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RESEARCH ARTICLE
Effects of sodium nitroprusside (SNP) pretreatment on UV-B
stress tolerance in lettuce (Lactuca sativa L.) seedlings
Aslıhan Esringu
1
&Ozkan Aksakal
2
&Dilruba Tabay
2
&Ayse Ay d a n K a r a
2
Received: 19 June 2015 /Accepted: 21 August 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Ultraviolet-B (UV-B) radiation is one of the most
important abiotic stress factors that could influence plant
growth, development, and productivity. Nitric oxide (NO) is
an important plant growth regulator involved in a wide variety
of physiological processes. In the present study, the possibility
of enhancing UV-B stress tolerance of lettuce seedlings by the
exogenous application of sodium nitroprusside (SNP) was
investigated. UV-B radiation increased the activities of super-
oxide dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX), peroxidase (POD) and total phenolic concentrations,
antioxidant capacity, and expression of phenylalanine ammo-
nia lyase (PAL) gene in seedlings, but the combination of SNP
pretreatment and UV-B enhanced antioxidant enzyme activi-
ties, total phenolic concentrations, antioxidant capacity, and
PAL gene expression even more. Moreover, UV-B radiation
significantly inhibited chlorophylls, carotenoid, gibberellic
acid (GA), and indole-3-acetic acid (IAA) contents and in-
creased the contents of abscisic acid (ABA), salicylic acid
(SA), malondialdehyde (MDA), hydrogen peroxide (H
2
O
2
),
and superoxide radical (O
2
•
−
) in lettuce seedlings. When SNP
pretreatment was combined with the UV-B radiation, we ob-
served alleviated chlorophylls, carotenoid, GA, and IAA inhi-
bition and decreased content of ABA, SA, MDA, H
2
O
2
,and
O
2
•
−
in comparison to non-pretreated stressed seedlings.
Keywords Antioxidant enzyme .Lettuce .Phenylalanine
ammonia lyase .Sodium nitroprusside .Ultraviolet-B
Introduction
Ultraviolet-B (UV-B) is one of the most detrimental environ-
mental stresses that hinder plant growth and development.
Several studies have shown deleterious effects of UV-B stress
on plants via reduced photosynthesis and biomass production
(Tossi et al. 2011,2012; Zlatev et al. 2012; Hideg et al. 2013).
On the other hand, UV-B not only inhibits the growth and
development of plants, but also induces the overproduction
of reactive oxygen species (ROS), including superoxide anion
(O
2
•
−
) and hydrogen peroxide (H
2
O
2
) (Mittler 2002). The
accumulation of ROS causes lipid peroxidation, which will
ultimately lead to plant cell death. To mitigate the damage
from ROS, plants have developed enzymatic antioxidant, such
as superoxide dismutase (SOD), peroxidase (POD), ascorbate
peroxidase (APX), and catalase (CAT) and also possess non-
enzymatic antioxidants that include reduced ascorbate, gluta-
thione, α-tocopherol, and flavonoids.
Nitric oxide, a small highly diffusible bioactive molecule,
is believed to play an important role in a wide range of phys-
iological processes in plants, such as germination, iron ho-
meostasis, mitochondrial functionality, fruit ripening, floral
regulation, and programmed cell death (Khan et al. 2012;
Xu et al. 2014). Many studies show that exogenous nitric
oxide (NO) improves the growth and yield of a number of
plants by enhancing growth and development of plant tissue
(Mur et al. 2012). Furthermore, a number of studies indicated
that NO in low concentration regulates key physiological pro-
cesses associated with plant growth under various biotic and
abiotic stresses, including UV (Tossi et al. 2011), low and high
temperature (Song et al. 2006), salinity (Khan et al. 2012),
Responsible editor: Philippe Garrigues
*Ozkan Aksakal
oz_aksakal@yahoo.com
1
Narman Vocational Training School, Atatürk University,
Erzurum, Turkey
2
Department of Biology, Science Faculty, Atatürk University,
Erzurum, Turkey
Environ Sci Pollut Res
DOI 10.1007/s11356-015-5301-1
drought (Boogar et al. 2014), and heavy metals (He et al.
