r ^

V

Physiological and Biochemical Studies of NaCI-Salinity Stress

in Crop Plants

Chapter 5

DISCUSSION

DISCUSSION

Effect of NaCl-salinity stress on growth of plants

Salinity (NaCi) stress can cause ionic toxicity and imbalance, membrane damage,

reduced uptake of CO2 as a result of stomatal closure, decreased hydrolytic enzyme

activity and increased lipid peroxidation level, it may stimulate formation of reactive

oxygen species (ROS) such as superoxide (O2'), hydrogen peroxide (H2O2), hydroxy!

radicals (OH) and etc.

The decrease in the fresh weight and dry weight as observed in the results showed that

the effect of salt stress on the performance and growth are mediated through decrease

in stimulating conduction and photosynthesis. This is attributed to the rate of CO2

assimilation which is generally reduced in response to salinity and this reduction is

partially due to reduced stomatal conductance and due to the direct effects of NaCl on

the photosynthetic apparatus independently of stomatal closure has been reported for

several plant species, both halophytes and non-halophytes (Seeman' and Critchley

1985, Allarcon et al. 1994, Torrcellas 1995, Hajar 1,996, Steduto et al. 2000, Garg et

al 2001, Garg and Singla 2004). It is known that the degree to which the growth is

reduced by salinity differs greatly with species and than cultivars within a species

(Khan and Panda 2002b). Fresh and dry weight though decreased with an increase in

the cultivar Lunishree in some of the concentrations showing a modulatory role

(Rascio et al. 1988). The growth of Pancratium seedlings in terms of fresh weight of

the shoot was reduced by salt stress (Khedr et al. 2003). Reduction of plant growth

and dry-matter accumulation by salinity has been reported in several important grain

legumes, including Phaseolus vulgaris (Delgado et al. 1994). Decrease in fresh and

dry weight has also been reported in rice roots (Demiral and Tukan 2005). The salt

effects on shoot dry weight were more pronounced than on root dry weigh, thereby

increasing the root / shoot ratio. A decrease in shoot dry matter accompanied by a

decline in root dry matter is a normal growth phenomenon (Hawkins and Lewis 1993,

Crammer 1994). Shoot growth is a complex process and several factors other than

reduced root growth are involved. According to Cheeseman (1988), salinity stress

imposes additional energy requirements on plant cells and diverts metabolic carbon to

storage pools so that less carbon is available for growth. Depletion in plant growth

76

(FW and DW) under saline stress is attributed to decreased water uptake followed by

limited hydrolysis of food reserves from storage tissue, as well as due to impaired

translocation of food reserves from storage tissue to the developing embryo axis

(Olmos et al. 1994, Meneguzzo et al. 1999, Misra and Gupta 2006). Reduction of

plant grovrth and dry-matter accumulation by salinity has been reported in several

important grain legumes, including Phaseolns vulgaris (Delgado et al. 1994). The

shoot is more sensitive to salinity than the root, as previously reported (Cordovilla et

al. 1994, Soussie/a/. 1998).

Plant responses to salinity stress and thereby water stress include morphological and

biochemical changes that lead first to acclimation and later, as water stress become

more severe, to fiinctional damage and the loss of plant parts (Chaves et al. 2002).

During the acclimation phase, water stress typically results in slower growth rates

because of the inhibition of cell expansion, the reduction in carbon assimilation

(Osorio et al. 1998) and the resultant effect on carbon partitioning (Hsiao and Xu

2000). The ability of bean Orfeo to retain water under drought appeared to result in

less photoinhibition compared with bean Arroz. Furthermore, there appears to be an

inherent difference between the susceptibility-^bf these varieties to photoinhibition,

even in well-watered conditions. Thus, the leaves of bean Orfeo seem generally to be

more resistant to stress, not only exhibiting an increased ability to retain water but a

higher photosynthetic capacity under high light conditions and an increased resistance

to photoinhibition. This was confirmed by contrasting levels of anthocyanin

accumulation, indicative of acclimation to stress, and MDA, indicative of membrane

damage by stress, in the two varieties (Lizana et al. 2006).

