Investigating physiological and biochemical

Study 3 
3.3: Physiological and biochemical characterization of linseed 
genotypes in response to NaCl stress 
3.3.1. Introduction 
The nature of the damage due to high salt concentrations on plants is not entirely 
clear. The integrity of cellular membranes, the activities of various enzymes, nutrient 
acquisition and function of photosynthetic apparatus are all known to be prone to the 
toxic effects of high salt stress. Salinity stress is known to affect various growth 
processes including photosynthesis, stomatal conductance, water relations, synthesis 
and transport of organic compounds (Ashraf, 2004) and ultimately growth of the plant 
is reduced. An important cause of damage might be reactive oxygen species (ROS) 
generated by salt stress. Plants subjected to salt stress display complex molecular 
responses including the production of stress proteins and compatible osmolytes (Zhu 
et al., 1997). Many of the osmolytes and stress proteins with unknown functions 
probably detoxify plants by scavenging ROS or prevent them from damaging cellular 
structures (Zhu, 2001). 
The inhibitory effects of salinity on plant growth are also attributed to specific ion 
toxicity, low external osmotic potential and nutrients deficiencies (Parida and Das, 
2005). Ion toxicity is caused by the replacement of K
+
by Na
+
in biochemical 
reactions and by the loss of function of proteins, as Na
+
and Cl
-
ions penetrate the 
hydration shells and interfere with the non-covalent interaction among the amino 
acids (Zhu, 2002). Sodium translocation from the leaves and lower leaf accumulation 
of Na
+
could result in the maintenance of higher K
+
/Na
+
ratios, which would be 
suitable for the metabolic processes occurring within the plants (Ashraf and Khanum, 
1997). Hence, the ability of plants to maintain a high cytosolic K
+
/Na
+
ratio is 
considered to be one of the important physiological mechanisms contributing to salt 
tolerance in many oat species (Chen et al., 2005; Akram et al., 2010).
Salinity poses several undesirable effects on several plant processes, leading to 
membrane disorganization, increase in reactive oxygen species (ROS) levels and 
metabolic toxicity (Hasegawa et al., 2000). High concentration of salts disturbs 
several biochemical processes and enzyme activities, particularly of CO
2
and nitrate 
assimilation. The enzyme carbonic anhydrase (CA) is found in abundance in the 
photosynthesizing tissues of both C
3
and C
4
plants and regulates the availability of 

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95 
CO
2
to ribulose bisphosphate carboxylase (rubisco) by catalyzing the reversible 
hydration of CO
2
(Badger and Price, 1994). Whereas nitrate reductase (NR) is the 
enzyme that catalyses the first step of nitrate assimilation, which appears to be a 
rate-limiting process in the acquisition of nitrogen (Flores et al., 2002). Limited 
uptake of CO
2
results in decreased carbon reduction by Calvin cycle, which in turn 
leads to non-availability of oxidized NADP
+
for acceptance of electrons during 
photosynthesis, stimulating the formation of ROS such as superoxide (O
2
-
), hydrogen 
peroxide (H
2
O
2
) and hydroxyle radicals (Peltzer et al., 2002). The toxic effects of O
2
-
and H
2
O
2
generate hydroxyl radicals and other destructive species such as lipid 
peroxides (Vaidyanathan et al., 2003). 
For ameliorating salt stress, plants have evolved complex mechanisms that 
contribute to the adaptation to both osmotic and oxidative stresses caused by salinity. 
The mechanisms that include osmotic adjustment is usually accomplished by either 
uptake of organic ions from external solution or by de novo synthesis of some 
compatible solutes (osmoprotectants) such as amino acids and soluble sugars which 
act as osmolytes (Shabala et al., 2000; Rontein et al., 2002; Ghoulam et al., 2002; 
Sakamoto and Murata, 2002; Ashraf and Harris, 2004). Osmoprotectants are neutral 
molecules that stabilize proteins and membranes against denaturation effect of high 
concentration of salts (Munns, 2002). Moreover, plant cell must adjust osmotic 
potential to prevent water losses, maintaining cell turgor under salt stress (Naidoo and 
Naidoo, 2001). 
To minimize the effect of oxidative stress, plant cell have evolved a complex 
antioxidant system, which is composed of antioxidant compounds (glutathione, 
ascorbate, β-carotene and α-tocopherol) as well as ROS scavenging enzymes such 
as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and 
peroxidase (POD) (Apel and Hirt, 2004). When ROS production suppresses the 
antioxidant system capacity, oxidative stress occurs, resulting in protein, DNA, 
damage and lipid peroxidation (Shalata and Neumann, 2001). These enzymes play 
significant roles in detoxifying ROS. SOD dismutases superoxide radical to H
2
O
2,. 
H
2
O
2 
reacts with various targets inducing damage to proteins and DNA and also cause 
lipid peroxidation. Thus CAT and POD are involved in converting H
2
O
2
into water 
and oxygen (Hussain et al., 2007). Ascorbate peroxidase (APX) is the most important 

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96 
peroxidase, catalyzing the reduction of H
2
O
2
to water using the reducing power of 
ascorbate. Glutathione reductase (GR) plays a crucial role in catalyzing the last and 
rate-limiting step of the Halliwell-Asada enzymatic pathway (Bray et al., 2000). 
Malondialdehyde (MDA) contents are considered as the general indicator of lipid 
peroxidation (Meloni et al., 2003; Wang and Zhou, 2006). Thus antioxidants and 
compatible solutes may provide a strategy to enhance salt tolerance in plants.
The present study was designed to: 
1. Investigate the effect of salt stress on some specific processes having functional 
significance in C-assimilation and nitrogen status of linseed under stress 
conditions. 
2. Elucidate the effects of salt stress on the activity of anti oxidative enzymes and 
lipid peroxidation in leaves. 
3. Assess the mechanism of osmotic adjustment in linseed under salt stress.