Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

nitrate as described by Pareek et al., (1995). Standard
protein markers (Sigma, USA) of 118, 79, 47, 33, and
25 kD each of 2 microgram were loaded in a separate
well. The polypeptides in each well of the gel were
characterized for their molecular weight using Gene
Tool Software with SYNGENE (UK) Gel
Documentation and imaging system.
Western detection
Western detection was carried out as suggested by
Towbin et al., (1979) and Singla and Grover (1994).
Stable proteins were resolved on 10% SDS-PAGE and
electroblotted on to nitrocellulose membrane. Blots
were blocked with blocking buffer (5% non-fat milk
protein dissolved in potassium bisulphite). Later blots
were probed with primary antibodies for 2h at room
temperature with a dilution of 1:25000 (Primary
antibodies were raised against rice Hsp 104). Antigen
and antibodies complex were then detected by antirabbit
horse radish peroxidase linked secondary
antibodies (dilution 1:1000). Subsequently the blots
were washed three times with 0.3% mixture of
potassium bisulphate and tween-20 (PBST) and then
in 0.1% PBST respectively. The blots were then
developed using diaminobenzidine solution
containing 1ml of 1% CoCl 2 and 0.1% H 2O 2. Prestained
molecular weight markers (Sigma,USA) were
employed to check the efficiency of protein transfer.
Metabolic changes and protein patterns in salinity 33
Biochemical estimations
Contents of proline, sucrose, total soluble sugars,
malondialdehyde (MDA) and activity of superoxidedismutase
(SOD) were estimated in NaCl stressed and
non-stressed seedlings as described earlier (Gehlot et
al., 1989 and 2003a). The methods used for total
sugars, sucrose and proline were of Mc Cready et al.,
(1950), Sadasivam and Manickam (1992) and Bates et
al., (1973) respectively. MDA contents were
determined using thiobarbutyric acid (TBA) following
the method of Heath and Packer (1968). MDA
contents were calculated using extinction coefficient
of 155 mM -1 cm -1 . The activity of superoxidedismutase
was measured according to the method of
Giannopolitis and Ries (1977).
Results
(i) Growth and metabolism
We evaluated responses of four cultivars (cvs. RT-46,
RT-54, RT-125 and RT-127) of Sesamum indicum to
low and high concentration of NaCl in terms of
seedling growth, alterations in the levels of
cytosolutes, membrane damage and activity of
superoxide scavenging enzyme –superoxidedismutase.
Table-1: Root and shoot length (cm) of Sesamum indicum seedlings under salinity (values with in parenthesis are percent
reduction over control).
Cultivars Root length Shoot length
Control 30 mM 50 mM Control 30 mM 50 mM
RT-46 4.7±0.16 4.0±0.06 2.7±0.12 3.3±0.10 2.3±0.22 1.2±0.10
(100) (15.0) (42.56) (100) (30.31) (57.28)
RT-54 3.2±0.26 3.0±0.88 2.0±0.21 3.2±0.39 2.4±0.16 1.5±0.07
(100) (6.25) (37.5) (100) (25.0) (53.13)
RT-125 2.6±0.58 2.0±0.09 1.4±0.09 2.3±0.37 1.3±0.20 0.78±0.16
(100) (23.00) (46.16) (100) (43.48) (66.09)
RT-127 5.2±0.07 4.9±0.07 3.4±0.12 3.2±0.16 2.5±0.16 1.6±0.12
(100) (5.77) (34.6) (100) (21.8) (50.00)
Values are mean of the ten seedlings which were in triplicate (± values are SD)