Effect of NaCI-salinity stress on photosynthetic pigments in leaves

Plant biomass production depends on the accumulation of carbon products through

photosynthesis, but elevated salinity can adversely affect photosynthesis (Munns and

Termaat 1986, Macler 1988). Decreased chlorophyll and carotenoid content were

observed in the resuhs under NaCl stress. Photosynthetic capacity of many plant

species is reduced in the presence of salinity, which is associated with stomatal

closure (Seemann and Critchley 1985, Delfine et al. 1998), increased mesophyll

resistance for CO2 diffusion (Delfine et al. 1998), reduced efficiency of Rubisco for

carbon fixation (Delfine et al. 1998), damage to photosynthestic systems by excessive

77

energy (Brugnoii and Bjorkman 1992) and structural disorganization (Flowers et al.

1985, Delfine et al. 1998, Romero-Aranda 1998, Gandul-Rojas 2004). In addition to

reduced photosynthesis, reduced growth of glycophytes exposed to salinity may be

the result of accelerated respiration and relatively higher photorespiration, which

leads to faster consumption of photosynthates that are otherwise used to support

growth (Waisel 1972, Burchett et al. 1989, Gersani et al. 1993, Singh and Singh

1999a). Bahaji et al. (2002) verified the effect of salinity on chlorophyll content.

Although water content (WC) is a very sensitive parameter of plant development,

chlorophyll formation is impaired at slightly more severe stresses (Hsiao 1973).

Judging from the changes in WC and chlorophyll content, salinity is probably a more

severe stress than the pure osmotic and thus chlorophyll decrease can be attributed to

the other parameters of salinity and not to osmotic stress. Bahaji et al. (2002)

supported that most of the saline-related responses in rice seedlings were due to

osmotic stress and not due to the ionic component of salinity.

Carotenoids are a group of isoprenoid pigments which are widely distributed in

nature. Carotenoids are synthesized by all photosynthetic organisms and some non-

photosynthetic bacteria and fiingi. Carotenoids protect the photosynthetic apparatus

from photo-oxidation and represent structural components of light-harvesting antenna

and reaction-center complexes. Carotenoids confer their colour to many flowers and

fruits, contributing substantially to plant-animal communication (Cunningham and

Gantt 1998, Hirschberg 2001, Fraser and Bramley 2004, Al-Babili et al. 2006). In

addition, some 9-cisepoxycarotenoids serve as precursors of the phytohormone

abscisic acid. That evergreen leaves are able to adapt to a wide range of

environmental conditions must be due, in part, to high antioxidant and xanthophyll

cycle pools (Garcya-Plazaola et al. 2000, 2003). In parallel with the synthesis of

photoprotective compounds, leaves of this species also accumulate red (retro)-

carotenoids during stress conditions (Hormaetxe et al. 2004, 2005). Decreased

carotenoid content were observed in the leaf under salt stress. There is an evolutionary

convergence to accumulate red compounds (mainly anthocyanins, but also

retrocarotenoids and betacyanins) in photosynthetic organs under unfavourable

conditions such as suboptimal temperatures, pathogen attacks, nutrient deficiencies, or

ultraviolet radiation (Steyn et al. 2002). Despite the wide distribution of this

characteristic among higher plants, there is still great controversy regarding its

78

photoprotective function. Most studies point to two main hypotheses: (i) a potential

role as passive light fihers that would reduce light intercepted by chlorophyll

(Chalker-Scott 1999, Feild et al. 2001, Close and Beadle 2003, Manetas et al. 2003,

Neil and Gould 2003, Williams et al. 2003) and (ii) the protection from reactive

oxygen species (Rice-Evans et al. 1997, Neil et al 2002a, Steyn et al. 2002).

Carotenoids can effectively quench the excited triplet state of chlorophyll and 'O2

(Knox and Dodge 1985). Lowered amount of carotenoids would lead to increased

formation of O2. Unavoidably, this directly leads to increased peroxidation reaction,

which is indicated by an increased content of MDA.