2012).
In the present study, lettuce was used as a model plant,
since it is one of the most important dietary leafy vegetables
which is primarily consumed worldwide fresh or fresh-cut
(Putnam et al. 2000). In addition, lettuce is an important
source of dietary antioxidants especially considering its high
ROS scavenging activity (Ramos et al. 2011). It contains a
number of nutritive and health-promoting compounds such
as phenolic components; Vitamin A, C, and E; calcium; lutein;
and dietary fiber (Ramos et al. 2011). Furthermore, it was also
reported that lettuce was able to eliminate the stress factors
such as drought or UV-B irradiation by activating genes re-
sponsible for phenylalanine ammonia lyase (PAL) biosynthe-
sis (Oh et al. 2010) or triggering the synthesis of anthocyanin
and other flavonoids (Tsormpatsidis et al. 2010).
The objective of this study was to investigate the interactive
effect of UV-B radiation and exogenous sodium nitroprusside
(SNP) (a nitric oxide donor) treatments on the ROS, photo-
synthetic pigments, antioxidant enzymes, total phenolic con-
centration, antioxidant capacity, some endogenous hormones,
and soluble sugars of lettuce seedlings.
Materials and methods
Plant material
Lettuce seeds were supplied from the Agricultural Faculty of
Atatürk University. They were sterilized with 5 % sodium
hypochlorite for 15 min and rinsed thoroughly with distilled
water, then germinated on moist filter paper in the dark at
25 °C for 3 days. Germinated seedlings were grown on
soil/vermiculite (3:1, v/v) at 25 °C in an environment-
controlled chamber at a light intensity of 120 μmol
photons m
−2
s
−1
and a 14/10 h light/dark photoperiod.
SNP and UV-B treatment
Thirteen-day-old healthy seedlings were used in this experi-
ment. When the second pair of leaves were fully expanded,
seedlings were sprayed with 100 μM of SNP (Sigma Aldrich
St. Lois, MO, USA) or H
2
O. After 24 h, they were exposed to
UV-B radiation for 18 h by using UV-B lamp (Philips
TL100W/12) at an irradiance of 3.3 Wm
−2
. After completion
of UV-B treatment, leaves were immediately sampled for var-
ious analyses.
Determination of lipid peroxidation
Lipid peroxidation was determined by the method of Heath
and Packer (1968). This method is based on the determination
of the content of malondialdehyde produced by thiobarbituric
acid reacting substances (TBARS).
Determination of hydrogen peroxide and superoxide
radical
H
2
O
2
concentration was determined according to Patterson
et al. (1984). The assay was based on the absorbance change
of the titanium peroxide complex at 415 nm. Absorbance
values were quantified using standard curve generated from
known concentrations of H
2
O
2
. The rate of superoxide pro-
duction was measured by the method of Elstner and Heupel
(1976).
Determination of photosynthetic pigments
Fresh leaf tissues (0.1 g) were homogenized in chilled 80 %
(v/v) acetone. The homogenate was centrifuged at 8800gfor
10 min at 4 °C in dark. The absorbance of the acetone extract
was measured at 663, 645, and 470 nm using a spectrometer
(Shimadzu UV mini 1240). The contents of chlorophyll a,
chlorophyll b, and total carotenoids were calculated according
to Arnon (1949).
Determination of antioxidant enzyme activity
To determine the activities of antioxidant enzymes, fresh
leaves (0.5 g) were ground with a mortar and pestle under
chilled conditions in the presence of phosphate buffer
(0.1 M, pH 7.5) containing 0.5 mM EDTA. The homogenate
was centrifuged at 12,000gfor 10 min at 4 °C, and the
resulting supernatant was used for the enzyme assay.
SOD activity was assayed using the method of Agarwal
and Pandey (2004) that spectrophotometrically measures in-
hibition of the photochemical reduction of nitroblue tetrazoli-
um at 560 nm.
