全 文 ：第 45 卷 第 2 期
2 0 0 9年 2 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol.45 , No.2
Feb., 2 0 0 9
NaCl胁迫对构树组培苗抗氧化酶
活性及同工酶谱的影响＊
李荣锦1 , 2　张　敏1, 2　蒋泽平2　黄利斌2　王宝松2　季永华2　方炎明1
(1.南京林业大学森林资源与环境学院　南京 210037;2.江苏省林业科学研究院　南京 211153)
摘　要:　构树组培苗在 50 , 75 , 100 , 125 和 150 mmol·L-1 NaCl胁迫下 , SOD, POD, CAT活性和这 3 种抗氧化酶同工
酶谱的变化情况 ,以及不同浓度 NaCl对可溶性蛋白 、脯氨酸和 MDA 含量的影响。结果表明:NaCl 浓度小于 100
mmol·L-1时可溶蛋白含量升高 ,随着 NaCl浓度的增加可溶蛋白含量降低。低浓度 NaCl使SOD活性略有降低 , 随着
浓度升高活性逐渐增强;SOD同工酶谱在NaCl胁迫后变化明显。 POD活性变化趋势与 SOD相似 ,而 POD同工酶谱
略有变化。CAT活性明显提高 , 而其同工酶谱变化甚微。此外 , NaCl胁迫还导致脯氨酸含量明显升高 , 而 MDA 含
量呈现先升高后降低的趋势。
关键词:　构树;NaCl胁迫;抗氧化酶;同工酶
中图分类号:S718.43　　　文献标识码:A　　　文章编号:1001-7488(2009)02-0040-08
Received on July 10 , 2009.
Foundat ion project:This research was supported by National Science &Technology Pillar Program in the Eleventh Five-year Plan(No.2006BAD09A04 and
No.2006BAD03A0507), Science and Technology Plan Project in Jiangsu Province(BC2005372), and Scientifi c Research Fund for Young Scholars in Jiangsu
Academy of Forestry (JAF-2005-2).
＊ Corresponding author:Fang Yanming.Thank the help of Professor Li Qiurong and M.D.Zhang Qiang for experimental assistance and critical reading of
the manuscript.
Effect of NaCl Stress on Antioxidant Enzyme Activities and
Isoenzyme Pattern of Broussonetia papyrifera Plantlets
Li Rongjin1 , 2　Zhang Min1 , 2 　Jiang Zeping2 　Huang Libin2
Wang Baosong2　Ji Yonghua2　Fang Yanming1
(1.College of Forestry Resources and Environment , Nanjing Forestry University　Nanjing 210037;
2.Jiangsu Academy of Forestry 　Nanjing 211153)
Abstract:　The activities and isoforms of superoxide dismutase peroxidase(POD)and catalase(CAT), as well as the contents
of proline , soluble protein (SOD)and malondialdehyde (MDA)in plantlets of Broussonetia papyrifera subjected to salt stress
treatments of 50 , 75 , 100 , 125 and 150 mmol·L-1 NaCl were studied.Our results demonstrated that soluble protein content
increased with NaCl concentration at low level of NaCl concentration , and it decreased when NaCl concentration exceeds 100
mmol·L-1.SOD activity showed a slight decrease in low concentration of NaCl and then an increase with further increase in
NaCl concentration.Moreover , the isoforms of SOD were affected by salt stress.POD exhibited a similar trend in enzyme activity
as compared to SOD , However , the POD isoformswereminimally affected by NaCl treatment.CAT activity was increased by salt
treatment.Moreover , proline contents markedly increased while the contents of MDA was increased at first and decreased
subsequently in response to salt stress.
Key words:　Broussonetia papyrifera ;salt stress;antioxidant enzyme;isoenzyme
　 　 Salinitization of soil is becoming a critical
environmental problem.It was reported that about 20% of
the world s cultivated land and nearly half of all irrigated
lands are affected by salinity (Rhoades et al., 1990).