Effect of NaCI-salinity stress on Na* and K" ions compositions in plants

Salinization of irrigated lands is an increasing threat to agriculture (Epstein 1961,

Tanji 1990). In saline soils Na^ is the principal toxic ion, the concentrations of which

often exceed 25 mM (Greenway and Munns 1980). Many cellular activities are

sensitive to Na^ inhibition (Greenway and Munns 1980). Thus, maintaining a low

cytosolic Na^ concentration is important for many plants growing in NaCl-affected

environments. Plant cells maintain a low cytosolic Na^ concentration through Na^

exclusion, extrusion, or compartmentation (Niu et al. 1995). Compartmentation of

Na^ into the vacuole is likely through the action of Na*-H* antiporters in the tonoplast

(Barkla and Blumwald 1991). Na' extrusion via Na^-ATPase contributes the major

portion of NaCl tolerance in the yeast Saccharomyces cerevisiae (Haro et al. 1991). In

plants no Na^ pump activity has been detected, and Na^ extrusion is thus likely

achieved through Na^-H^ antiporters on the plasma membrane (DuPont 1992).

Whenever in a saline environment the passive Na* flux into the cell (the apparent

permeability coefficient PNa+ of the plasma membrane of marine prototrophs is in the

order of 0.05-3 x 10' m s"', increases the cytoplasmic Na^ concentration above a

critical level, Na^ re-export into the environment is initiated (Na^-homeostasis). One

obvious possibility of Na^ retranslocation is the coupling to inverse H*- gradients

created by H'-ATPases. In many plants, a large component of tolerance to long-term

exposure to Na" can be attributed to the ability of plants to exclude Na^ from the

shoot (Munns 2002). The first step in the movement of Na" from the soil solution to

the shoot is the initial entry of Na^ into the cells of the root epidermis and cortex. This

is termed (unidirectional) Na' influx, and it is distinct from net influx (or

79

accumulation), which is the end result of the processes of both influx and efflux. It is

proposed that the initial influx step is a key determinant of overall shoot Na^

accumulation (Schubert and Lauchli 1990, Davenport and Tester 2000). Na^

exclusion requires restricting Na^ uptake at the plasma membrane. The molecular

mechanisms of Na* uptake are poorly understood (Niu et al. 1995). Physiological

studies suggest that Na* influx occurs through the mechanism 2 (low-affinity)

potassium-uptake system (Rains and Epstein 1965, 1967). Cloned potassium

channels, which are presumed to function in low-affinity potassium uptake, however,

are a very selective against Na" (Schroeder et al. 1994). It is possible that other

potassium channels exist that are less selective between K* and Na^. Schachtman et

al. (1991) have suggested that Na^ entry into plant cells may be via outward-

rectifying cation channels. Under saline conditions Na^ depolarizes the plasma

membrane, which increases the open probability of outward-rectifying cation

channels, thereby allowing Na* influx to occur down its steep electrochemical

gradient. More recently, Rubio et al. (1995) reported that the high-affinity potassium

transporter HKTl (Schachtman and Schroeder 1994) from wheat contributes to Na^

influx because it functions as a K^-Na* symporter. It was demonstrated that at

physiologically toxic Na^ concentrations, high-affinity potassium uptake through

HKTl was blocked and low-affinity Na"" uptake occurred. Therefore, mechanism 1

(high-affinity) potassium-uptake systems may also mediate Na^ influx.

Analysis of the basis for salinity toxicity has of en shown specific toxic effects of Na^

ions (Kingsbury and Epstein 1986, Munns 1993). Excessive Na^ influx results in toxic

levels of Na^ building up in the cytoplasm, bringing about a range of detrimental

cellular effects (Volkmar et al. 1999, Singh and Singh 1999b, Munns 2002, Tester

and Davenport 2003, Murthy and Tester 2006). Roots, stems and leaves showed

increased Na^ and decreased K^ ions content in the rice and greengram under salt

stress. The increase in the Na^ ion content and decrease in K^ ion uptake which

disturbed ionic imbalance as observed in most species exposed to salt stress (Al

Zahrani and Hajar 1998, Jafari 1998, Heuer 2003). A linear increase in the Na"

. concentration in the shoots (Asch et al. 1999) which, in plants treated with 50 mmol/L

