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ORIGINAL PAPER
Effects of exogenous nitric oxide on cadmium toxicity, element
contents and antioxidative system in perennial ryegrass
Quanhui Wang •Xue Liang •Yuanjie Dong •
Linlin Xu •Xiuwei Zhang •Jun Hou •
Zhenyi Fan
Received: 11 January 2012 / Accepted: 8 August 2012 / Published online: 19 August 2012
ÓSpringer Science+Business Media B.V. 2012
Abstract The effects of sodium nitroprusside (SNP, a
donor of NO) on cadmium (Cd) toxicity in ryegrass seed-
lings (Lolium perenne L.) were studied by investigating the
symptoms, plant growth, chlorophyll content, lipid perox-
idation, H
?
-ATPase enzyme and antioxidative enzymes.
Addition of 100 lM CdCl
2
caused serious chlorosis and
inhibited the growth of ryegrass seedlings, and dramati-
cally increased accumulation of Cd in both shoots and
roots, furthermore, the absorption of macro and micronu-
trients were inhibited. Addition of 50, 100, 200 lM SNP
significantly decreased the transport of Cd from roots to
shoots, alleviated the inhibition of K, Ca, Mg and Fe, Cu,
Zn absorption induced by Cd, reduced the toxicity symp-
toms and promoted the plant growth. The accumulation of
reactive oxygen species (ROS) significantly increased in
ryegrass seedlings exposed to Cd, and resulted in the lipid
peroxidation, which was indicated by accumulated con-
centration of thiobarbituric acid-reactive substances.
Addition of 50, 100, 200 lM SNP significantly decreased
the level of ROS and lipid peroxidation. Activities of
antioxidant enzymes also showed the same changes.
Addition of 50, 100, 200 lM SNP increased activities of
superoxide dismutase, peroxidase, catalase and ascorbate
peroxidase in ryegrass seedlings exposed to Cd. Addition
of 100 lM SNP had the most significant alleviating effect
against Cd toxicity while the addition of 400 lM SNP had
no significant effect with Cd treatment.
Keywords Antioxidative enzyme Cd stress
Lolium perenne L. Mineral content SNP
Abbreviations
APX Ascorbate peroxidase
CAT Catalase
H
2
O
2
Hydrogen peroxide
MDA Malondialdehyde
NO Nitric oxide
O
2
-
Superoxide anion radical
POD Peroxidase
SNP Sodium nitroprusside
SOD Superoxide dismutase
Introduction
Cadmium (Cd) is one of the most toxic elements that nega-
tively affects plant growth and development (Macek et al.
2002). Since the 1950s, the Cd contamination has aroused
people’s concern because of the ‘‘Itai-itai disease’’ which
first emerged in Japan at the start of the twentieth century.
The approval of China’s ‘‘Heavy Metal Pollution Integrated
Control’’ and ‘‘Cd rice’’ event raises the high importance of
Cd pollution once again. With the rapid development of
industry, wastewater irrigation and the widespread use of
chemical fertilizer have been an increasingly serious envi-
ronmental problem (Xu and Ji 2001), which lead to serious
Cd pollution. It has been reported that Cd disturbs the
metabolism of plants and inhibits plant growth. A chemical
balance of inorganic nutrients is a basic condition for proper
growth and development of plants. Some studies have
Q. Wang X. Liang Y. Dong (&)L. Xu X. Zhang
J. Hou Z. Fan
College of Resources and Environment, Shandong Agricultural
University, Tai’an 271018, People’s Republic of China
e-mail: yjdong@sdau.edu.cn
Y. Dong
Chinese National Engineering Research Center for Slow/
Controlled Release Fertilizers, Tai’an 271018, Shandong,
People’s Republic of China
123
Plant Growth Regul (2013) 69:11–20
DOI 10.1007/s10725-012-9742-y
indicated that excess Cd causes deficiency of potassium (K),
calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn),
copper (Cu) and zinc (Zn) (Rivetta et al. 1997; Sanita di
Toppi and Gabbrielli 1999; Sharma and Dubey 2005) and
induces inhibition of chlorophyll biosynthesis and a decline
in the photosynthetic rate (Prasad et al. 1999). The toxic
effects of heavy metals, both essential and nonessential
elements, have been linked to the production of ROS, such as
1
O
2
,O
2
-
,
OH and H
2
O
2
(Stochs and Bagchi 1995). As such
oxidative damage to vital molecules can result in the dis-
ruption of the cellular metabolism living organisms have
evolved various enzymatic and non-enzymatic means to
rapidly detoxify ROS (Ekmekci et al. 2008; Smeets et al.
2008). It has been suggested that excess Cd
2?
induces oxi-
dative stress, since this ion is involved in various processes
including the change of some enzyme activity related to ROS
and lipid peroxidation in common Medicago truncatula and
rice (Xu et al. 2010; Liu et al. 2003).
