25 mM incre-
ments every day until a final concentration of 75 mM was reached.
The experimental plots included four treatments: (a) Cont; 0 mM
NaCl þ 0 mM Spd, (b) Spm; 0 mM NaCl þ 0.3 mM Spm, (c) NaCl;
75 mM NaCl þ 0 mM Spd and (d) NaCl þ Spm; 75 mM
NaCl þ 0.3 mM Spm. The containers were arranged in completely
randomized blocks with three replicates per treatment, so each
treatment involved a total of 36 plants. The nutrient solutions were
renewed every 2 days. After 7 days of the final concentration salt
treatment, the third fully expended leaf, numbered basipetally
starting at the uppermost fully expended leaves, was used to
measure the photosynthesis rate, chlorophyll fluorescence, chlo-
rophylls analysis, and observe ultrastructure of chloroplasts.
4.3. Determination of growth parameters
For determination of fresh weight, shoots and roots were
separated and weighed after being washed with sterile distilled
water. The dry weight was obtained after the shoots and roots were
dried at 75  C in an oven for 72 h.
4.4. Chlorophyll analysis
Chlorophylls were extracted from the third fully expanded leaf
(10 disks, Ø 4 mm) with a mixture of ethanol, acetone and water at
the volume ratio of 4.5:4.5:1 and quantified following the method
by Holden [29].
4.5. Chlorophyll fluorescence parameters
Chlorophyll fluorescence was measured using a portable fluo-
rometer (PAM-2100, Walz, Germany) as described by Lu et al. [30].
The maximum quantum yield of PSII (Fv/Fm) was determined after
dark adaptation for 30 min. Fluorescence parameters were
measured on light-adapted leaves using the equations of Genty
et al. [31] as follows: the photochemical efficiency of open PSII
centers [Fv0 /Fm0 ¼ (Fm0  Fo0 )/Fm0 ], photochemical quenching coef-
ficient [qP ¼ (Fm0  Fs)/(Fm0  Fo0 )], non-photochemical quenching
[NPQ ¼ (Fm  Fm0 )/Fm0 ], actual photochemical efficiency of PSII
[VPSII ¼ (Fm0  Fs)/Fm0 ], and relative electron transport rate
[rETR ¼ VPSII  PPFD  0.5  0.84], where PPFD is photosynthetic
photon flux density incident on the leaf, 0.5 is factor that assumes
equal distribution of energy between PSII and PSI, and 0.84 is
assumed for leaf absorbance.
4.6. Isolation of intact chloroplasts
Intact chloroplasts were isolated using the method described by
Seigneurin-Berny et al. [32] with a slight modification. After 7 days
of the final concentration salt treatment, the third and fourth fully
expanded leaves, numbered basipetally starting at the uppermost
leaves, were removed for chloroplast isolation. Leaves (30 g) were
homogenized for 3 s in a Polytron blender with 60 mL of 330 mM
sorbitol, 30 mM Mes, 2 mM ascorbate, and 0.1% BSA, adjusted to pH
6.5 with Tris. The homogenate was filtered through four layers of
cheesecloth, and the dark green filtrate was centrifuged at 1800  g
for 2 min. The pellets were resuspended in 2 mL of buffer (330 mM
215
sorbitol, 30 mM Hepes and 0.2% BSA, adjusted to pH 7.6 with Tris),
put into a tube containing 8 mL of resuspension medium plus 40e
80% (v/v) percoll and centrifuged for 3 min at 2000  g. The
interlayer between 40% and 80% Percoll contained intact chloro-
plasts. All procedures were carried out at 4  C. The percentage of
intactness of chloroplasts was about 85% according to the method
checked by Aravind and Prasad [33].
4.7. Determination of O2  and MDA contents in the chloroplasts

MDA was measured according to the method of Xu et al. [34].
O2  generation rate was determined according to the method of

Elstner and Heupel [35] with a slight modification. The equal
chlorophyll of the chloroplasts supernatant was mixed with
0.675 mL of 65 mM phosphate buffer (pH 7.8) and 0.1 mL of 10 mM
hydroxylamine chlorhydrate and was placed at 25  C for 20 min;
0.375 mL of 17 mM sulfanilamide and 0.375 mL of 7 mM a-naph-
thylamine were added, and the mixture was placed at 25  C for
another 20 min before being mixed with 2.25 mL pure ether. The
absorbance was measured at 530 nm and O2  generation rate was

calculated from a standard curve of NaNO2.