Salinity severely limits vegetative and reproductive growth
of plants by inducing severe physiological dysfunctions
and causing widespread direct and indirect harmful effects
(Shannon et al., 1994).The stress induced by salinity
on plants is the results of two mechanisms:hyperosmotic
stress , which causes water deficit and reduces water
adsorption , and ion imbalance resulting in accumulation
of Na
+
and Cl
+(Beritognolo , 2007).As a consequence
of these primary effects , secondary stresses such as
oxidative damage often occur(Zhu , 2001).
In recent years , considerable attention has been
drawn to mechanisms enabling plants to cope with salt
stress.Several studies have established the relationship
between metabolic changes and salt stress (Adams et
al., 1992;Delauny et al., 1993).Elavumoottil et al.
(2003)reported that callus and cell suspension cultures
of Brassica oleracea accumulated sucrose and reducing
sugars to cope with NaCl treatment.Woody plants may
adapt to salinity by variously tolerating or avoiding salt , or
both.In some plants , osmotic adjustment results from
synthesis in the cytoplasm of compatible organic solutes
including proline and other amino acids in addition to
sugars(Kozlowski , 1997).Other researchers have aimed
to identify components involved in salt-stress tolerance by
identifying mRNAs and proteins selectively induced under
such condition (Claes et al., 1990;Holland et al.,
1993).And a lot of salt stress induced proteins have
been found in many plant species(Sadka et al., 1991;
Bishnoi et al., 2006;Elavumoottil et al., 2003).
An important cause of damage that high salt
concentrations inflict on plants might be reactive oxygen
species(ROS)generated by salt stress (Zhu , 2001).
These reactive oxygen species are highly deleterious for
cell structures and functions(Hideg et al., 1996;Foyer
et al., 1997), thus they need to be scavenged to prevent
the harmful effects caused by these stresses.A major
safeguard mechanism against free radicals is provided by
superoxide dismutase (SOD), which catalyzes the
conversion of O
-2
to H2O2 and then H2O2 is decomposed
in the presence of catalase(CAT)and peroxidase (POD)
(Guo et al., 2004).A large body of evidence has
accumulated from various plant systems showing that
drought and salt stress alter the amounts and activities of
enzymes involved in scavenging oxygen radicals (Gueta-
Dahan et al., 1997;Zhang et al., 1994).
Broussonetia papyrifera widely distributes in China.
The roots , leaves and fruits are used in folkloric medicine
for treating many diseases.It is also an excellent and
financial plant for making paper and firewood , feeding
stuff B .papyrifera adapts to most situations and can be
used in sprigging operation.A previous study showed that
this plant species had a moderate degree of salt tolerance
(Wang et al., 2004).
A better understanding of the mechanisms that
enable plants to adapt to salt stress could help in selecting
salt resistant species. In the present study , the
responding mechanism of plants to salt stress was
investigated using the plantlets of B.papyrifera.The
effect of salinity on the content of total soluble protein and
the activities of antioxidant enzyme were analyzed.In
addition , the accumulation of malondialdehyde (MDA)
and proline were also measured.
1　Materials and methods
1.1　Plant material and NaCl treatments
Callus culture was induced from scion of B.
papyrifera , and then the calli were transferred to bud-
inducing medium (MS medium supplemented with 1
mg·L-1 6-BA , 0.1 mg·L-1 IBA , 30 g·L-1 sucrose and
6.5 g·L-1 argrose , pH 5.8).The obtained plantlets
were maintained by transferring an inoculum to fresh
medium.Salt stress was carried out by transferring the
plantlets to the same medium with the addition of 50 , 75 ,
100 , 125 , 150 mmol·L-1 NaCl.All the cultures were
incubated at 25 ℃ and photoperiod of 16 h.Plantlets
were harvested on WhatmanNo.1 paper filters to soak up
the water , immediately frozen in liquid nitrogen and
stored at-80 ℃until use.For the determination of plant
dry weight , the plantlets were dried at 70 ℃ until the
constant mass was reached.