POD activity was measured according to the method of
Zhang and Kirkham (1994). The enzyme extract (20 mL)
was added to the reaction mixture containing 20 mL guaiacol
solution and 10 mL H
2
O
2
solution in 3 mL of phosphate
buffer solution (pH 7.0). The addition of enzyme extract
started the reaction, and the increase in absorbance was re-
corded at 470 nm for 5 min. Enzyme activity was quantified
by the amount of tetraguaiacol formed using its molar extinc-
tion coefficient (26.6 mM
−1
cm
−1
).
CAT activity was performed according to Qiu et al. (2011);
the reaction mixture in a total volume of 2 ml contained 0.1 ml
enzyme extract, 100 mM phosphate buffer (pH 7), 0.1 μM
EDTA, and 0.1 % H
2
O
2
.
APX activity was determined according to Nakano and
Asada (1981); the reaction mixture in a total volume of 3 ml
consisted of 50 mM phosphate buffer (pH 7), 0.1 ml enzyme
extract, 0.1 mM EDTA, 0.5 mM ascorbate, and 0.1 mM H
2
O
2
.
Environ Sci Pollut Res
APX was assayed as a decrease in absorbance at 290 nm of
ascorbate, and enzyme activity was quantified using the molar
extinction coefficient for 2.8 mM
−1
cm
−1
.
Determination of total phenolic concentration
and antioxidant capacity
The concentrations of phenolic compounds in the extracts of
lettuce leaves were measured according to the Folin–
Ciocalteu reagent method.
The antioxidant capacity of lettuce leaves was measured by
the modified 2,2
’
-azino-bis (3-ethylbenzthiazoline-6-
sulphonic acid) or ABTS method (Miller and Rice-Evans
1996).
PAL gen e e x p ress i o n
Total RNA from the leaves of lettuce was isolated using
RNeasy Plant Mini Kit (Qiagen) according to the manufac-
turer’s instructions. cDNA was synthesized using the
RevertAidTM First Strand cDNA Synthesis Kit (Fermentas
catalog no: K1622). The analysis of the PAL and γ-TMT gene
expressions were carried out according to Lee et al. (2014)
using lettuce-specific primers. The primers used for the PAL
(forward: ACGAAATGGACCGTTACAG, reverse:
TTCCCTCTCGATCATTTTGG) and γ-TMT (forward:
TGTTGACGCAATACCACCAC, reverse: GCCATTG
TCATCGGAGGAAC) were designed by Primer 3 software,
referring to accessed sequencing in the gene bank. For nor-
malization, the lettuce β-actin gene (forward:
AGCAACTGGGATGACATGGA, reverse: GGGTTG
AGAGGTGCCTCAGT) as endogenous control was used.
Real-time PCR was performed with a Rotor-gene Q instru-
ment using SYBR Green detection chemistry (Quantifast
SYBR Green PCR Kit, Qiagen). The relative ratio of thresh-
old cycle (ct) values between the endogenous control and the
specific genes was calculated for each sample.
Determination of phytohormones
Extraction and purification of phytohormones were done by
some modifications of the methods of Kuraishi et al. (1991)
and Battal and Tileklioglu (2001). First, methanol (80 % and
−40 °C) was added to fresh leaf samples (Davies 1995).
Material was homogenized for 10 min with Ultra Turrax and
incubated for 24 h in the dark. The final samples were filtered
through Whatman No. 1 filter paper, and the supernatants
were filtered again through a 0.45-μm pore filter (Cutting
1991). Then, the supernatants were dried at 35 °C using an
evaporator pump. Powder supernatants were dissolved in
0.1 M KH
2
PO
4
(pH 8.0) and centrifuged at 3600gfor1hat
4 °C for the separation of fatty acids (Palni et al. 1983). One
gram of polyvinylpyrrolidone (PVPP) was added to the
supernatant to remove the phenolic and colored materials
(Chen 1991; HernandezMinana 1991;Qamaruddin1996). It
was filtered with Whatman No. 1 paper to remove the PVPP
(Cheikh and Jones 1994). On the other hand, for further spe-
cific separation, a Sep-Pak C-18 (Waters) cartridge was used.