4.8. Assay of antioxidant enzymes in the chloroplasts
The equal chlorophyll of chloroplast supernatants were mixed in
3 mL of ice-cold Hepes buffer (25 mM, pH 7.8) containing 0.2 mM
EDTA and 2% (w/v) PVP. The mixed solution was centrifuged at 4  C
and 12,000  g for 20 min, and the supernatants were used for
assay antioxidant enzyme activity and determination of antioxidant
contents (metabolism of ascorbate and glutathione).
SOD was assayed by monitoring its ability to inhibit the
photochemical reduction of nitro blue tetrazolium (NBT) [36]. POD
was analyzed by the increase in absorbance at 470 nm recorded
40 s after adding H2O2 [37]. APX was assayed following the method
of Nakano and Asada [38] by monitoring the rate of ascorbate
oxidation at 290 nm. GR activity was measured by following the
decrease at 340 nm in 1 min due to NADPH oxidation [39]. The
activities of MDAR and DHAR were assayed according to the
method described by Duan et al. [40].
4.9. Determination of antioxidant content in the chloroplasts
AsA was determined according to the methods of Arakawa et al.
[41] with a small modification. The reaction mixture contained 5%
TCA, ethanol, 0.4% H3PO4-ethanol, 0.03% FeCl3-ethanol, 0.5% BP-
ethanol, and 0.2 mL of enzyme extract at 534 nm. The level of
DAsA was equal to the total AsA minus contents of AsA. Total AsA
was determined as above. Enzyme extract of 0.2 mL was activated
with 60 mM DTT-ethanol and 0.2 M Na2HPO4, incubated for 10 min.
The solution was then mixed with the reaction mixture containing
5% TCA, ethanol, 0.4% H3PO4eethanol, 0.03% FeCl3eethanol, and
0.5% BPeethanol; The absorbance then recorded at 534 nm. The
levels of AsA and total AsA were calculated from a standard curve of
ascorbic acid. The levels of total glutathione and GSSG were
measured at 412 nm according to Griffiths et al. [42]. The content of
GSH was estimated from the difference between total glutathione
and GSSG.
4.10. Observation of ultrastructure of the chloroplasts
The ultrastructure of the chloroplasts was observed as described
by Shu et al. [26]. Leaf segments were cut into pieces of approxi-
mately 1 mm2. The leaf pieces were immersed in a mixture of 3%
glutaraldehyde and 1% formaldehyde in a 0.1 M phosphate buffer
(pH 7.4) for 2 h (primary fixation) and then 2% osmic acid in the same
216 S. Shu et al. / Plant Physiology and Biochemistry 63 (2013) 209e216
buffer for 2 h (secondary fixation). After dehydration in acetone and
embedding in Durcupan ACM (Fluka), ultra-thin sections were cut,
stained with uranium acetate and lead citrate in series, and exam-
ined under a Hitachi transmission electron microscope (Carl Zeiss,
Göttingen, Germany) using an 80 kV acceleration voltage.
4.11. Statistical analysis
All biochemical analyses were conducted at least three times. All
data were statistically analyzed with SAS software (SAS institute,
Cary, NC) using Duncan’s multiple range test at the P < 0.05 level of
significance.
Acknowledgments
This work was funded by National Basic Research Program of
China (973 Program, No.2009CB119000) and National Natural
Science Foundation of China (No. 30900995; No. 31071831; No.
31272209). This work was supported by the China earmarked fund
for Modern Agro-industry Technology Research System (CARS-25-
C-03) and the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).
References
[1] J.H. Liu, H. Inoue, T. Moriguchi, Salt stress-mediated changes in free polyamine
titers and expression of genes responsible for polyamine biosynthesis of apple
in vitro shoots, Environ. Exp. Bot. 62 (2008) 28e35.
[2] S. Shu, J. Sun, S.R. Guo, J. Li, C.J. Liu, C.Y. Wang, C.X. Du, Effects of exogenous
putrescine on PSII photochemistry and ion distribution of cucumber seedlings
under salt stress, Acta Hortic. Sin. 37 (2010) 1065e1072.
[3] D.A. Meloni, M.A. Oliva, C.A. Martinez, J. Cambraia, Photosynthesis and activity
of superoxide dismutase, peroxidase and glutathione reductase in cotton
under salt stress, Environ. Exp. Bot. 49 (2003) 69e76.
[4] S. Mittal, N. Kumari, V. Sharma, Differential response of salt stress on Brassica
juncea: photosynthetic performance, pigment, proline, D1 and antioxidant
enzymes, Plant Physiol. Biochem. 54 (2012) 17e26.
[5] R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston, Polyamines in
plants: an overview, J. Cell. Mol. Biol. 2 (2003) 1e12.