1.2　Extraction of proteins
Frozen material (1 g fresh weight , FW) was
homogenized with an ice-cooled mortar and pestle in 5 mL
of potassium phosphate buffer (pH 7.5) containing 1
mmol·L-1 EDTA , 3 mmol·L-1 DTT and 5%(w v)
insoluble PVP (Pereira et al., 2002).The homogenate
was then centrifuged at 10 000 g 4 ℃ for 20 min.Total
soluble protein was determined by the method of Bradford
(1976)using bovine serum albumin as a standard and
protein content was expressed as mg protein·g-1 FW.
1.3　Enzyme activity assays
For enzyme assay , frozen plant materials were
homogenized with phosphate buffer (pH 6.8)in a pre-
cooled mortar and pestle.The homogenate was filtered
through two layers of muslin cloth , and centrifuged at
10 000 g for 20 min at 4 ℃.The supernatants were used
for the assays of superoxide dismutase(SOD), peroxidase
(POD)and catalase (CAT)activity.
SOD activity was determined using the
spectrophotometric method according to Cho and Park
41　第 2期 李荣锦等:NaCl胁迫对构树组培苗抗氧化酶活性及同工酶谱的影响
(2000).The reaction medium comprised 50 mmol·L-1
sodium phosphate buffer (pH 7.8), 0.1 mmol·L-1
EDTA-Na2 , 2.2 μmol·L-1 riboflavin , 14.3 mmol·L-1
methionine , and 82.5 μmol·L-1 nitroblue tetrazolium
(NBT)and addition of the enzyme extract.One unit of
SOD activity was defined as the amount of enzyme
required to produce a 50% inhibition of NBT reduction
measured at 560 nm.
POD activity was measured using the procedure of
MacAdam et al.(1992)with minor modification.The
enzyme activity was measured with guaiacol as the
substrate.The oxidation of guaiacol into tetraguaiacol was
measured by the increase in absorbance at 470 nm.
Enzyme activity was calculated in terms of mmol of
guaiacol oxidized min
-1
mg
-1
protein (Guo et al.,
2004).
The activity of CAT was analyzed following Tang et
al.(1984)by measuring the consumption of H2O2 at 240
nm using a UV spectrophotometer.Briefly , the reaction
mixture contained 1.5 mL 50 mmol · L-1 sodium
phosphate buffer (pH 7.0)and 1 mL 0.2%H2O2 , and
the reaction was initiated by adding 500 μL of the enzyme
extract.The results were expressed as U·mg-1 protein.
1.4　PAGE analysis of antioxidant enzymes
The electrophoresis was performed under native
condition in 10% polyacrylamide mini-gels for SOD ,
POD and CAT activity staining.Electrophoresis running
was conducted at 4 ℃.Equal amounts of protein were
loaded on to each lane.
SOD activity was determined on native PAGE gels
according to Pereira et al.(2002).Briefly , the gels
were washed with distilled and incubated in the dark for
30 min at room temperature in 50 mmol·L-1 potassium
phosphate buffer (pH 7.8) containing 1 mmol·L-1
EDTA , 0.05 mmol ·L-1 riboflavin , 0.1 mmol·L-1
nitroblue tetrazolium and 0.3%(v v)N , N , N′, N′′-
tetramethylethylenediamine (TEMED).At the end , the
gels were rinsed with distilled water and placed in
distilled-deionized water and exposed to light for 10 min
until the bands of SOD activity was visible.
Staining for peroxidase was achieved following the
method described by Khedr et al.(2000).Gels were
washed with distilled water and then incubated in 1mmol·
L
-1
3-amino-9-ethyl carbazole in 100 mmol·L-1 acetate
buffer(pH 5.0)for 45 min.After incubation , the gels
were soaked in 0.3%(v v)H2O2 and allowed to stand
for a few minutes at room temperature until the bands
appeared.
CAT isoenzymes were separated on nondenaturating
acrylamide gels following the procedure of Jebara et al.
(2005).Gels were washed with distilled water for twice ,
and then soaked in 0.3%H2O2 for 10 min.After a brief
rinse , gels were incubated in a solution of 2%potassium
ferricyanide and 2% ferric chloride until the bands
appeared.