Adsorbed hormones were transferred to vials using 80 %
methanol and analyzed by high-performance liquid chroma-
tography (HPLC) using a Zorbax Eclipse-AAA C-18 column
(Agilent 1200 HPLC) and by absorbance at 265 nm in a UV
detector. Flow speed was set to 1.2 mL min
−1
at a column
temperature of 25 °C. For the determination of phytohor-
mones (abscisic acid (ABA), gibberellic acid (GA), and
indole-3-acetic acid (IAA)), 13 % acetonitrile (pH 4.98) was
used as the mobile phase.
Determination of contents of soluble sugars
The soluble sugar contents of plants were determined accord-
ing to the method of Rosa et al. (2004)withsomemodifica-
tions. For this purpose, the plant tissue was powdered with a
mortar. Soluble sugars were extracted from 0.7 g of powdered
tissue by homogenization in 2 mL of 80 % (v/v)ethanol.The
homogenate was heated in water bath at 75 °C for 10 min and
was centrifuged at 5000gfor 10 min. It was cooled and pre-
cipitate was reextracted using 2 mL of 80 % (v/v) ethanol and
centrifuged again. Under a hot air stream, the supernatant was
evaporated and the residue was suspended in distilled water.
The final material was subjected to the desalination procedure
and applied to determine individual (glucose, fructose, su-
crose, and maltose) soluble sugars.
Statistical analysis
All data presented are mean values. Each value was presented
as the mean± SE with a minimum of three experiments. Data
were subjected to a one-way analysis ANOVA procedure, and
significant differences among treatments were determined by
Duncan’s multiple range test (p<0.05). All statistical analyses
were conducted using SPSS version 20.0 (SPSS Inc./IBM
Corp.).
Results
MDA and ROS
UV-B radiation significantly increased malondialdehyde
(MDA) and H
2
O
2
contents and the rate of O
2
•
−
production
in lettuce seedlings over the control (Fig. 1). SNP pretreatment
did not significantly affect MDA content and the rate of O
2
•
−
production (Fig. 1a, b). But SNP pretreatment significantly
affects H
2
O
2
content (Fig. 1c). The combination of UV-B
and SNP induced an important decrease in MDA (24.5 %)
Environ Sci Pollut Res
and H
2
O
2
(20.5 %) contents and the rate of O
2
•
−
production
(31.8 %) compared with UV-B treatment alone.
Antioxidant enzyme activities
As shown in Fig. 2a, SOD activity in leaves increased mark-
edly when plants were subjected to UV-B. Application of
100 μM SNP alone had no statistically important effect on
SOD activity. On the other hand, SNP pretreatment further
activated activity SOD under UV-B radiation.
As compared with plants grown in control, POD activity
under UV-B radiation was remarkably higher (Fig. 2b).
Exogenously applied SNP further activated POD under UV-B
radiation.
Exposure to UV-B increased CAT activity as well. Compared
with the UV-B-only treatment, CAT activity under UV-B radia-
tion with SNP increased notably and significantly (Fig. 2c).
Similarly, APX activity in leaves increased under UV-B
radiation. SNP pretreatment significantly enhanced the APX
activity under UV-B stress (Fig. 2d).
A
C
MDA concentration (nmol g
-1
FW)
H
2
O
2
content (µmol g
-1
FW)
c
0
2
4
6
8
10
12
14
16
c
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
c
a
d
a
a
b
a
b
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
O
2-.
concentration (nmol g
-1
FW)
cc
a
b
Fig. 1 Effect of SNP
pretreatment on aMDA, bO
2
•
−
,
and cH
2
O
2
content in lettuce
seedlings exposed to 3.3 Wm
−2
UV-B for 18 h. Bars represent a
mean±standard error of three
independent experiments.
Different letters indicate
significant differences between
treatments (p<0.05)
Environ Sci Pollut Res
Photosynthetic pigments
Compared with the control, under UV-B radiation alone, Chl
a,Chlb, and Car contents were reduced by 27, 21, and 8 %,
respectively (Table 1). However, Chl a,Chlb, and Car con-
tents in leaves treated with SNP increased by 7, 20, and 1 %,
respectively. Under the combination of UV-B and SNP, Chl a,
Chl b, and Car contents were more than those in treatment
with UV-B alone.