[6] A.C. Childs, D.J. Mehta, E.W. Gerner, Polyamine-dependent gene expression,
Cell. Mol. Life Sci. 60 (2003) 1394e1406.
[7] H.P. Bais, G.A. Ravishankar, Role of polyamines in the ontogeny of plants and their
biotechnological applications, Plant Cell. Tissue Organ Cult. 69 (2002) 1e34.
[8] P.J. Zapata, M. Serrano, M.T. Pretel, M.A. Botella, Changes in free polyamine
concentration induced by salt stress in seedlings of different species, Plant
Growth Regul. 56 (2008) 167e177.
[9] J.J. Duan, J. Li, S.R. Guo, Y.Y. Kang, Exogenous spermidine affects polyamine
metabolism in salinity-stressed Cucumis sativus roots and enhances short-
term salinity tolerance, J. Plant Physiol. 165 (2008) 1620e1635.
[10] J. Li, X.H. Gao, S.R. Guo, R.H. Zhang, X. Wang, Effects of exogenous spermidine
on photosynthesis of salt-stressed cuellmis sativus seedlings, Chin. J. Ecol. 10
(2007) 1595e1599.
[11] R.H. Zhang, J. Li, S.R. Guo, T. Tezuka, Effects of exogenous putrescine on gas-
exchange characteristics and chlorophyll fluorescence of NaCl-stressed
cucumber seedlings, Photosynth. Res. 100 (2009) 155e162.
[12] E. Navakoudis, K. Vrentzou, K. Kotzabasis, A polyamine-and LHCII protease
activity-based mechanism regulates the plasticity and adaptation status of the
photosynthetic apparatus, BBA-Bioenerg. 1767 (2007) 261e271.
[13] G. Demetriou, C. Neonaki, E. Navakoudis, K. Kotzabasis, Salt stress impact on
the molecular structure and function of the photosynthetic apparatus-The
protective role of polyamines, BBA-Bioenerg. 1767 (2007) 272e280.
[14] M.K. Chattopadhayay, B.S. Tiwari, G. Chattopadhayay, A. Bose, D.N. Sengupta,
B. Ghosh, Protective role of exogenous polyamines on salinity-stressed rice
(Oryza sativa) plants, Physiol. Plant. 116 (2002) 192e199.
[15] L.X. He, K. Nada, Y. Kasukabe, S. Tachibana, Enhanced susceptibility of
photosynthesis to low-temperature photoinhibition due to interruption of
chill-induced increase of S-adenosylmethionine decarboxylase activity in
leaves of spinach (Spinacia oleracea L.), Plant Cell. Physiol. 43 (2002) 196e206.
[16] S. Mapelli, I.M. Brambilla, N.L. Radyukina, A.V. Ivanov, YuV. Kartashov,
R. Reggiani, V.l.V. Kuznetsov, Free and bound polyamines changes in different
plants as a consequence of UV-B light irradiation, Gen. Appl. Plant Physiol. 34
(2008) 55e66.
[17] H.G. Duan, Y. Shu, D.H. Liu, D.H. Qin, H.G. Liang, H.H. Lin, Effects of exogenous
spermidine on photosystem II of wheat seedlings under water stress, J. Integr.
Plant Biol. 48 (2006) 920e927.
[18] J.C. Yiu, L.D. Juang, Y.T. Fang, C.W. Liu, S.J. Wu, Exogenous putrescine reduces
flooding-induced oxidative damage by increasing the antioxidant properties
of Welsh onion, Sci. Hortic. 120 (2009) 306e314.
[19] D. Unal, I. Tuney, A. Sukatar, The role of external polyamines on photosyn-
thetic responses lipid peroxidation, protein and chlorophyll a content under
the UV-A (352 nm) stress in Physcia semipinnata, J. Photochem. Photobiol. B 90
(2008) 64e68.
[20] Y. Naka, K. Watanabe, G.H.M. Sagor, M. Niitsu, M.A. Pillai, T. Kusano,
Y. Takahashi, Quantitative analysis of plant polyamines including thermo-
spermine during growth and salinity stress, Plant Physiol. Biochem. 7 (2010)
527e533.
[21] O.H. Sayed, Chlorophyll fluorescence as a tool in cereal crop research, Pho-
tosynthetica 3 (2003) 321e330.
[22] P. Mehta, A. Jajoo, S. Mathur, S. Bharti, Chlorophyll a fluorescence study
revealing effects of high salt stress on photosystem II in wheat leaves, Plant
Physiol. Biochem. 48 (2010) 16e20.
