African Journal of Biotechnology Vol. 11(24), pp. 6476-6483, 22 March, 2012
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Salinity induced changes in cell membrane stability,
protein and RNA contents
Muhammad Jamil1*, Muhammad Ashraf2, Shafiq ur Rehman4,
Mushtaq Ahmad5 and Eui Shik Rha3*
1Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000,
2Department of Botany, University of Agriculture, Faisalabad, Pakistan.
3College of Agriculture and Life Sciences, Sunchon National University, Suncheon 540-742, Republic of Korea.
4Department of Plant Science, Kohat University of Science and Technology, Kohat 26000, Pakistan
5Department of Plant Science, Quid-i-Azam University, Islamabad, Pakistan
Accepted 8 February, 2012
Seedlings of sugar beet (Beta vulgarus L.) were used at salinity levels of 0 (control), 4.7, 9.4 and 14.1 dS
m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability,
protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and
roots decreased significantly with increasing salt concentration. Salinity significantly reduced leaf area
and relative water content while cell membrane injury increased with increasing salt concentration. Leaf
protein content decreased significantly and sodium dodecyl sulphate-polyacrylamide gel electrophresis
(SDS-PAGE) analysis showed significant change in protein profiles in salt treated samples, which
suggests that NaCl altered protein pattern. Salinity induced RNA degradation with increasing salt level.
Cell membrane stability exhibited negative correlation with fresh and dry weight, leaf area, leaf water
content and total protein content. There was also a significant positive correlation between cell
membrane injury and RNA degradation.
Key words: Salt stress, membrane injury, growth, RWC, protein activity, RNA, Beta vulgarus L.
Abiotic stress is a main factor in limiting plant growth and
food production in many regions of the world (Osakabe et
al., 2011; Jamil et al., 2010). In general, salinity limits
plant growth and productivity (Munns, 2002; Ashraf and
Foolad, 2007). Salt tolerance in plants is a complex
mechanism involving morphological, physiological and
biochemical processes. Water deficiency is one of the
most common example of salt stress (Tabaei-Aghdaei et
al., 2000) that results in malfunctioning of the cellular
membranes by increasing their ion leakage. Plasma
membrane may be the primary site of salt injury (Maas
and Nieman, 1978; Mansour, 1997) the harmful effect of
salinity on the plasma membrane is basically due to the
action of salt ions (Mansour, 1997).
*Corresponding author. E-mail: firstname.lastname@example.org,
It has well known fact that salinity decreased
performance of RNA machinery. Na+ initiated RNA
degradation in vitro but in vivo RNA stability depended on
the relative Amount of the ion accumulated (Rauser and
Hanson, 1966; Aspinall, 1986; Munns and Termaat,
1986). Therefore, the reduction in RNA content will
ultimately reduce the protein content, since RNA is
required for the process of protein synthesis through
transferring the amino acids into protein synthesis
centers (Udovenko et al., 1971).Sugar beet is a member
of the Cheno-podiaceae. Sugar beet is a salt-tolerant
crop that is common grown in saline soil. Sugar beet is
well established in the saline soil to tolerate at high salt
level (Bernstein, 1964) and soil water stress (Hills et al.,
1990). It has been reported that sugar yield of sugar beet
was not affected by salt stress up to an electrical
conductivity value of f 7dSm-1 (Roades and Loveday,
1990).The objective of this study was to determine the
effect of salinity on growth, cell membrane stability,
protein synthesis and RNA of sugar beet and whether cell
membrane injury was attributable to biological (fresh and
dry weights, leaf area) and biochemical changes such as
total protein content and RNA.
MATERIALS AND METHODS
Sugar beet seeds (Beta vulgaris L. cv Tianjin qing pielan) used in
this experiment were obtained from Institute of Soils and Fertilizers,
A green-house experiment was initiated to investigate the effect of
salt stress on vegetative growth, relative water content (RWC), cell
membrane stability (CMS), and protein and RNA contents.