Total phenolic concentration, antioxidant capacity,
and PAL gene expression
The changes in the total phenolic concentration and antioxi-
dant capacity in lettuce seedlings exposed to UV-B, SNP, and
UV-B + SNP were measured and shown in Table 1.Ascanbe
seen in Table 1, total phenolic concentrations and antioxidant
capacity were significantly affected by SNP, UV-B, and UV-
B+ SNP. UV-B alone and in combination with SNP promoted
A
SOD Activity (EU g
-1
FW)
CAT Activity (EU.g
-1
FW)
c
0
10
20
30
40
50
60
.
d
0
100
200
300
400
500
600
)
c
b
c
b
a
a
B
C D
0
10
20
30
40
50
60
70
80
90
POD Activity (EU.g
-1
FW)
0
2
4
6
8
10
12
14
16
18
APX Activity (EU.g
-1
FW)
cc
d
c
b
b
a
a
Fig. 2 Effect of SNP
pretreatment on aSOD, bPOD, c
CAT, dAPX, and activities in
lettuce seedlings exposed to
3.3 Wm
−2
UV-B for 18 h. Bars
represent a mean± standard error
of three independent experiments.
Different letters indicate
significant differences between
treatments (p<0.05)
Environ Sci Pollut Res
both total phenolic concentrations and antioxidant capacity
significantly (P<0.05); however, SNP alone had no statisti-
cally significant influence on these parameters compared to
the control. Total phenolic concentrations and antioxidant ca-
pacity in the combined treatment of UV-B and SNP were
about 20 and 13 % more than those in UV-B treatment alone,
respectively.
The effect of SNP, UV-B, and UV-B + SNP on the PAL
mRNA abundance was shown in Fig. 3.Whencomparedwith
control, SNP application caused a small increment in PAL (not
significant) but the applications of UV-B and UV-B+SNP
increased the PAL mRNA abundance significantly (p<0.05).
The most effective application on PAL expression was UV-
B+SNP.
Phytohormones
In order to investigate the changes of hormonal contents under
UV-B stress with SNP or not, the effects of SNP on the ABA,
GA, IAA, and salicylic acid (SA) contents of lettuce seedlings
were examined (Table 2). The hormone content of lettuce
seedlings was affected by UV-B and SNP treatments. The
results indicated that UV-B induced ABA and SA contents
of seedlings. Compared to control, ABA and SA contents of
lettuce seedlings were increased by 25.1 and 30.41 % under
UV-B stress, respectively (Table 2). SNP pretreatment de-
creased ABA and SA contents. Through SNP treatment,
ABA and SA contents were decreased by 16.5 and 18.5 %
in lettuce than only UV-B-treated plants. As shown in Table 2,
UV-B decreased GA and IAA contents in lettuce seedlings
compared to control. SNP increased GA and IAA contents
of lettuce seedlings under UV-B stress. Application of SNP
alone resulted in the significant increase of GA and IAA
contents.
Soluble sugars
Soluble sugar (glucose, fructose, and sucrose) concentrations
were significantly affected by UV-B and UV-B+SNP
(Table 3). Compared with control, SNP treatment alone had
no significant effect of soluble sugars. On the other hand, UV-
B enhanced the content of glucose, fructose, and sucrose con-
centrations by 52.2, 39.3, and 18.3 %, respectively. The com-
bination of SNP and UV-B led to an increase in glucose fruc-
tose and sucrose concentrations of about 69.1, 69.6, and
32.2 % respectively, even higher than that of the UV-B alone.