[23] K. Asada, Production and scavenging of reactive oxygen species in chloro-
plasts and their functions, J. Plant Physiol. 141 (2006) 391e396.
[24] P. Stepien, G. Klobus, Antioxidant defense in the leaves of C3 and C4 plants
under salinity stress, Physiol. Plant. 125 (2005) 31e40.
[25] S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machinery in
abiotic stress tolerance in crop plants, Plant Physiol. Biochem. 12 (2010)
909e1026.
[26] S. Shu, S.R. Guo, J. Sun, L.Y. Yuan, Effects of salt stress on the structure and
function of the photosynthetic apparatus in Cucumis sativus L. and its
protection by exogenous putrescine, Physiol. Plant. 148 (2012) 285e296.
[27] A.F. Tiburcio, R.T. Besford, T. Capell, A. Borrell, P.S. Tes-tillano, M.C. Risueno,
Mechanisms of polyamine action during senescence responses induced by
osmotic stress, J. Exp. Bot. 45 (1994) 1789e1800.
[28] C.J. Pjon, S.D. Kim, J.Y. Pak, Effects of spermidine on chlorophyll content,
photosynthetic activity and chloroplast ultrastructure in the dark and under
light, J. Plant Res. 103 (1990) 43e48.
[29] M. Holden, Chlorophylls: Chemistry and Biochemistry of Plant Pigments,
Academic Press, London, 1965, p. 461.
[30] C.M. Lu, N.W. Qiu, B.S. Wang, J.H. Zhang, Salinity treatment shows no effects
on photosystem II photochemistry, but increases the resistance of photo-
system II to heat stress in halophyte Suaeda salsa, J. Exp. Bot. 54 (2003)
851e860.
[31] B. Genty, J.M. Briantais, N.R. Baker, The relationship between the quantum
yield of photosynthetic electron transport and quenching of chlorophyll
fluorescence, BBA-Gen. Subjects 990 (1989) 87e92.
[32] D. Seigneurin-Berny, D. Salvi, A.j. Dorne, J. Joyard, N. Rolland, Percoll-puried
and photosynthetically active chloroplasts from Arabidopsis thaliana leaves,
Plant Physiol. Biochem. 46 (2008) 951e955.
[33] P. Aravind, M.N.V. Prasad, Zinc protects chloroplasts and associated poto-
chemical functions in cadmium exposed Ceratophyllum demersum L. a fresh
water macrophyte, Plant Sci. 166 (2004) 1321e1327.
[34] P.L. Xu, Y.K. Guo, J.G. Bai, L. Shang, X.J. Wang, Effects of long-term chilling on
ultrastructure and antioxidant activity in leaves of two cucumber cultivars
under low light, Physiol. Plant. 132 (2008) 467e478.
[35] E.F. Elstner, A. Heupel, Inhibition of nitrate formation from hydrox-
ylammonium chloride: a simple assay for superoxide dismutase, Anal. Bio-
chem. 70 (1976) 616e620.
[36] C.N. Giannopotitis, S.K. Ries, Superoxide dismutase in higher plants, Plant
Physiol. 59 (1977) 309e314.
[37] W. Tan, J. Liu, T. Dai, Q. Jing, W. Cao, D. Jiang, Alterations in photosynthesis and
antioxidant enzyme activity in winter wheat subjected to post-anthesis
water-logging, Photosynthetica 1 (2008) 21e27.
[38] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific
peroxidase in spinach chloroplasts, Plant Cell. Physiol. 22 (1981) 679e690.
[39] A.S. Gupta, R.P. Webb, A.S. Holaday, R.D. Allen, Overexpression of superoxide
dismutase protects plants from oxidative stress (Induction of ascorbate
peroxidase in superoxide dismutase- overexpressing plants), Plant Physiol.
103 (1993) 1067e1073.
[40] J.J. Duan, S.R. Guo, Y.Y. Kang, G.X. Zhou, X.E. Liu, Effects of exogenous sper-
midine on active oxygen scavenging system and bound polyamine contents in
chloroplasts of cucumber under salt stress, Acta Ecol. Sin 29 (2009) 653e661.
[41] N. Arakawa, K. Tsutsumi, N.G. Sanceda, A rapid and sensitive method for the
determination of ascorbic acid using 4, 7-diphenyl-1, 10-phenanthroline,
Agric. Biol. Chem. 45 (1981) 1289e1290.
[42] O.W. Griffiths, Determination of glutathione and glutathione disulphide
using glutathione reductase and 2-vinylpyridine, Anal. Biochem. 106 (1980)
207e212.