Seedlings of sugar beet were grown in plastic pots in sand culture
irrigated with full strength Hoagland's nutrient solution (Hoagland
and Arnon, 1950). Plants were irrigated for 2 weeks with full
strength Hoagland's nutrient solution. Salt treatments in Hoagland’s
nutrient solution were applied two week days after the start of the
experiment. The NaCl treatments used were 0 (control), 4.7, 9.4
and 14.1 dS m-1 in full strength Hoagland's nutrient solution. The
experiment was designed in three replicates with average
temperature of 25/15°C for day/night and photoperiod for the
day/night cycle was 16/8 h. The plants were uprooted carefully after
30 days of treatments, washed with distilled water, and then the
fresh weight of plants, shoots and roots was recorded. Dry weights
were determined by drying plant samples in an oven at 80°C.
Leaf area and relative water contents (RWC)
Area meter (AM-200, ADC Bio Scientific Ltd., England) was used
for the measurement of individual plant leaves area. Relative water
content was determined by detaching fresh leaves from each repli-
cation and weighed instantly to note fresh weight (FW), followed by
dipping in DW for 12 h. The leaves were blotted dry to remove
surplus water and weighed to note fully turgid weight (TW). The
leaves were then dried in the oven at 80°C for 24 h to note the dry
weight (DW). The RWC were calculated by using the equation
developed by Turner (1986) that is RWC = [FW−DW]×100/[TW–
Measurement of cell membrane stability or cellular injury
Cell membrane stability (CMS) was investigated from each
treatment and replication by using fully expanded young plant
leaves. Twenty pieces (1 cm diameter) were cut from leaves and
sunken in DW contained in test tubes. The tubes were kept in
cooled chamber for 24 h at 10°C, followed by warming at 25°C and
measuring the electrical conductivity (C1) of the samples. The
leaves samples were then autoclave for 15 min at 121°C and the
electrical conductivity (C2) of the samples was measured again.
CMS was calculated by using the formula: (C1/C2)*100 where C
represents conductivity one and two.
Isolation of total cell RNA
RNA was extracted by the method of TRIzol Reagent (MRC, USA).
The material was ground to a fine powder with liquid nitrogen and
Jamil et al. 6477
homogenized in 1 ml (1 ml/50 - 100 mg tissue) of TRIzol solution.
The homogenized samples were incubated for 5 min at 15 to 30°C.
The material was extracted with 0.2 ml chloroform per 1 ml of
TRIzol. The two phases was separated by centrifugation for 15 min
at 12000´g at 2 to 8°C. RNA was precipitated with 500 µl
isopropanol overnight at 20°C and centrifuged for 10 min at
12000´g. The pellet was washed with 75% ethanol, resedimented
for 5 min at 10000 g and dried carefully. The RNA was re-dissolved
with diethylpyrocarbonate (DEPC) water to remove the supernatant.
Samples (10 µl) were subjected to electrophoresis on 1.1% agarose
gel in TAE buffer. RNA was stained with 0.5 µg ml −1 ethidium
bromide for 30 min.
Total soluble protein
Total leaf protein was determined by using the procedure described
by Santoni et al. (1994). The protein of the supernatant was
determined with SmartSpec™ 3000 Spectrophotometer according
to Bradford (1976) method using bovine gamma globulin as
SDS-PAGE analysis of protein
SDS polyacrylamide gel electrophoresis (PAGE) was used for
protein synthesis of control and NaCl treated plants by following the
procedure of Laemmli (1970). Fifty microgram (50 µg) protein with
sample buffer [62.5 mM Tris–HCl, pH 6.8, 20% (w/v) glycerol, 2%
(w/v) SDS, 5% (v/v) 2-mercaptoethanol and 0.01% (w/v)
bromophenol blue] was loaded in each lane of 12.5%
polyacrylamide gel. Electrophoresis was done at 500 V for 30 min
by using Bio-Rad, Mini Protein II electrophoresis system. The gel
was stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma) for
2 h and destained with 50% methanol and 10% acetic acid.
Densitometer (GS-710, Bio-Rad, USA) was used for photographs
and scanning. Standard proteins were as follows: phosphorylase b
(97.4 kDa); bovine serum albumin (66.0 kDa); ovalbumin (43.0
kDa); carbonic anhydrase (29.0 kDa); soyabean trypsin inhibitor
(20.1 kDa); lysozyme (14.3 kDa).
Analysis of variance was calculated by using the Microsoft Excel
version 5.0. Means values for different plant characteristics were
compared through least significance difference (LSD) test (Li, 1964).