Discussion
Lettuce is one of the most consumed vegetables in the world
either fresh or produced. However, lettuce plants are also sen-
sitive to different adverse environmental conditions including
UV-B that provoke a significant reduction in growth and de-
velopment, and results of the current study further confirmed
Tabl e 1 Effect of SNP
pretreatment on
chlorophylls and
carotenoid content, total
phenolic concentrations,
and antioxidant capacity
in lettuce seedlings
exposed to 3.3 Wm
−2
UV-B for 18 h
Tot al ph eno lic
concentrations
(mg GAE g
−1
FW)
Antioxidant capacity
(μmol TEAC g
−1
FW)
Chl a
(mg/g fr wt)
Chl b
(mg/g fr wt)
Car
(mg/g fr wt)
Control 0.90± 0.04
c
37.9±2.27
c
2.77±0.08
ab
0.94±0.01
b
1.37±0.03
ab
100 μM SNP 0.94±0.02
c
40.8±1.21
c
2.97±0.04
a
1.13±0.02
a
1.39±0.05
ab
UV-B 1.35±0.05
b
51.0±1.8
5b
2.02±0.09
c
0.74±0.02
c
1.26±0.03
b
UV-B+ 100 μM SNP 1.62±0.03
a
58.7±1.13
a
2.58±0.06
b
0.95±0.06
b
1.43±0.04
a
Each value is the mean± standard error of three independent experiments. Different superscript letters indicate significant
differences between treatments (p<0.05)
mRNA abundance relative to actin
0
0.5
1
1.5
2
2.5
3
3.5
( LSPAL/LSACT)
b
b
a
a
Fig. 3 Effect of SNP pretreatment on PAL mRNA gene expression in
lettuce seedlings exposed to 3.3 Wm
−2
UV-B for 18 h. Bars represent a
mean±standard error of three independent experiments. Different letters
indicate significant differences between treatments (p<0.05)
Environ Sci Pollut Res
the negative effect of UV-B treatments on lettuce metabolism.
On the other hand, data presented in this study indicated that
SNP application through foliar spray protected lettuce seed-
lings from the damaging effects of UV-B stress.
Stress conditions cause the overproduction of ROS, which
damage lipid membranes and increase MDA contents
(Perveen et al. 2013). In the present study, UV-B stress not
only increased the levels of O
2
•
−
and H
2
O
2
, but it also in-
creased the MDA contents in lettuce seedlings. Pretreatment
with exogenous 100 μM SNP decreases the levels of O
2
•
−
,
H
2
O
2
, and MDA in high-light-stressed tall fescue leaves (Xu
et al. 2014). Similarly, in the present study, SNP pretreatment
reduced the MDA content under UV-B stress, which is in
accordance with the reduced levels of O
2
•
−
and H
2
O
2
in the
SNP-pretreated UV-B-stressed seedlings (Fig. 1). These re-
sults suggest that pretreatment with exogenous SNP alleviates
UV-B stress by reducing the accumulation of O
2
•
−
and H
2
O
2
and reducing MDA levels in lettuce seedlings.
To protect against oxidative stress, plants evolutionally de-
veloped enzymatic and non-enzymatic ROS scavenging sys-
tems. Non-enzymatic compounds include reduced forms of
ascorbate and glutathione, as well as tocopherol, flavonoids,
and alkaloids, etc. Enzymatic scavenging mechanism includes
SOD, POD, CAT, and APX, etc. Among antioxidant enzymes,
SOD detoxifies superoxide radicals (O
2
•
−
) by forming H
2
O
2
,
which is harmful to the chloroplast, nucleic acids, and proteins.
H
2
O
2
canbeeliminatedbyPOD,CAT,andAPX.Presentex-
periments demonstrated that UV-B stress increased remarkably
SOD, POD, CAT, and APX activities in lettuce leaves when it
was applied alone. Furthermore, it was determined that co-
application of SNP pretreatment and UV-B gave rise to more
increments in the activities of these enzymes compared with
using UV-B alone. This finding should not be so surprising
because of the fact that SNP can induce the expression of some
antioxidant genes and enhance the activities of antioxidant en-
zymessuchasSOD,POD,CAT,andAPX(Fanetal.2013;
Siddiqui et al. 2011). Several reports indicated that SNP appli-
cation resulted in the enhancement of antioxidant enzymes’
activity under various stresses (Siddiqui et al. 2011). Khan
et al. (2012) and Xu et al. (2014) showed that SNP treatment
increased antioxidant enzyme activities in mustard leaves and
tall fescue leaves under stress conditions. Santa-Cruz et al.