Sodium nitroprusside (SNP) is the inorganic compound
with the formula Na
2
[Fe(CN)
5
NO]2H
2
O. The sodium salt
dissolves in water and to a lesser extent in ethanol to give
solutions containing the dianion [Fe(CN)
5
NO]
2-
(Butler
and Megson 2002). Nitroprusside is a complex anion that
features an octahedral ferrous centre surrounded by five
tightly bound cyanide ligands and one linear nitric oxide
ligand. The molecular symmetry is C
4v
. Linear nitrosyl
ligands are assigned a single positive charge, thus the iron is
assigned an oxidation state of 2
?
. As such it has a low-spin
d
6
electron configuration and is diamagnetic. Its chemical
reactions are mainly associated with the NO ligand
(Coppens et al. 2002). Delledonne et al. (1998) proved that
0.5 mM of SNP can approximately produce 2.0 lM NO.
NO is a natural signal molecule, which plays an important
role in regulating a number of physiological processes and
plant resistance to biotic and abiotic stresses (Lopez-
Carrion et al. 2008). There is also evidence that NO ame-
liorates the damaging effects of heavy metals by broad
but different effects (Pedroso and Durzan 2000; Guo and
Crawford 2005; Bright et al. 2006;Huetal.2007). The pro-
tective function of NO mainly includes the regulation of ROS
and antioxidants, induction of gene expression, and absorption
and distribution of elements (Hsu and kao 2007; Xiong et al.
2009;Xuetal.2010). However, NO is a reactive nitrogen
species, and studies have shown that its effects on different
cells are either protective or toxic, depending on its concen-
tration and the position of action (Lamattina et al. 2003).
Perennial ryegrass is an important and widespread
perennial cool-season turfgrass due to its massive root sys-
tem, superior regeneration and tillering ability (Hannaway
et al. 1999). Previous researches demonstrated that perennial
ryegrass has potential for rehabilitation of Cd-contaminated
soils (Arienzo et al. 2004). Based on the above studies, we
suppose that NO may ameliorate toxic effects of excess Cd
on ryegrass. The influence of different concentrations of
SNP on Cd-induced changes of growth and antioxidant
system, as well as on nutrient distribution in ryegrass seed-
lings has been investigated.
Materials and methods
Plant material and culture conditions
Ryegrass seeds were sterilized with 5 % sodium hypo-
chlorite for 15 min and washed extensively with distilled
water, then germinated on moist filter paper in the dark at
26 °C for 3 days. Initially, seedlings of uniform size were
transferred to plastic pots (volume 500 ml) filled with
perlite (50 plants per pot) and watered with half-strength
Hoagland nutrition solution for 7 days. The seedlings were
then watered with full-strength Hoagland solution. Three-
week-old uniform seedlings were transferred into 1,000 ml
black plastic containers with 20 seedlings per container.
The nutrient solution was renewed every 2 days.
The experimental design is given in Table 1. The
treatments were arranged in a randomized block design
with three replicates, giving a total of 18 containers.
The experiment was carried out under a controlled-
environment chamber at 14/10 light/dark photoperiod and
photon flux density 150 lmol m
-2
s
-1
at the leaf level,
day/night temperature of 25/18 °C and 65 ±5 % relative
humidity. After 2 weeks of growth with the above condi-
tions, the plants were harvested and the roots and shoots
were separated and washed with 5 mM CaCl
2
first and then
repeatedly washed with deionized distilled water. For the
estimation of plant dry matter, Cd and mineral nutrients
content, the plants were dried at 80 °C for 48 h. For the
enzyme determination, fresh plant material was frozen in
liquid nitrogen and stored at -70 °C until use.
Determination of chlorophyll content
The chlorophyll content was determined according to the
method of Knudson et al. (1977). Fresh ryegrass leaf
Table 1 The experimental design
No. Treatments
CK No added SNP and CdCl
2
Cd 100 lmol CdCl
2
T1 100 lmol CdCl
2
?50 lmol SNP
T2 100 lmol CdCl
2
?100 lmol SNP
T3 100 lmol CdCl
2
?200 lmol SNP
T4 100 lmol CdCl
2
?400 lmol SNP
12 Plant Growth Regul (2013) 69:11–20
123
(0.5 g) was extracted in 2 ml 95 % ethanol for 24 h in the
dark, and the extracted solution was analyzed. The amounts
of chlorophyll a,band carotenoid were determined spec-
trophotometrically (SHIMADZU UV-2450, Kyoto, Japan),
by reading the absorbance at 665, 649 and 470 nm. The
chlorophyll content results are expressed as unit’s mg per
gram-fresh weight (mg g
-1
FW).