NaCl rose up to 8-fold with respect to non-saline controls. The result shows that there

is lesser uptake of Na* and higher uptake of K' in Lunishree than in Begioibitchi. Due

to high uptake and accumulation of Na"" and antagonastically low uptake,

80

translocation and accumulation of K^ and also enhanced K^ efflux under salt stress

could suppress growth by decreasing the capacity of osmotic adjustment and turgor

maintenance or by inhibiting metabolic activities (Leigh and Wyn Jones 1984, He and

Cramer 1993). The diminution of K^ concentration in tissue may also be due to direct

competition between K* and Na^ at plasmalemma (Burgos et al. 1993), inhibition of

Na^ on K^ transport process in xylem tissues and / or Na" induced K* efflux from the

roots. The results shows that Limishree leaves have lower accumulation of Na" as

compared to Begunbiichi showing that Lunishree is capable of avoiding Na^ toxicity

by better K^ levels in the tissue (Qadar 1991, Pandey and Srivastava 1991,

Sreenivasulu et al. 2000, Panda and Khan 2003, Racagni et al. 2003/4, Mandhania et

al. 2006).

Because, the K^ requirement applies to every cell in muhicellular organisms, after

entering a plant, K^ has to be transported to distant organs through the xylem. K*

moves from the root symplast to the xylem sap and from this to the apoplastic space

outside the bundle sheath, a process that involves many types of cells. Although, in

most cells, the cytoplasmic K" concentrations are quite similar in non stressing

conditions, around 100 mM NaCl (Walker et al. 1996, Cuin et al. 2003), the external

K^ concentrations and pH values to which root and internal cells are exposed are

considerably variable (Leigh and Wyn Jones 1984). Salinity affects the elongation

zone and mature zone, and epidermis and mesophyll cells, differently in their K* and

Na^ relationships. At the same time, the basic distribution of Na^, K , and CI" between

epidermis and mesophyll was not affected by stress, with concentrations being either

non-significantly different or significantly higher in the epidermis than the mesophyll

(Fricke et al. 2006). During the first hours of stress, K^ accumulated substantially in

all tissues, probably as a result of increased import via the phloem (Wolf and Jeschke,

1987). By contrast, Na^ accumulated significantly only in epidermal cells of the

elongation zone. As stress continued, between 20 h and 72 h, Na^. K^ ratios were

affected differentially in epidermis and mesophyll. This was due primarily to changed

K" relationships of tissues. Whereas K^ increased significantly in mesophyll cells, it

started to decrease significantly in epidermal cells, particularly in the elongation zone

(Fricke et al. 2006). It appears that transport properties for Na"" and K^ differ between

leaf tissues and change with the cell developmental stage. Na" increasingly replaced K

as the main inorganic cation counter balancing CI", yet osmolarity hardly changed

81

between 20 h and 72 h of stress. This suggests that growing leaf cells are able to sense

turgor or osmolarity and use this information to control solute transport in a similar

way to roots (Tyerman et al. 1999, Fricke et al. 2006). Osmotic adjustment at the

physiological level, is an adaptive mechanism involved in drought and salinity

tolerance, which permits the maintenance of turgor under conditions of water deficit,

as it can counteract the effects of a rapid decline in leaf water potential (Hsiao et al.

1973, Cutler and Rains 1978). The ability to maintain low Na^ and high K^

concentrations in leaves, is correlated with salt tolerance (Francois et al. 1986,

Gorham etal. 1987, Shah et al. 1987, Maas and Grieve 1990, Dvorjak et al. 1994,

Munns and James 2003, Poustini and Siosemardeh 2004, Garthwaite et al. 2005,

Colmer et al. 2006). There is little genetic variation in CI" accumulation in leaves

within cultivated wheat (Gorham 1990, Husain et al. 2004). However, considerable

variation exists in some wild species in the Triticeae in capacity to restrict the rate of

Cr accumulation in leaves (Garthwaite et al. 2005). The NaCl-hypersensitive mutant

of Arabidopsts thaliana, sosl, is defective in high-affinity potassium uptake (Wu et al.

1996). NaCl-stressed sosl seedlings contain less potassium than the wild type. Ding

and Zhu (1997) reported that sosl also contains less Na^ when treated with NaCl and

suggested that the reduced Na"" accumulation is due to a decreased Na^ influx into sosl

seedlings. Thus, the SOSZ gene is probably also involved in Na" uptake into plant

cells. Lin and Kao (2001b) have shown that increasing concentrations of NaCl from

50 to 150 mM progressively increased Na"" and CI' levels in roots of rice seedlings.