(2010) reported that SNP-enhanced antioxidant enzyme activ-
ities play an important role in UV-B tolerance.
It is well documented that the plants exposed to stressful
environments such as UV-B resulted in decreased chlorophyll
concentration thereby leading to overall retarded growth
(Zlatev et al. 2012). In the present study, lettuce seedlings
treated with UV-B exhibited a significant reduction in chloro-
phyll content (Table 1). Under UV-B condition, chlorophyll
content reduction might be due to instability of protein com-
plexes and destruction of chlorophyll by increased activity of
chlorophyll-degrading enzyme chlorophyllase. However, the
results presented here show that foliar application of 100 μM
SNP to lettuce seedlings led to a significant increase in Chl a,
Chl b, and Car concentration under UV-B stress. Tossi et al.
(2011) reported that exogenous 100 μM SNP significantly
increased the total chlorophylls content in maize under UV-
B stress, and Santa-Cruz et al. (2010)alsoreportedthatexog-
enous 1.2 mM SNP significantly increased the leaf chloro-
phyll concentration in soybean under UV-B stress, which fur-
ther supported the present results that exogenous SNP treat-
ment significantly increased Chl a,Chlb, and Car concentra-
tion in lettuce seedlings under UV-B stress. These results
Tabl e 2 Effect of SNP
pretreatment on some
phytohormones in lettuce
seedlings exposed to 3.3 Wm
−2
UV-B for 18 h
Plant hormone ABA (ng/μl) GA (ng/μl) IAA (ng/μl) SA (ng/μl)
Control 0.131± 0.02
ab
106.9±2.50
a
2.61±0.17
a
8.58±0.74
ab
100 μM SNP 0.118±0.01
b
112.0±2.54
a
2.87±0.20
a
7.68±0.80
b
UV-B 0.164±0.01
a
88.03±2.14
b
2.02±0.13
b
11.19±1.30
a
UV-B+ 100 μM SNP 0.137±0.01
ab
101.20 ± 1.73
ab
2.33±0.13
ab
9.13±0.63
ab
Each value is the mean± standard error of three independent experiments. Different superscript letters indicate
significant differences between treatments (p<0.05)
Tabl e 3 Effect of SNP
pretreatment on glucose, fructose,
and sucrose concentrations in
lettuce seedlings exposed to
3.3 Wm
−2
UV-B for 18 h
Soluble sugar Glucose (nmol g
−1
FW) Fructose (nmol g
−1
FW) Sucrose (nmol g
−1
FW)
Control 982± 56.0
b
94± 5.3
c
2926±71.6
c
100 μM SNP 1196 ± 96.7
b
110±6.0
c
3038±58.1
c
UV-B 1495±53.1
a
131± 5.6
b
3462±61.2
b
UV-B+ 100 μM SNP 1666±65.6
a
159± 4.7
a
3871±66.5
a
Each value is the mean± standard error of three independent experiments. Different superscript letters indicate
significant differences between treatments (p<0.05)
Environ Sci Pollut Res
suggested that exogenous SNP treatment could alleviate the
negative effect of UV-B stress that allows plants to increase
their tolerance to unfavorable conditions.
Reddy et al. (2004) have reported that higher plants exhibit
a unique capability to synthesize non-enzymatic secondary
metabolites including phenolic compounds. Phenolic com-
pounds have an antioxidative role in scavenging ROS. On
the other hand, the synthesis and release of phenolic com-
pounds are induced by various biotic and abiotic stress factors
(Oh et al. 2009). Table 1shows that total phenolic concentra-
tions were significantly increased under UV-B stress com-
pared to their corresponding controls. The foliar spraying of
SNP concentration resulted in significant increases in total
phenolic concentration compared to the control. Moreover,
lettuce leaves treated with an application of SNP under UV-
B stress showed significant increases in phenolic concentra-
tions compared to controls. In this regard, it can be speculated
that phenolic contents protect cells from potential oxidative
damage, increase the stability of cell membranes, and mitigate
UV-B stress injuries. Besides, the accumulation of phenolic
compounds and other antioxidants in response to abiotic stress
would be attributed to the activation of phenylalanine ammo-
nia lyase (PAL) (Rivero et al. 2001;Ohetal.2009). PALs,
which are involved in phenylpropanoid pathway and led to
accumulation of phenolic compounds in plants, were stimu-
lated by UV radiation (Caldwell and Britz 2006; Oh et al.