Determination of plant growth, cadmium and mineral
element concentrations
At harvest, the roots and shoots were separated and oven-
dried for 30 min at 105 °C, then at 70 °C till the materials
reach their constant weights. The dried tissues were
weighed and grinded into powder for the determination of
cadmium and mineral element concentrations, which was
measured by flame atomic absorbance spectrometry (SHI-
MADZU AA-6300, Kyoto, Japan) after digested with
mixed acid [HNO
3
?HClO
4
(3:1, v/v)] (Ali et al. 2002).
Determination of O
2
-
generation rate
For the measurement of O
2
-
generation rate, 0.3 g fresh
samples were ground in liquid N
2
and extracted in 3 ml of
ice-cold 50 mM sodium phosphate buffer (PBS) (pH 7.0).
The O
2
-
generation rate was determined by monitoring the
A530 of the production of the hydroxylamine reaction
following the modified method of Elstner and Heupel
(1976) as described by He et al. (2005). One milliliter
supernatant of fresh samples extraction was added to
0.9 ml 65 mM phosphate buffer solution (pH 7.8) and
0.1 ml 10 mM hydroxyl ammonium chloride. The reaction
was incubated at 25 °C for 35 min. Solution (0.5 ml) from
above reaction mixture was then added in 0.5 ml 17 mM
sulfanic acid and 0.5 ml 7.8 mM a-naphthylamine solu-
tion. After 20 min of reaction, 2 ml of ether was added into
the above solution, and then mixed well. The solution was
centrifuged at 1,5009gat 4 °C for 5 min. The absorbance
of the pink supernatant was measured at 530 nm with the
spectrophotometer. Absorbance values were calibrated to a
standard curve generated with known concentrations of
HNO
2
.
Determination of H
2
O
2
concentration
Fresh samples (1 g) were homogenized in 2 ml ice-cold
acetone. Titanium reagent (2 % TiCl
2
in conc. HCl) was
added to a known volume of extract supernatant to give
a Ti (IV) concentration of 2 %. The Ti–H
2
O
2
complex,
together with unreacted Ti, was then precipitated by adding
0.2 ml 17 M ammonia solution for each 1 ml of extract.
The precipitate was washed five times with ice acetone by
resuspension, drained, and dissolved in 1 M H
2
SO
4
(3 ml).
The absorbance of the solution was measured at 410 nm
against blanks, which had been similarly prepared but
without plant tissue (Patterson et al. 1984).
Determination of lipid peroxidation
Lipid peroxidation was determined by measuring mal-
ondialdehyde (MDA), a major thiobarbituric acid reactive
species thiobarbituric acid-reactive substances (TBARS)
and product of lipid peroxidation (Heath and Packer 1968).
Fresh samples (0.2 g) were ground in 3 ml of trichloro-
acetic acid (0.1 %, w/v). The homogenate was centrifuged
at 10,0009gfor 10 min and 1 ml of the supernatant fraction
was mixed with 4 ml of 0.5 % thiobarbituric acid (TBA) in
20 % TCA. The mixture was heated at 95 °C for 30 min,
chilled on ice, and then centrifuged at 10,0009gfor 5 min.
The absorbance of the supernatant was measured at 532 nm.
The value for non-specific absorption at 600 nm was sub-
tracted. The amount of MDA was calculated using the
extinction coefficient of 155 mM
-1
cm
-1
and expressed as
nmol g
-1
FW.
Determination of enzymatic activities
For extraction of antioxidative enzymes, leaves and roots
were homogenized with 50 mM Na
2
HPO
4
–NaH
2
PO
4
buf-
fer (pH 7.8) containing 0.2 mM EDTA and 2 % insoluble
polyvinylpyrrolidone in a chilled pestle and mortar. The
homogenate was centrifuged at 12,0009gfor 20 min and
the resulted supernatant was used for determination of
enzyme activities. The whole extraction procedure was
carried out at 4 °C. All spectrophotometric analysis was
conducted on a SHIMADZU UV-2450 spectrophotometer
(Kyoto, Japan).
Superoxide dismutase (SOD) activity was assayed by
measuring its ability to inhibit the photochemical reduction
of nitroblue tetrazolium following the method of Stewart
and Bewley (1980). Catalase (CAT) activity was measured
as the decline in absorbance at 240 nm due to the decrease
of extinction of H
2
O
2
according to the method of Patra
et al. (1978). Peroxidase (POD) activity was measured by
the increase in absorbance at 470 nm due to guaiacol
oxidation (Nickel and Cunningham 1969). Ascorbate per-
oxidase (APX) activity was measured by the decrease in
absorbance at 290 nm as ascorbate was oxidized (Nakano
and Asada 1981).