1.5　Measurement of malondialdehyde(MDA)
MDA content was measured by the method of Heath
et al.(1968).
1.6　Determination of proline
Proline concentration was determined as described by
Guo et al.(2004).In brief , approximately 0.5 g of
frozen plant material was ground in 5 mL 3%
sulfosalicylic acid to extract free proline.2 mL of the
extract was mixed with 2 mL water and then reacted with
2 mL glacial acetic acid and 4 mL 2.5%(w v)acid
ninhydrin (dissolved in 3∶2 glacial acetic acid and 6mol·
L
-1
phosphoric acid , V∶V)at 100 ℃ for 1 h.After
being cooled , the reaction mixture was extracted with
toluene and optical density was measured at 520 nm.
1.7　Statistical analysis
Statistical analysis was carried out by one-way
ANOVA using SPSS 10.0 software to determine the
different significance.When the ANOVA was significant
at P<0.05 , the Duncan multiple range test was used for
mean comparison.Data presented were mean ±SD of
three experiments.
2　Results
2.1　Effect of NaCl on the growth of B.papyrifera
The data in Tab.1 showed that stem height , plant
fresh weight and dry matter accumulation decreased
significantly with increasing of salinity.The rate of
decline in these growth parameters was greater in higher
salinity.These results indicated that the growth of B.
papyrifera was inhibited by salt stress.
2.2　Soluble protein content
The content of total soluble protein in whole plantlet
was examined.As shown in Fig.1 , the protein content
increased with increasing of NaCl concentrations until
100 mmol·L-1 NaCl.And then the protein content
42 林 业 科 学 45 卷　
decreased when NaCl concentration was elevated to 125
and 150 mmol·L-1.The maximum increase in protein
content was found at 100 mmol·L-1 NaCl(4.92 mg·g-1
FW), and it was increased by 17.88% as compared to
control(4.04 mg·g-1 FW).
Tab.1　Effect of NaCl on stem height , plant fresh weight
and dry weight of Broussonetia papyrifera①
NaCl
(mmol·L-1)
Stem height
cm
Plant f resh weight
(mg·plant-1)
Plant dry weight
(mg·plant -1)
0 4.33 a±0.38 343.67 a ±13.01 54.67 a±7.09
50 3.98 a±0.18 325.33 a±13.80 47.00 a±3.61
75 2.84 b±0.13 284.33 b±14.64 34.33 b±4.51
100 2.68 b±0.17 249.67 bc±11.68 26.17 b ±3.20
125 2.62 b ±0.18 246.00 c±21.70 21.00 b ±2.65
150 2.12 c±0.15 197.33 c±12.50 19.73 c±3.41
① Data were presented as mean ±SD from thee replicates.Different letters
indicate significant diff erence by Duncan s multiple range test at 0.05 leve1.
Fig.1　Effects of salt stress on protein
content of Broussonetia papyrifera
Each measurement was conducted with three replicates ,
and data were mean ±SD.Different letters indicate
signif icant difference by Duncan s multiple range
test at 0.05 leve1.FW represents fresh weight.The same below.
2.3 　Effect of NaCl on antioxidant enzyme activity
and isoenzyme patterns
SOD , POD and CAT are important antioxidant
enzymes.In our study the activities of these three
enzymes under salt stress were investigated.The total
SOD activity was slightly decreased at low concentration of
NaCl.SOD activity increased when NaCl concentration
reached 75 mmol·L-1 , and then the activity was declined
but it was still higher than the control by 100 mmol·L-1.
Thereafter the enzyme activity significantly increased with
the increasing of NaCl concentration(Fig.2A).In Fig.
2B , the isoenzyme pattern of SOD showed six bands in
plantlets of control and treatment with 50 mmol·L-1
NaCl.However , band V disappeared when NaCl
concentration increased.Moreover , gel staining analysis
also demonstrated that SOD activity increased dramatically
with increasing of NaCl concentration.