When in growing in saline soils, roots also encounter osmotic stress resulting from

salt concentration in the soil that results in lowered water potential and consequence

loss of cell turgor in roots (Tsai et al. 2004). Increasing concentrations of NaCl from

50 to 150 mM progressively increased both Na" and CI' levels in roots of rice

seedlings (Lin and Kao 2001b).

Plants vary in their ability to tolerate salt stress. This is particularly problematic for

many crop plants, which often fail to tolerate soil salinization caused by irrigation.

Determining the mechanism of salt tolerance requires an understanding of the

transport phenomena that prevent the toxic accumulation of salts in plant cells. The

salt tolerant cultivar Lunishree showed lower Na* accumulation as compared to salt

sensitive cultivar Begioibitchi. It has been proposed that the major deleterious effects

of high salinity are caused by Na" accumulation in the cytoplasm (Brady et al. 1984,

82

Gibson et al. 1987, Flowers and Yeo 1988). Plant cells must maintain low

cytoplasmic Na^ if they are going to be tolerant of salt stress. To prevent the

accumulation of Na^, a plant may limit its uptake, sequester it in a vacuole, or remove

it via the plasma membrane. Influx of Na^ occurs by a mechanism that is not fully

understood (Niu et al. 1995). In most cases, particularly at moderate to high salinities,

there is a strong driving force for Na" to enter the cell. K^ competes with Na^ entry,

which suggests that the two share a pathway (Schachtman et al. 1991, Whittington

and Smith 1992). Salt tolerance may be enhanced by decreasing the Fwa (permeability

of the plasma membrane to sodium), but since the PH (permeability of the plasma

membrane to P\\) cannot be reduced to zero, removal from the cytoplasm is also

required. The plant cell has two options for Na^ removal. Sequestration in a vacuole is

a useful option when tissues are expendable. In the above-ground organs of terrestrial

plants, accumulation in the vacuole is accomplished by the action of a tonoplast

Na"/H' antiport (Blumwald and Poole 1987, Gabarino and DuPont 1988). Another

option is direct removal from the cell across the plasma membrane. Although this may

not be feasible for above-ground organs such as leaves, recent reports suggest that

plasma membrane Na^ efflux may contribute to the salt tolerance of root cells in

higher plants. In plasma membrane vesicles isolated from tomato roots, Wilson and

Shannon (1995) observed Na^ efflux driven by the establishment of a pH gradient

across the vesicles, this gradient was not sensitive to amiloride. Allen et al. (1995)

showed an amiloride-sensitive Na^ / H^ antiport in plasma membrane vesicles isolated

from wheat roots. Aquatic plants are able to use the efflux of Na^ through the plasma

membrane to minimize the irreversible accumulation of Na* in the cytoplasm, since

this efflux results in the removal of the ions to the aqueous medium surrounding the

plant. Kiegle et al. (1996) suggested that the plasma membrane efflux is important in

conveying salt tolerance to C. longifolia and that this efflux is up-regulated by saline

culture and discussed possible mechanisms for Na^ transport across the plasma

membrane and the thermodynamic driving forces behind them. A major part of the

sensitivity of glycophytic plants to salinity may result from Na ^ replacing K " within

the cell, particularly the cytosol (Maathius and Amtmann 1999). The importance of

effluxing Na* from the cytosol was indicated recently by the demonstration that plants

over expressing a vacuolar H* / Na * exchanger had improved salt tolerance when

compared with wild type plants (Apse et al. 1999). According to Blumwald et al.

(2000) the decrease of K" concentration due to NaCl may be attributed to high

83

external Na" concentration. Since the two ions have similar hydrated ionic radii,

transport proteins find difficult to discriminate them, making easy the Na^ entry to the

cell through low-affinity and high affinity K^ carriers, excluding K^ uptake (Maathuis

and Amtmann 1999). Furthermore, translocation of Na^ to the leaves leads to a

displacement of the apoplastic Ca * causing depolarization of membrane systems

(Nakamura et al. 1992). Subsequently, the ability of membranes to selectively absorb

some ions is impaired and ion imbalance is inevitable.