Plasma membrane preparation
A membrane fraction enriched in plasma membrane vesi-
cles was prepared as described by Briskin et al. (1987) with
minor changes. Excised roots were homogenized (1/2, w/v)
with a mortar and pestle in a cold grinding medium
Plant Growth Regul (2013) 69:11–20 13
123
containing: 25 mM HEES-Tris (pH 7.2), 250 mM manni-
tol, 5 mM EDTA, 1 mM DTT and 1.5 % (w/v) PVP. The
whole isolation procedures were carried out at 4 °C. The
homogenate was filtered through four layers of cheesecloth
and centrifuged at 5609gfor 12 min, then the supernatant
was centrifuged at 10,0009gfor 15 min, and the super-
natant was centrifuged at 60,0009gfor 30 min to yield a
crude membrane fraction. The resulting pellet was resus-
pended with 1 ml in a gradient buffer containing: 20 mM
HEPES-Tris (pH 7.5), 5 mM EDTA and 0.5 mM EGTA.
The supernatant was layered on top of a step gradient
consisting of 1 ml of 45, 33 and 15 % (w/w) sucrose,
respectively, and then centrifuged for 2 h at 70,0009g.
Measurement of H
?
-ATPase in PMs
ATP hydrolysis assays were performed as described by
Briskin et al. (1987). 0.5 ml the reaction medium con-
taining: 36 mM Tris-Mes (pH 6.5), 30 mM ATP-Na
2
,
3 mM MgSO
4
, 1 mM NaN
3
, 50 mM KNO
3
,1mM
Na
2
MoO
4
, 0.02 % (v/v) Triton X-100, in the presence or
absence of 2.5 mM Na
3
VO
4
. The reaction was started by
adding 50 ll PM vesicles. After 30 min incubation at
37 °C, the reaction was quenched by the addition of 55 %
(w/v) TCA. The H
?
-ATPase activity was determined by
measuring the release of Pi (Ohinishi et al. 1975).
Statistics
Statistical analyses were carried out by analysis of variance
(ANOVA) using SAS software (SAS Institute, Cary NC).
Differences between treatments were separated by the least
significant difference (LSD) test at a 0.05 probability level.
Results
Symptoms and plant growth
Symptoms of Cd toxicity in ryegrass leaves became
apparent 5 days after imposing Cd. In the beginning, the
leaf apex showed brown, and with the treatment time
similar colored markings were observed along the veins of
the leaves which gradually became desiccated. Addition of
different concentrations of SNP inhibited the appearance of
Cd toxicity, especially 100 lM SNP (Fig. 1).
Figure 2shows that Cd exposure significantly decreased
the shoot and root biomass of ryegrass seedlings, however,
this inhibition was significantly alleviated by 50, 100,
200 lM SNP additions, especially in the 100 lM treat-
ment. Compared with Cd treatment, the shoot and root
biomass of T3 treatment was increased by 66.4 and
113.6 %, and T4 treatment had no significant effect. The
shoot and root biomass of addition of SNP treatments was
found in a general trend of T2 [T3 [T1 [T4. This
revealed that exogenous NO could alleviate the effect of
Cd toxicity. However, it needed proper concentration.
Chlorophyll content
Ryegrass seedlings treated with Cd showed a significant
decrease in total chlorophyll (Fig. 3a), chl a(Fig. 3b), chl
CK Cd T1
T2 T3 T4
Fig. 1 Effects of different concentrations of SNP supply on ryegrass plants growth in nutrient solutions without or with 100 lmol CdCl
2
14 Plant Growth Regul (2013) 69:11–20
123
b(Fig. 3c) and car (Fig. 3d) content as compared to CK.
With adding different concentrations of SNP under Cd
stress, the chlorophyll content has different changes. The
T1, T2 and T3 treatments increased total chlorophyll by
34.5, 53.8 and 58.6 % than Cd treatments. And the T4
treatment decreased total chlorophyll by 4.1 %. The T2
treatment had the best effect on increasing total chlorophyll
content. Similar findings were found for the chl a, chl b,
and car content.