Correlation between morphological parameters and biochemical
attributes were developed by using Minitab statistical software
Increased salt concentration caused a significant
reduction in the vegetative growth of sugar beet (Figure
1). Fresh and dry weights of sugar beet decreased
significantly with increasing salt concentration in the
growth medium (Figure 1). However, significant reduction
was observed at high salinity levels. Furthermore, root
was found to be more sensitive towards salt stress than
that of shoot as decrease in fresh weight was more
pronounced as compared to that of shoot at 9.4 and 14.1
dS m–1 (Figure 1A). A significant reduction in leaf area
was also observed with increasing salt concentration but
6478 Afr. J. Biotechnol.
Figure 1. Effect of various concentrations of salinity on fresh (A) and dry (B) weights of sugar
the magnitude of decrease was more pronounced at 9.4
and 14.1 dS m–1(Figure 2A).
Considerable differences were observed for RWC and
cell membrane injury (Figures 2B and 3). Salinity induced
significant decrease in RWC, but the pattern of decrease
varies at different salinity levels. Maximum reduction was
observed at high salinity levels as compared to the
control (Figure 2B). Increased salt concentration caused
an increased in cell membrane injury (Figure 3). Cellular
injury increased significantly at high salt concentrations,
but the enormity of increase was more intense at 9.4 and
14.1 dS m–1 as compared to the control (Figure 3).
There was a decrease in the amount of total soluble
protein content with the consequent raise in salinity level
(Figure 4). Decrease in total protein content was more at
high salt concentration (Figure 4). SDS-PAGE analysis
showed significant changes in protein profiles of in all
treated samples. It was observed that molecular weight
markedly decreased with increasing salt stress (Figure 5).
Biochemical analysis revealed that nuclear RNA
commenced disintegration after salt stress (Figure 6).
Fragmentation of RNA was clearly detected at higher
NaCl stress, but not in the control. With the increase of
NaCl concentration, more nuclear RNA degraded and the
degraded genomic RNA formed a smear in the gel and
the genomic RNA band became undefined. The RNA
band intensity also increased with the increased in salt
concentration (Figure 6).
There was a significant negative correlation between
cellular injury and growth parameters (Table 1). Corre-
lation also revealed a strong (R2 = 0.99, P = 0.001)
significant negative relationship between cellular injury
and leaf area. Table 1 also shows a weak (R2 = 0.95, P =
0.047) significant negative relationship between cellular
Jamil et al. 6479
Figure 2. Effect of various concentrations of salinity on leaf area (A) and relative water content
(RWC)(B) of sugar beet.
injury and fresh plant weight. Cellular injury was also
significantly (P=0.05) and negatively correlated with RWC
(R2 = 0.95) and with total protein content (R2 = 0.95).
Cellular injury and RNA also exhibited positive (R2 = 0.95)
correlation and the value was also significant (P=0.05).