Effect of NaCl-salinity stress on water relations in plants

Both salt and water stresses have a common osmotic effect that induces the plants to

decrease their internal water potential to avoid desiccation. Nevertheless, salinity

provokes ionic stresses other than osmotic stress, therefore physiological mechanisms

that plants use to respond to salinity or drought may partly differ on a case-by-case

basis (Erdei et al. 1990, Lefevre et al. 2001, Hernandez and Almansa 2002). The

results showed a decrease in the relative water content (RWC) in both cultivars of rice

and greengram. In the roots the water content decreased in response to salt stress

(Khedr et al. 2003). Working with rice, Srivalli et al. (2003) reported that RWC

decreased under water stress, however, the water status was recovered after watering

the plants. It is known that salt stress affects both leaf growth and water status

(Allarcon et al. 1994, Torrecilas et al. 1995, Hernandez et al. 1999, 2000). The

osmotic effect resulting from soil salinity may cause disturbances in the water balance

of the plant, reducing turgor and inhibiting growth as well as provoking stomatal

closure and reducing photosynthesis (Poljakoff-Mayber 1982, Sanchez-Bianco et al.

1991, Allarcon et al. 1993, Jose et al. 2002). Plants respond by means of osmotic

adjustment, normally by increasing the concentrations of Na" and CI" in their tissues,

although such accumulation of inorganic ions may produce important toxic effects

and cell damage (Flowers and Yeo 1986) and inactivate both photosynthetic and

respiratory electron transport (Allahkhverdiev et al. 1999). This limited osmotic

adjustment was not sufficient to avoid water stress in the treated plants, and thus there

was a decrease in the roots water content after 24h of sak stress. Wilson et al. (1989)

indicated that osmotic adjustment accounted for decreases in the fresh weight / dry

weight ratio, increases in apoplastic water content and direct solute accumulation.

Inhibition of growth and a decrease in water content induced by salt / water stress has

been universally observed even in tolerant plants (Bartels and Salamini 2001, Mittler

84

et al. 2001). AJthough growth is the visible indicator of plant performance under

stress, it is considered to result from the sum of the adaptive mechanisms that are

adapted by a given species (Khedr ei al. 2003).

Effect of NaCI-salinity stress on peroxide content and lipid peroxidation levels in plants

Environmental stresses are known to cause oxidative stress within plant cells. The

accumulation of hydrogen peroxide (H2O2) has been observed in response to chilling

(Okuda et al. 1991, Prasad et al. 1994, Fadzilla et al. 1996, O'Kane et al. 1996), heat

(Dat et al. 1998, Gong e/ al. 2001), UV radiation (Murphy and Huerta 1990), excess

light (Karpinski et al. 1997), and anoxic stress (Blokhina et al. 2001). The resuhs

showed that increased H2O2 levels in all the tissues under salt stress. The

accumulation of H2O2 has been observed in response to NaCl in Lycopersicon

esculentum (Mittova et al. 2004), Pistim sativum (Hernandez et al. 1993, 2001),

Morns alba (Sudhaker et al. 2001), and Lens adinaris (Bandeogolu et al. 2004),

Oryza sativa (Bhattacharjee and Mukherjee 1997, Khan and Panda 2002a, 2004b,

Khan et al. 2002, Panda and Khan 2003). Lee et al. (2001) showed that NaCl

treatment resulted in an accumulation of H2O2 in the leaves but not in the roots of rice

plants. There is increasing evidence that NaCl salinity is one factor leading to

oxidative stress in plant cells (Van Camp et al. 1996, Hernandez et al. 1999, 2000a,

2000b). Tsai et al. (2004) showed that H2O2 levels increased in NaCl-treated roots of

rice seedlings. NaCl-induce accumulation of H2O2 in rice leaves has been suggested

to be due to NaCl-enhanced SOD and NaCl-deactivated CAT activities (Lee et al.

2001). This does not seem to be the case in the roots of rice seedlings, because had no

effect on SOD and CAT activities (Tsai et al. 2004). In several model systems

investigated in plants, the accumulation of H2O2 appears to be mediated by the

activation of a plasma-membrane bound NADPH oxidase complex (Orozco-Cardenas

and Ryan 1999, Pei et al. 2000, Zhang et al. 2001, Jiang and Zhang 2002).