Cd concentrations
As shown in Fig. 4, Cd content was significantly increased
in both leaves and roots compared to CK. The T2 treatment
had the lowest Cd content in leaves and highest Cd content
A
a
e
d
b
c
e
0.00
2.00
4.00
6.00
8.00
CK Cd T1 T2 T3 T4
Shoot biomass [g (10plant)
-1
]
B
a
b
c
d
c
b
0.00
0.40
0.80
1.20
1.60
CK Cd T1 T2 T3 T4
Root biomass [g (10plant )
-1
]
Fig. 2 Effects of different concentrations of SNP supply on biomass
production of 14 days treatment ryegrass plants biomass in nutrient
solutions without or with 100 lmol CdCl
2
. Values are the mean of
three replicates. Each replicate has 20 plants. Bars with different
letters are significantly different at P\0.05
A
a
d
c
bb
de
0.00
1.00
2.00
3.00
4.00
CK Cd T1 T2 T3 T4
Total Chlorophyll conten
(mg g
-1
FW)
B
d
bb
c
d
a
0.00
1.00
2.00
3.00
CK Cd T1 T2 T3 T4
Chlorophyll a content
(mg g -1
FW)
C
a
d
c
bb
e
0.00
0.20
0.40
0.60
0.80
CK Cd T1 T2 T3 T4
Chlorophyll b content
(mg g-1 FW)
D
ab
c
b
a
ab
c
0.00
0.20
0.40
0.60
CK Cd T1 T2 T3 T4
Carotenoids content
(mg g-1
FW)
Fig. 3 Effects of different concentrations of SNP supply on the
chlorophyll contents in leaves of 14 days treatment ryegrass plants
grown in nutrient solutions without or with 100 lmol CdCl
2
. Values
are the mean of three replicates. Each replicate has 20 plants. Bars
with different letters are significantly different at P\0.05
d
d
a
a
b
b
c
c
b
b
a
c
-5.00
5.00
15.00
25.00
35.00
rootsshoots
Cd content (mg kg-1)
CK Cd T1 T2 T3 T4
Fig. 4 Effects of different concentrations of SNP supply on Cd
concentrations in leaves and roots of 14 days treatment ryegrass
plants grown in nutrient solutions without or with 100 lmol CdCl
2
.
Values are the mean of three replicates. Each replicate has 20 plants.
Bars with different letters are significantly different at P\0.05
Plant Growth Regul (2013) 69:11–20 15
123
in roots. This observation suggested that proper concen-
tration exogenous NO supply could influence Cd uptake
and transport. Cd content in roots varied more extensively
than in leaves, which indicated that Cd is inclined to
accumulate in roots. These results confirm the potential of
Lolium perenne as a suitable species for revegetation of
contaminated soils. And, SNP as a donor of NO has an
important role in rehabilitation of heavy metal contami-
nated soils, especially in Cd-contaminated soils.
K, Ca and Mg concentrations
Table 2shows the K, Ca and Mg content in the shoots and
roots. Compared to CK, Cd treatments significantly
decreased K and Ca content in ryegrass shoots and roots.
The application of exogenous NO increased K and Ca
content in shoots and aggravated the decreases of K content
in roots. At the addition of SNP treatments, T1, T2, T3 and
T4 treatments increased K content in shoots by 94.8, 116.9,
102.1, 18.2 % and decreased K content in roots by 28.8,
50.7, 29.7, 19.7 % than treatment with Cd. The T2 treatment
had highest K content in shoots. However, T4 treatments
had no significant effect compared with Cd treatment. Cd
treatment significantly decreased the Mg content in the
shoots and roots. Adding different concentrations of
exogenous NO, the Mg content of ryegrass in shoots sig-
nificantly increased compared to the Cd treatments, how-
ever, the Mg content in roots had no significant influence.
Both Ca and Mg content in the shoots and roots were the
highest at T2 treatment.
Fe, Cu and Zn concentrations
As shown in Table 3, Cd treatment significantly decreased
Fe, Cu and Zn content in shoots and Fe and Zn content in
roots. The application of exogenous NO increased Fe
content in shoots and roots. At the addition of SNP treat-
ments, T1, T2, T3 and T4 treatments increased Fe content
in shoots by 65.4, 73.2, 66.0, 9.5 % and by 144.3, 157.5,
147.0, 25.7 % in roots than treatment with Cd. And, the T2
treatment had highest the Fe content in shoots and roots.
Similar findings were found for the Cu and Zn content.