Decrease in plant growth under different salinity concen-
tration has been reported in various plants by many
scientists (Alpaslan and Gunes, 2001; Greenway and
Munns, 1980). Salinity significantly reduced fresh and dry
weights of plants, shoots and roots and leaf area (Figures
1 and 2A). It has been documented that the effect of salt
stress on leaf area was more pronounced than on dry
weight because salt accumulation in the shoot occurs
through transpiration stream, which is highest in old
leaves (Greenway and Munns, 1980). Decrease in plant
growth under saline soil condition is a common process
in mesophytes (Ashraf and Harris, 2004), but such
decrease occurs differently in different plant parts. For
example in the present study, fresh root weight was
affected more than fresh shoot weight (Figure 2). Our
results are similar in line with the results earlier reported
by Jamil et al. (2005). They investigated that salinity
inhibited the growth of shoot more than root in Brassica
Our results indicate continue increase in cellular
membrane injury and decrease in RWC with increasing
salt concentration (Figures 3 and 2B). Cellular membrane
injury exhibited a negative correlation with fresh weight of
6480 Afr. J. Biotechnol.
0 4.7 9.4 14.1
Salinity (dS m-1)
Cell membrane injury (%)
Figure 3. Effect of various concentrations of salinity on cell membrane stability (CMS) of sugar
0 4.79.4 14.1
Salinity (dS m-1)
Protein Content (mg g-1 FW)
Figure 4. Effect of various concentrations of salinity on total protein content of sugar
Jamil et al. 6481
Salinity (dS m-1)
M.W (kDa) M C 4.7 9.4 14.1
leaf as analyzed by SDS-PAGE. Lanes C and 4.7, 9.4 and 14.1 represent proteins extracted from
the control and NaCl treated plants after 30 days of treatment and lane M represents molecular
Figure 5. Effect of various concentrations of salinity on polypeptide patterns of total protein from
plants, roots, shoots, leaf area, and relative water
contents (Table 1): the parameters that are equally
affected by salt stress (Munns, 2002). However, water
deficiency one of the most common examples of salt
stress (Tabaei-Aghdaei et al., 2000) results in the mal-
functioning of the cellular membranes by increasing their
ion leakage. The deleterious effect of salinity on the
plasma membrane is essentially due to the action of salt
ions (Mansour, 1997). It has been also reported that
salinity induced decrease in RWC (Gadallah, 1999) and
cell membrane stability (Bhattacharjee and Mukherjee,
Specific expression of stress proteins is an important
adaptive manifestation in maintaining the integrity, native
configuration and topology of cellular membranes
components to ensure their normal functioning under
salinity stress (Wahid et al., 2007). The decrease in
protein content and protein molecular weights in sugar
beet suggests that salinity exposure affect protein
activities in this plant (Figures 4 and 5). A significant
negative relation was found between cell membrane
stability and total protein content (Table 1). Changes in
the expression of proteins occur due to stress, yet it is
probable that only some of these proteins are directly
involved in stress tolerance. It is possible that in some
cases the synthesis of a protein indicates sensitivity to a
stressor rather than being part of a tolerance mechanism.
Ashraf and Waheed (1993) reported that leaf soluble
proteins decreased due to salt stress in all lentil lines,
irrespective of their salt tolerance. It has been well
documented that salinity decreased protein contents of
leaves in glycophytes (Alamgir and Ali, 1999; Gadallah,
1999; Wang and Nil, 2000). In case of Rhizobium, certain
outer membrane proteins of molecular weight 22, 38, 40,
42, 62 and 68 kDa noticeably decreases with increasing
NaCl levels (Unni and Rao, 2001).
Salinity induced RNA degradation with increasing salt
stress (Figure 6). Na+ initiated RNA degradation in vitro
but in vivo, RNA stability depended on the relative
amount of the ion accumulated (Rauser and Hanson,
1966; Aspinall, 1986; Munns and Termaat, 1986). RNA
exhibited a negative relationship with total protein content
and positive relation with cell membrane stability (Table
1). The reduction in RNA content will ultimately reduce
the protein content, since RNA is required for the process
of protein synthesis through transferring the amino acids
into protein synthesis centers (Udovenko et al., 1971).
Structural changes of nuclei caused by salt stress have
6482 Afr. J. Biotechnol.
Salinity (dS m-1)
0.0 4.7 9.4 14.1
Fig. 6. Effect of various concentration of salinity on RNA of sugar beet. Histograms
represent the relative intensity/mm2 of the RNA bands.
Table 1. Relationships among cell membrane injury and growth, RWC, total protein content (TPC) and RNA (Band intensity/mm2) of
sugar beet under various level of salt concentration.
Parameter FPW FRW FSW DPW DRW
FSW 0.993** 0.959*
DPW 0.952* 0.993** 0.981*
0.963* 0.986* 0.988* 0.999**
DSW 0.969* 0.981* 0.980* 0.980* 0.979*
LA 0.951* 0.985* 0.981* 0.998** 0.999**
CMS -0.953* -0.977* -0.982* -0.995** -0.997**
RWC 0.975* 0.968* 0.980* 0.969* 0.970*
TPC 0.838 ns 0.979* 0.894 ns 0.965* 0.951*
RNA - 0.864 ns -0.982* -0.917 ns -0.977* -0.967*
**, * Significant at P = 0.01 and P = 0.05, respectively; ns = Non-significant. FPW; Fresh plant weight, FRW; fresh root weight, FSW; fresh
shoot weight, DPW; dry plant weight, DRW; dry root weight, DSW; dry root weight, LA; leaf area, CMS; cell membrane stability, RWC;
relative water content, TPC; total protein content.
0 4.79.4 14.1
Salinity (dS m-1)
Figure 6. Effect of various concentration of salinity on RNA of sugar beet. Histograms
represent the relative intensity/mm2 of the RNA bands.
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