Diphenyleiodonium chloride (DPI) and imidazole are known to inhibit plasma-

membrane NADPH oxidase (Cross 1990, Orozco-Cardenas and Ryan 1999, Jiang and

Zhang 2002). It appears that NaCl-induced H2O2 accumulation is mediated through

the activation of NADPH oxidase in rice roots (Tsai et al. 2004). NADPH oxidase

does not appear to be the only source of H2O2 generation in rice roots, because NaCl-

85

induced ceil wall-bound NADH peroxidase and diamine oxidase activities, which are

devoted to H2O2 generation, have been detected in the roots of rice seedlings (Lin and

Kao 2001a). In the last few decades an increasing body of evidence has suggested that

salt stress is associated with oxidative stress, through altering antioxidant molecule

levels and inducing antioxidative enzymes (Gossett et al. 1994, Meneguzzo et al.

1998, 1999, Hernandez et al. 2000, Pacoda et al. 2004). High NaCl concentrations

seem to impair electron transport in chloroplasts and mitochondria, and lead to the

formation of ROS such as 'O2, H2O2, 02' and HO^ (Hernandez et al. 1995, Asada

1999, Foyer and Noctor 2000). During salt-induced oxidative stress, excess reactive

oxygen species (ROS) were generated in chloroplasts, mitochondria and peroxisomes

via a number of metabolic pathways such as photosynthesis, respiration and

photorespiration (Noctor and Foyer 1998). Because of chloroplast and mitochondrial

membrane damage, the ROS is released into the cytoplasm. The presence of cytotoxic

ROS, such as 'O2, O2", H2O2 and OH in different compartments pose a threat to cells

by unrestricted oxidation of various cellular molecules such as nucleic acids, proteins,

and lipids (Fridovich 1986, Del Rio el al. 1998, Foyer and Mullineaux 1994, Leprince

et al. 2000).

The involvement of ROS, mainly H2O2 having functions as signalling molecules, has

been described for other plant systems mediating responses to several stimuli (Neill et

al. 2002a, 2002b Hung and Kao 2004) such as stomatal closure (Pei et al. 2000),

programmed cell death (Beers and McDowell 2001) or as being involved in plant

senescence (Puppo et al. 2005). The salt treatment increased lipid peroxidation or

induce oxidative stress in plant tissues. Lipid peroxidation measured as amount of

MDA is produced when polyunsaturated fatty acids in the membrane undergo

peroxidation by the accumulation of free oxygen radicals. There are reports that salt

treatment increases lipid peroxidation or induce oxidative stress in plant tissues

(Hernandez et al. 1994, 1995, Cosset et al. 1996, Gomez et al. 1999, Jain et al. 2001,

Khan and Panda 2002a, Khan et al. 2002, Panda and Khan 2003, Demiral and Turkan

2005, Mandhania et al. 2006). Increasing evidence exists that membrane injury under

salt stress is related to increased production of highly toxic ROS (Fadzilla et al. 1997,

Gomez et al. 1999, Savoure et al. 1999, Hernandez et al. 2000, Brausegem et al.

2001, Khan and Panda 2002a, Khan et al. 2002, Panda and Khan 2003, Upadhaya et

al. 2003). Lipid peroxidation could be a resuh of light dependent formation of singlet

86

oxygen in rice during water stress rather than due to H2O2 (Boo and Jung 1999,

Srivalli et al. 2003). Increased accumulation of MDA was observed in all the tissues

under salt stress. The results reported here show that the degree of acccumulation of

MDA was higher in rice cultivar Begimbiichi than in Lutiishree roots and leaves,

indicating a high rate of lipid peroxidation in Begunbitchi due to salt stress (Mittal and

Dubey 1991, Gosset et al. 1994, 1996, Hernandez et al. 1995, 2001, Gomez et al.

1999, Meloni et al. 2003, Bor et al. 2003). The post stress recovered roots and leaves

of Begunbitchi. and Lunishree showed lesser accumulation of MDA than the stressed

roots and leaves showing the ability of the plants to recover to some extent the

damage occurring during the stressed conditions. Similar results were found in

recovered greengram plant parts. Increasing evidence exists that membrane injury

under salt is related to increased production of highly toxic ROS (Hernandez et al.