O
2
-
generation rate, H
2
O
2
and MDA content
Compared to the CK, Cd treatment significantly increased
O
2
-
generation rate (Fig. 5a) by 43.5 % in leaves and by
137.2 % in roots. The application of exogenous NO could
decrease O
2
-
generation rate under Cd stress. T1, T2, T3
and T4 treatments decreased O
2
-
generation rate by 12.1,
Table 2 Effects of different concentrations of SNP on concentrations of K, Ca and Mg in shoots and roots of ryegrass seedings under Cd stress
Items Treatments
CK Cd T1 T2 T3 T4
K(gkg
-1
) Shoot 35.42 ±3.95 a 15.37 ±1.88 c 29.96 ±2.32 b 33.34 ±4.52 ab 31.06 ±2.63 b 18.16 ±1.30 c
Root 48.37 ±3.68 a 35.35 ±2.78 b 25.17 ±3.51 c 17.41 ±1.87 d 24.84 ±2.56 c 28.38 ±1.07 c
Ca (g kg
-1
) Shoot 5.41 ±0.35 a 3.83 ±0.30 d 4.91 ±0.13 bc 5.14 ±0.64 ab 4.80 ±0.31 c 4.09 ±0.28 d
Root 1.53 ±0.18 a 0.74 ±0.08 c 1.21 ±0.04 b 1.41 ±0.07 a 1.20 ±0.07 b 0.88 ±0.06 c
Mg (g kg
-1
) Shoot 8.11 ±0.13 a 6.65 ±0.11e 7.37 ±0.34 c 7.84 ±0.25 b 7.46 ±0.61 c 7.04 ±0.65 d
Root 6.67 ±0.14 ab 6.64 ±0.23 ab 6.74 ±0.14 ab 6.79 ±0.28 a 6.77 ±0.21 ab 6.70 ±0.16 ab
Values are the mean ±SD of three replicates. Each replicate has 20 plants. The values followed by the different letter show statistically
significant differences at P\0.05
Table 3 Effects of different concentrations of SNP on concentrations of Fe, Cu and Zn in shoots and roots of ryegrass seedlings under Cd stress
Items Treatments
CK Cd T1 T2 T3 T4
Fe (mg kg
-1
) Shoot 378.67 ±10.42 a 216.48 ±5.29 d 358.14 ±4.02 b 374.89 ±9.91 a 359.43 ±7.44 b 237.15 ±11.02 c
Root 413.09 ±12.89 c 186.59 ±9.84 e 456.00 ±15.92 b 480.48 ±19.26 a 460.92 ±16.37 b 234.56 ±10.25 d
Cu (mg kg
-1
) Shoot 38.23 ±6.48 a 20.32 ±4.10 d 30.53 ±2.89 bc 30.92 ±6.31 b 28.91 ±3.37 bc 23.20 ±2.11 cd
Root 68.33 ±5.27 c 76.69 ±8.23 c 92.58 ±10.46 b 113.19 ±9.64 a 90.92 ±7.59 b 71.14 ±8.26 c
Zn (mg kg
-1
) Shoot 89.13 ±7.83 a 54.43 ±6.97 c 74.44 ±9.58 b 82.44 ±8.26 a 73.19 ±3.62 b 60.80 ±7.93 c
Root 58.84 ±4.84 a 32.16 ±3.22 c 48.90 ±6.74 b 54.00 ±5.39 ab 48.34 ±3.83 b 36.95 ±3.83 c
Values are the mean ±SD of three replicates. Each replicate has 20 plants. The values followed by the different letter show statistically
significant differences at P\0.05
16 Plant Growth Regul (2013) 69:11–20
123
28.0, 12.7, 2.3 % in shoots and by 31.4, 40.7, 34.9, 2.61 %
in roots than Cd treatment. The T2 treatment had the lowest
O
2
-
generation rate in shoots and roots. Similar findings
were found for the H
2
O
2
content (Fig. 5b).
When plants were subjected to environmental stress,
oxidative damage resulted in membrane lipid peroxidation,
which could be estimated by MDA content (Fig. 5c).
Similar to O
2
-
and H
2
O
2
change, Cd treatment significantly
increased MDA contents in ryegrass leaves and roots, T1,
T2 and T3 treatments significantly reduced MDA contents
in leaves and roots, while T4 treatment did not reach to
statistically significant differences in leaves. T2 treatment
had the best effect of alleviation.
Antioxidant enzymes
As shown in Fig. 6, Cd treatment significantly decreased
SOD activity (Fig. 6a) by 48.9 % in leaves and by 55.2 % in
roots. The T1, T2, T3 and T4 treatments increased SOD
activity by 66.3, 84.3, 69.7, 4.4 % in leaves and by 46.5, 95.1,
A
d
c
a
a
b
b
c
cb
b
a
a
0
1
2
3
4
leaves roots
O
2
•-
generation rate
(nmol g
-1
FW min
-1
)
CK Cd T1 T2 T3 T4
B
de
a
b
b
cd
c
c
b
a
a
0
2
4
6
rootsleaves
H
2
O
2
content (µmol g
-1
FW)
CK Cd T1 T2 T3 T4
C
e
d
b
a
c
bd
cc
ba
a
0
10
20
30
40
rootsleaves
MDA content (nmol g
-1
FW)
CK Cd T1 T2 T3 T4
Fig. 5 Effects of different concentrations of SNP supply on O
2
-
generation rate (a), H
2
O
2
(b), MDA (c) content in leaves and roots of
14 days treatment ryegrass plants grown in nutrient solutions without
or with 100 lmol CdCl
2
. Values are the mean of three replicates.