2009). Figure 3shows that an increased mRNA level of
PAL was observed in lettuce leaves in response to UV-B
stress. This response was consistent with the higher accumu-
lation of phenolic matters in lettuce leaves. The results obtain-
ed in this study clearly suggest that UV-B stress can activate
key genes involved in the biosynthesis of secondary metabo-
lites in lettuce. Our results have also shown that SNP and UV-
B applications increased the antioxidant capacity of lettuce,
and these results may be related with the increasing of pheno-
lic compounds. Thus, phenolic compounds and some second-
ary metabolites are largely responsible for antioxidant capac-
ity in plant tissues (Larson 1988).
Phytohormones play critical roles in regulating plant re-
sponses to stress (Yang et al. 2013). Under the effect of UV-
B, the endogenous growth hormones GA and IAA content
decreased, while ABA and SA content increased. The results
appeared that UV-B stress led to sharp changes in the balance
of endogenous hormones which associated with the accumu-
lation of ABA and SA and decrease in the level of GA and
IAA. These results are in a good agreement with those of Peng
and Zhou (2009) who showed that treatment of soybean with
UV-B caused changes in ABA, GA, and IAA; similar results
were obtained by Yang et al. (2004) working on tomato. GA
and IAA contents were decreased by UV-B radiation due to
photooxidation free radical damage, reinforcing harm from
free radical induced by UV-B stress. In addition to this, the
decrease of GA and IAA contents in lettuce seedlings under
UV-B stress is likely associated with its low UV-B tolerance.
On the other hand, synthesis of ABA and SAwere affected by
stress conditions. Li et al. (2010) reported that UV-B radiation
damage chlorophyll and cell membrane structure, cause a de-
creasing in Mg-ATPase activity of membranes and pH in chlo-
roplast, and this phenomenon may cause an increase in ABA
content. The increases in ABA and SA contents help to im-
prove the stress tolerance in plants. As seen from Table 2,
there were no significant increases in the amounts of GA
and IAA when SNP was applied alone. In contrast, the
amounts of ABA and SA were found to be significantly de-
creased in SNP-pretreated plants as compared with untreated
ones. Data presented in Table 2also clearly show that al-
though combined UV-B and SNP treatment resulted in note-
worthy increases in GA and IAA contents, it significantly
decreased ABA and SA contents, as compared with the UV-
B treatment alone. The similar findings were also shown in the
previous report of He et al. (2012) who demonstrated that SNP
increased in GA and IAA but decreased in ABA contents in
rye in response to Al stress. These results indicate that SNP
can ameliorate UV-B stress by increasing the secretion of GA
and IAA and decreasing of ABA and SA.
In the present study, low level of SNP application signifi-
cantly enhanced soluble sugar contents in lettuce leaves
(Table 3). Our results also showed that SNP induced the ac-
cumulation of glucose, fructose, and sucrose under UV-B
stress, which were much higher than under UV-B stress alone
in lettuce leaves. It is well known that soluble sugars have an
essential role in plant metabolism. They act as typical
osmoprotectants and stabilize cellular membranes, maintain
turgor. They are also signal molecules in sugar sensing and
signaling system. It has been also reported that there is a rela-
tion between sugar accumulation and ROS balance (Couee
et al. 2006). The present results also suggest that the protective
effect of ALA on UV-B stress might be related to its regulative
roles on soluble sugar levels in lettuce leaves.
In conclusion, the present study revealed that SNP pretreat-
ment alleviate the negative effect of UV-B radiation through
reducing MDA and ROS, improving antioxidant system, and
regulating hormonal balance.
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