Fig.2　Effect of salt stress on superoxide dismutase(SOD)
activity and isoenzyme patterns in plantlets of B.papyrifera
(A)SOD activity measured by spectrophotometric method;
(B)Gel staining analysis.
Like SOD activity , salt stress resulted in an increase
of POD activity especially at 150 mmol·L-1 NaCl , which
increased the enzyme activity by more than 162%
compared to the control (Fig.3A).There were three
bands appeared on the gel of POD staining , and the three
bands were differentially regulated by stress treatments
(Fig.3B).
Activity gels showed that there was only one catalase
band.The band intensity , as a measure of CAT activity ,
showed the same trend as the spectrophotometric
measurements(Fig.4A and B), which may indicate that
this band is probably responsible for most of the change in
catalase activity.
2.4　Lipid peroxidation
Membrane lipid peroxidation was examined by
measuring MDA content in plantlets with salt stress and
control.As shown in Fig.5 , the MDA content increased
until 100 mmol·L-1 salinity and then declined.The
increase of MDA content was more pronounced at
100 mmol·L-1 NaCl , and it was increased by about 80%
compared to control.
43　第 2期 李荣锦等:NaCl胁迫对构树组培苗抗氧化酶活性及同工酶谱的影响
Fig.3　Changes in peroxidase(POD)activity and
isoenzyme patterns of B .papyrifera
plantlets after treatment with NaCl
(A)POD activity measured by spectrophotometric method;
(B)Gel staining analysis.
Fig.4　Changes in catalase(CAT)activity and isoenzyme
patterns in plantlets of B .papyrifera
(A)CAT activity measured by spectrophotometric method;
(B)Gel staining analysis.
2.5 　Proline content increased in response to salt
stress
The results shown in Fig.6 demonstrated that the
internal proline content increased in response to salt
stress.The accumulation of proline in plantlets of B .
papyrifera was in a concentration-dependent manner.The
increase of proline content was more evident when treated
with higher concentration of NaCl (100 ～ 150
mmol·L-1).The accumulation of proline in response to
150 mmol·L-1 NaCl was maximal , and it was increased
Fig.5　Effect of salt stress on MDA content
Fig.6　Proline contents of B.papyrifera
plantlets after NaCl treatment
Data represent mean ±SD(n=3).
by 300% compared to control.
3　Discussion
　　In order to learn more about the response of woody
plants to salt stress , we examined the physiological and
biochemical changes in plantlets of B.papyrifera under
NaCl stress.We found that NaCl treatment caused a lot of
changes in plant growth , protein content , antioxidant
enzyme activities and the content of proline and MDA.
The growth of B .papyrifera plantlets including stem
height , fresh weight and dry weight was reduced by salt-
stress (Tab.1).The effect of salt stress on soluble
protein content manifested a concentration-dependent
manner.At lower levels of NaCl , the protein content
increased with elevation of NaCl concentration , but higher
concentrations caused it to decline.This suggested that
protein synthesis was increased in the initial response to
salt stress but it was prevented when the stress became too
severe.Accumulation of soluble proteins in plants grown
under saline condition has been reported previously.The
accumulated proteins may provide a storage form of
nitrogen which could be reutilized when stress was over
(Singh et al., 1987), and they may play a critical role
in osmotic adjustment (Qasim et al., 2003).These
44 林 业 科 学 45 卷　
proteins may be synthesized de novo in response to salt
stress or the expression of formerly present proteins is
increased when plants are exposed to salt stress.
The accumulation of reactive oxygen species (ROS)
may be an important cause of damage to plants under salt
stress(Zhu , 2001).Plant cells are equipped with several
free radical detoxifying enzymes to protect them against
oxidative damage.These enzymes include superoxide
dismutase (SOD), peroxidase (POD) and catalase
(CAT)etc (Chien et al., 2001).Many studies have
shown that environmental stresses , especially drought and
salt stress , altered that activities of enzymes involved in
scavenging oxygen radicals(Gueta-Dahan et al., 1997;
Khedr et al., 2003;Zhang et al., 1994).In this
study , SOD activity showed an increase with the
increasing of NaCl concentration , and the SOD isoforms
were affected by salt treatment. Like superoxide
dismutase , peroxidase exhibited a consistent increase in
enzyme activity even under severe salt stress.However ,
the isoforms of POD were slightly changed under salt
stress.We found there was only one band in activity gels
of CAT , and the band intensity showed the same trend as
the spectrophotometric measurements , which may indicate
that this band is probably responsible for most of the
change in catalase activity.