Each replicate has 20 plants. Bars with different letters are
significantly different at P\0.05
A
a
ac
e
b
d
ab
b
b
c
c
e
0
50
100
150
200
250
rootsleaves
SOD activity (U g
-1
FW)
CK Cd T1 T2 T3 T4
B
a
a
c
c
b
b
a
a
b
b
c
c
0
5
10
15
20
leaves roots
POD activity (U mg
-1
FW min
-1
)
CK Cd T1 T2 T3 T4
C
a
a
e
e
c
b
b
a
c
c
d
d
0
2
4
6
8
leaves roots
CAT activity
(µmol H
2
O
2
mg
-1
FW min
-1
)
CK Cd T1 T2 T3 T4
D
a
a
c
c
b
b
a
a
b
b
c
c
0
1
2
3
4
5
rootsleaves
APX activity
(µmol ASA mg
-1
FW min
-1
)
CK Cd T1 T2 T3 T4
Fig. 6 Effects of different concentrations of SNP supply on activities
of SOD (a), POD (b), CAT (c), APX (d) in leaves and roots of
14 days treatment ryegrass plants grown in nutrient solutions without
or with 100 lmol CdCl
2
. Values are the mean of three replicates.
Each replicate has 20 plants. Bars with different letters are
significantly different at P\0.05
Plant Growth Regul (2013) 69:11–20 17
123
74.6, 6.0 % in roots than Cd treatment. And the T4 treatment
did not reach to statistically significant differences in leaves
and roots compared with Cd treatment. It was similar in
activities of POD (Fig. 6b), CAT (Fig. 6c) and APX
(Fig. 6d). All of antioxidant enzyme activities were found to
be in a general trend of CK [T2 [T1, or T3 [T4 [Cd.
H
?
-ATPase activity in plasma membrane
The H
?
-ATPase transports protons out of the cell across the
plasma membrane, thus establishing the proton electro-
chemical gradient that contributes to the maintenance of the
intracellular and extracellular pH and plays a major role in
the activation of ion and nutrient transport (Fre
´de
´ric et al.
2007). As shown in Fig. 7, Cd stress markedly inhibited H
?
-
ATPase activity both in leaves and roots. The T1, T2, T3 and
T4 treatments increased H
?
-ATPase activity by 38.2, 63.9,
36.6, 8.7 % in leaves and by 21.4, 63.1, 17.6, 7.2 % in roots
than treatment with Cd. And, the T2 treatment had the
highest H
?
-ATPase activity in shoots and roots.
Discussion
NO as a signaling regulatory molecule and reactive oxygen
scavenger, can regulate the biotic and abiotic stress adap-
tation of plants (Zhao et al. 2008; Song et al. 2006; Singh
et al. 2008). However, studies have shown that its effects on
different cells are either protective or toxic, depending on its
concentration and the position of action (Lamattina et al.
2003). In the present experiment, compared to CK, the
growth of ryegrass plants in the presence of Cd was signif-
icantly delayed. However, the inhibitory effects were sig-
nificantly alleviated by low concentrations of NO (T1, T2,
and T3). The mitigation effect of higher concentrations of
NO (T4) on ryegrass reduced significantly (Fig. 2). These
results demonstrated the dual nature of NO on plants that was
consistent with the results of Tu et al. (2003). The optimum
concentration of NO can act directly on cell wall components
through apoplast including cell walls relax, and on the
membrane lipid bilayers enhancing membrane fluidity and
promoting cell expansion and growth (Xiong et al. 2009).
It has been reported that Cd toxicity to plants is affected
by other ions in the nutrient solution (Sharma and Dubey
2005). Increasing Ca, Mg and Fe levels reduce the
absorption and transport of Cd (Liu et al. 2003). Cd leads to
a deficiency of macro and micronutrients, which may be
both a cause and a consequence induced by Cd (Tables 2,
3). The chlorosis induced by Cd has considered due to Fe
deficiency (Prasad et al. 1999). In the present experiment
similar results were observed, however, the addition of low
concentrations of NO alleviated the Fe absorption. There-
fore the alleviation of inhibitory growth by NO may depend
on increasing the absorption of macro and micronutrients.
Changes of Ca, Mg and Zn concentrations in ryegrass
seedlings indicate that Cd greatly disturbs ionic homeo-
stasis, and addition of low concentrations of NO stimulates
their maintenance. Until now only a few experiments data
showed that exogenous NO influence nutrient absorption
under heavy metal toxicity, furthermore, the results were
significantly different, therefore, the mechanism needs
further investigation and clarification. In the experiment,
Ca and Mg concentration in ryegrass leaves decreased
under the influence of Cd, and addition of low concentra-
tions of NO induces its transport from nutrient solution to
ryegrass seedlings. It is well known that H
?
