Investigating physiological and biochemical

2.3.2.2. Antioxidative defense system 
A complex antioxidative defense system comprising of enzymatic and 
non-enzymatic components is present in plants to counteract the destructive oxidative 
damage of reactive oxygen species (Prochazkova et al., 2001). Superoxide dismutases 
are the plant metalloenzymes responsible for the detoxification of superoxide ra dical 
and its conversion to H
2
O
2
(Mittler et al., 2004). Although H
2
O
2
by itself is less 
damaging but it can form even more toxic species so its level must be monitored. In 
leaf peroxisomes at relatively higher concentration H
2
O
2
is scavenged by catalase 
without any reducing power and thus provides plant with an energy efficient way. 
However, lower levels of H
2
O
2
are eliminated form chloroplast by ascorbate 
peroxidase along with other peroxidases with the help of various reductants like 
ascorbate and glutathione (Asada, 2006). APX, DHAR and GR are the main enzymes 
involved in the ascorbate-glutathione cycle. In the ascorbate-glutathione cycle 
(Halliwell-Foyer cycle) (Noctor and Foyer, 1998) APX uses ascorbate as electron 
donor to reduce H
2
O
2
to 
water. The monodehydroascorbate (MDHA) 
disproportionates spontaneously to ascorbate and dehydroascorbate or is regenerated 
to ascorbate by the NADPH dependent monodehydroascorbate reductase respectively. 

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46 
The re-reduction of dehydroascorbate to ascorbate is coupled to oxidation of 
glutathione by dehydroascorbate reductase (DHAR), which is regenerated by the 
NADPH dependent glutathione reductase (GR). Enzymes of this cycle are present in 
the cytosol, mitochondria, peroxisomes and in the stroma and thylakoid lumen of 
chloroplasts (Chew et al., 2003; Shingeoka et al., 2002).
Regarding defense against singlet oxygen in the thylakoid membranes, plants 
have evolved two strategies. The first is the regulation of the light-harvesting 
apparatus to diminish triplet chlorophyll production. The second is the rapid 
quenching of either singlet oxygen directly or indirectly preventing its formation by 
quenching the triplet chlorophyll production by membrane bound quenchers like 
carotenoids and tocopherols (Asada, 2006). Unfortunately, cells do not possess any 
enzymatic mechanisms for the detoxification of highly active hydroxyl radical and 
only rely on mechanisms that prevent its formation. These mechanisms include the 
preceding elimination of superoxide radical and H
2
O
2
and/or sequestering metal ions 
that catalyze the Haber-Weiss reaction with specific metal binding proteins (Hintze 
and Theil, 2006).
Therefore, salt tolerant plants, in addition to being able to regulate water and 
ionic relations, should also have an efficient antioxidative system for effective 
removal of the ROS (Rout and Shaw, 2001). Several works have provided evidence 
for an effective protector role of antioxidant enzymes against oxidative stress in 
diverse plant species (Mittler, 2002; Vaidyanathan et al., 2003; Jung, 2004). The 
effect of salinity (100 mM NaCl) and different nitrogen sources (NaNO
3
/ (NH
4
)
2
SO
4
) 
on the activity and spatial distribution of antioxidative enzymes (such as superoxide 
dismutase, guaiacol peroxidase, and catalase) was investigated in sunflower seedlings 
by Rios-Gonzalez et al. (2002). Their results indicated that salinity treated plants 
exhibited increased antioxidant enzyme activities. Moreover, these activities were 
comparatively higher in roots than in leaves as roots are the first to sense salinity and 
constitute the first line of adaptation reactions. Contrary to these findings the effects 
of 10 and 20% sea water studied in nutrient solutions in 30 day-old sunflower plants 

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47 
revealed that both APX and GR activities were significantly depressed at higher 
percentage of sea water. Moreover, a substantial increase in GR activity was exhibited 
in the leaves of plants grown in 10% sea water (Baccio et al., 2004). However, effect 
of long term soil salinity (ECe 5.4 and 10.6 dSm
-1
) in salt tolerant and moderately 
tolerant wheat cultivars revealed that salinity stress significantly increased 
thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), 
catalase (CAT) and glutathione reductase (GR) activity in both the genotypes and at 
all the stages (Sairum et al., 2002). Moreover, a higher activity of SOD, CAT and GR 
was recorded in tolerant cultivar compared with less tolerant cultivar. Results 
indicated that salinity tolerance of tolerant cultivar as manifested by lower decrease in 
biomass and grain yield was associated with higher antioxidant activity, and lower 
TBARS contents. Similar findings were reported again by Sairum et al. (2005) by 
using salt tolerant and susceptible wheat cultivars under higher salt stress. 
Later on, Mandhania et al. (2006) investigated the effect of salt stress on cell 
membrane damage, ion content and antioxidant enzymes in wheat seedlings of two 
cultivars salt tolerant and salt sensitive. 4 day old seedlings were irrigated with 0, 50 
and 100 mM NaCl. Observations recorded on the 3
rd
and 6
th
day after salt treatment 
revealed that the activities of catalase, ascorbate peroxidase and glutathione reductase 
increased with increase in salt stress in both the cultivars, however, superoxide 
dismutase activity declined. Anyhow, MDA contents were significantly increased 
indicating a high degree of membrane damage by salt stress. Similarly, the effects of 
salt stress on the activity of SOD, APX and GR enzymes studied by Stepein and 
Klobus (2005) in two wheat and two maize varieties cleared the role of these enzymes 
in defense mechanism. In the non-saline control plants, the antioxidant enzymes 
activities were significantly higher for maize than for wheat indicating that C
4
plants 
possess stronger defense mechanism as compared to C
3
plants. Adding salt to the 
nutrient solution significantly increased the level of SOD, APX and GR in leaves of 
both maize and wheat. In addition, lipid peroxidation analyses indicated an increase in 
TBARS contents in both plant species grown under salinity that corresponded to the 

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48 
damage that occurred in secondary oxidative stress. However, as a result of greater 
efficiency of antioxidant defense in maize, the TBARS quantities remained lower as 
compared to wheat plants.
The activity of antioxidant enzymes was also reported to increase under saline 
condition s in case of cotton (Meloni et al., 2003) and it was noted that salinity led to 
significant increase in SOD, POD and GR activities in salt tolerant cultivars but the 
activities remained unchanged in salt sensitive cultivars. Similarly, effect of salt stress 
on the activity of antioxidative enzymes and lipid peroxidation were also investigated 
in the leaves of two maize cultivars by Azevedo Neto et al. (2006). They reported that 
in the leaves of salt stressed plants, superoxide dismutase, ascorbate peroxidase, 
guaiacol peroxidase and glutathione reductase activities was more pronounced in the 
salt tolerant than in the salt sensitive genotypes. Salt stress had no significant effect on 
catalase activity in the salt tolerant, but it was reduced significantly in the salt 
sensitive genotypes.
It is thus apparent from forth going discussion that a combination of characters 
like higher antioxidant activity leading to lower oxidative stress, higher osmotic 
concentration and selective uptake of useful ions and prevention of over accumulation 
of toxic ions contribute to salinity tolerance in diverse crop species and using 
physiological and biochemical attributes as salt tolerance indicators is an indirect 
selection criteria whose success depends on the strong integration and relationship of 
such indicators with each other and with plant responses to salinity stress.