MDA is an oxidized product of membrane lipids
which reflect the extent of oxidative stress.In the present
study , we observed that MDA content increased at low
levels of salinity and then decreased at 125 mmol·L-1 and
150 mmol·L-1 NaCl concentration.The decrease of MDA
content probably results from up-regulation of the
antioxidative system in response to salt stress.Similar
results correlating lipid peroxidation to antioxidative
system activity were also reported by other researchers
(Azevedo-Neto et al., 2006;Hernández et al., 2002).
With Regard to MDA content reduction , our results were
in agreement with those of Azevedo-Neto et al.(2006).
These authors suggested the reduction of MDA content was
due to increased antioxidative enzyme activities , which
reduced H2O2 levels and membrane damage.In our
study , the decrease of MDA concentration appeared to be
associated with increase in SOD and POD activities ,
which could lead to reduction of H2O 2 concentration and
subsequent lipid peroxidation.
Plants under salt stress also need to establish water
or osmotic homeostasis. Plants accumulate various
compatible osmolytes in the cytosol , thus lowering the
osmotic potential to sustain water absorption from saline
soil solutions (Zhu et al., 1997).It has been reported
that several compounds such as proline and polyamines
increased with salt stress (Le Dily et al., 1991;Nanjo
et al., 1999).It has been suggested that proline protects
plant tissues against osmotic stress because it is an
osmosolute and a protectant for enzymes and cellular
structures(Stewart et al., 1974;Le-Rudulier et al.,
1984).In our study , there was a positive relationship
between salt concentration and proline content of tissues.
Proline content significantly increased in tissues with
increase in salinity.Our result was in consistent with the
data reported by Patel et al.(2007).The concomitant
increase in proline content of the tissues indicates that
proline accumulation may contribute to the alleviation of
NaCl stress in the plant.A negative relationship between
salt tolerance and proline accumulation was also reported
(Soussi et al., 1998;Petrusa et al., 1997).These
studies assumed that the accumulation of proline was more
a consequence of damage produced by salt stress than of a
protective strategy.In the past , most attention has been
concerned with the role of proline as a compatible
osmolyte(Yancey et al., 1982;Samaras et al., 1995)
and osmoprotectant (MacCue et al., 1990;Serrano et
al., 1994).However , Nanjo et al.(1999)suggested
that in addition to various known roles of proline , it was
also involved in the synthesis of key proteins that are
necessary for stress responses.In a recent study , Khedr
et al.(2003) reported that proline could increase
ubiquitin-conjugates and induce the expression of
dehydrins in Pancratium maritimum.They came to the
conclusion that , proline improved the salt-tolerance of P.
maritimum by protecting the protein turnover machinery
against stress-damage and up-regulating stress protective
proteins.Proline could act as a component of signal
transduction pathways that regulate stress responsive genes
in addition to its previously described osmoprotective
roles , thereby improving the tolerance to salt stress.
In conclusion , our study indicated that salt tolerance
occurred in plantlets of B.papyrifera.Salt stress
induced oxidative stress and the enhanced activity of anti-
oxidative enzymes (SOD , POD and CAT ) and
accumulation of proline may be responsible for the
45　第 2期 李荣锦等:NaCl胁迫对构树组培苗抗氧化酶活性及同工酶谱的影响
defenses against this oxidative stress.Our findings may be
helpful to understand the physiological and biochemical
mechanisms of salt stress adaptation of B.papyrifera.
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47　第 2期 李荣锦等:NaCl胁迫对构树组培苗抗氧化酶活性及同工酶谱的影响