-ATPase in
plasma membrane plays an important role in the transport
of multiple ions (Palmgren and Harper 1999), and there are
investigations indicating that NO could induce H
?
-ATPase
activity (Cui et al. 2010), which might be responsible for
NO increasing absorption of Ca, Mg under Cd stress. Cd
toxicity has been associated with an increase in indole-
acetic acid oxidase activity, which results in a deficiency of
auxin (IAA). It is well known that Zn is required for the
synthesis of IAA, addition of low concentrations of NO
increases the Zn concentration in ryegrass plants under Cd
stress. Furthermore, the protective effect of Zn has been
shown due to its ability to inhibit NADPH oxidation and
centered free radical generation (Cakmak 2000).
Most biotic and abiotic stresses activate a common
mechanism involving the production of ROS. Although Cd
does not generate ROS directly, it generates oxidative
stress via interference with the antioxidant defense system
(Foyer and Noctor 2005), and promotes MDA content in
plants due to increased lipid peroxidation (Krantev et al.
2008). These consequences together with oxidative stress
may be responsible for the influence of NO on avoiding the
toxic effects of Cd. Related results have been observed in
rice with metal toxicity (Yu et al. 2005). The observed
increases in H
2
O
2
and O
2
-
under Cd stress probably
account for the lipid peroxidation which is indicated by
excess accumulation of TBARS. Similar results have been
a
a
e
d
c
c
b
bcd
cde
d
0
5
10
15
20
25
30
rootsleaves
The activity of H
+
-ATPase
(µmol Pi mg
-1
protein h
-1
)
CK Cd T1 T2 T3 T4
Fig. 7 Effects of different concentrations of SNP supply on activity
of H ?-ATPase in leaves and roots plasma membrane of 14 days
treatment ryegrass plants grown in nutrient solutions without or with
100 lmol CdCl
2
. Values are the mean of three replicates. Each
replicate has 20 plants. Bars with different letters are significantly
different at P\0.05
18 Plant Growth Regul (2013) 69:11–20
123
observed in peanuts. (Shi et al. 2010). The addition of low
concentrations of NO alleviated H
2
O
2
and O
2
-
under Cd
stress, the influence can protect cell membrane from per-
oxiding and decrease the accumulation of TBARS (Dilek
and Kadiriye 2011). It has been well known that antioxi-
dant system plays important role in plant tolerance to stress
conditions, which is based on the fact that the activity of
one or more of these enzymes or antioxidant substances in
general increase in plants when exposed to stressful con-
ditions and these enhancements are related to increased
stress tolerance (Kadioglu et al. 2011). SOD constitutes the
first line of defense against ROS, which is crucial for the
removal of O
2
-
in the compartments where O
2
-
radicals
formed (Takahashi and Asada 1983). The decomposition of
O
2
-
is always accompanied by production of H
2
O
2
, which
rapidly diffuses across the membrane and is toxic because
it acts both as an oxidant and reductant (Foyer et al. 1997).
POD catalyses H
2
O
2
-dependent oxidation of substrate,
while CAT and APX eliminate H
2
O
2
by breaking it down
directly to form water and oxygen (Ekmekci et al. 2008). In
the present experiment, compared to CK, Cd treatment
significantly decreased SOD, POD, CAT and APX activi-
ties in leaves and roots (Fig. 6). Addition of low concen-
trations of NO increased SOD, POD, CAT and APX
activities under Cd stress, and therefore increasing the O
2
-
and H
2
O
2
scavenging. The similar results have been
observed in tomatos under Cu stress and in rice under Cd stress
(Zhang et al. 2009; Xiong et al. 2009). However, different
crop types, disposal methods and different levels of stress can
bring about different changes of antioxidant enzymes.
Conclusion
The present work demonstrates that low concentrations of
NO could significantly alleviate the effect of Cd toxicity
and high concentrations of NO had no significant effect.
The relief mechanism of low concentrations of NO to Cd
toxicity contains: (1) increased uptake of macro and
micronutrients and decreased the root-to-shoot transloca-
tion of Cd, (2) protection of plants under Cd stress induced
oxidative stress. Our results may have a potential value on
turf grass production and repair of heavy metal contami-
nated areas, but further study is required.
Acknowledgments The authors thank English Lecturer Mr Stuart
Craig MA (England, Taishan University of china, e-mail: stuartc-
raig269@msn.com), lecturer Xiujuan Wang (College of Foreign
Languages, Shandong Agricultural University) and Doctor Hongyi
Luo (Nanyang Technological University, Singapore) for their critical
reading and revision of the manuscript. Special acknowledgements
are given to the editors and reviewers. This research work was
financially supported by the Projects National Natural Science
Foundation of China (No. 40701094) and the Projects ‘‘948’’ of
Agriculture Ministry of China (No. 2011-G30).
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