1995, Gosset et al. 1996, Bhattacharjee and Mukherjee 1996, 1997, Gomez et al.

1999, Hernandez et al. 2001). As lipid peroxidation is the symptom mostly ascribed to

oxidative damage (Scandalios 1993, Zhang and Kirkham 1996), it is often used as an

indicator of increased damage (Halliwell 1982, Jagtap and Vargava 1995, Hernandez

et al. 1994, 1995, 2000, 2001, Gomez et al. 1999). Salt stress produced ion leakage

indicating injury to membrane integrity, which could be affected by ROS formed

during leaf photosynthesis or respiration, enhancing lipid peroxidation of the

membrane (Lechno et al. 1997, Savoure et al. 1999, Upadhaya et al. 2005). There are

reports of higher increase in amount MDA with the increase in salt stress in the sah-

sensitive cultivar as compared to tolerant cultivar of rice and in roots oiLemna minor

(Khan and Panda 2002a, Khan et al. 2002, Panda and Khan 2003, Panda and

Upadhyay 2004, Demiral and Turkan 2005, Mandhania et al. 2006).

Eflect of NaCI-salinity stress on ascorbate and glutathione content in plants

The non-enzymatic oxidants consist of ascorbate, glutathione, a-tocopherol,

carotenoids, and phenolic compounds (Alscher and Hess 1993, Foyer et al. 1994,

Jung 2004). Especially, carotenoid-dependent energy dissipation in the light

harvesting antennae is thought to play an important protective role by mitigating

oxidative damage.

87

Ascorbate (AsA) is a major antioxidant in photosynthetic and non photosynthetic

tissues which reacts directly with ROS in photosynthetic and non- photosynthetic

tissues, recycles a-tocopherol and protects enzymes with prosthetic transition metal

ions (Bartoli et al. 2000), and is utilized as a substrate for APX which catalyzes H2O2

detoxification (Asada 1994, 1999, Foyer et al. 1994, Foyer et al. 1997, Noctor and

Foyer 1998). AsA rapidly reacts with O2", OH and 'O2 (Asada, 1999) and, in addition,

can reduce the tocopheryl radical produced by a-tocopherol, working as a radical

scavenger in biological lipid phases (Smirnoff and Pallanca 1996). The results

observed shows a decreased AsA content in the salt stressed Begunbitchi (roots and

leaves) and Lunishree (roots), however, Lunishree leaves showed an increased AsA

content. The post stress recovered roots of both the cultivars showed an increased

AsA content with little or no change in the leaves allowing better antioxidant

protection as reported for other plants (Mishra et al. 1997, AJscher et al. 1997, Foyer

et al. 1997, Shalata et al. 2001, Ushimaru et al. 2002, Khan and Panda 2002a, Khan et

al. 2002). AsA decreased under water stress in rice, however the AsA content

increased following recovery of the plants and the increased was greater than the

control values control (Srivalli et al. 2003). Elevated ascorbate levels have been

measured in plants exposed to NaCl (Meneguzzo et al. 1999, Khan et al. 2002, Tsai et

al. 2004). However, there are reports that AsA levels decreased in plants in response

to NaCl stress (Hernandez et al. 1999, 2000a, 2000b, Shalata et al. 2001). It appears

that the increase in AsA levels in rice roots treated with NaCl depends on the rates of

it synthesis as well as on the rates of it regeneration (Tsai et al. 2004). In addition to

APX activity, ascorbate oxidase catalyzes the oxidation of AsA to DHA. The

definitive role of ascorbate oxidase is not clear, although it has been suggested that

the enzyme may participate in a redox system involving AsA (Weis 1975). It is most

likely that NaCl caused an increase in ascorbate oxidase activity in rice roots which in

turn resulted in an increase in DHA levels (Tsai et al. 2004).

Glutathione (GSH) is a major water-soluble antioxidant species. Glutathione as non-

enzymatic antioxidant to ROS (Noctor and Foyer 1998) plays a protective role by

increasing stress tolerance, in particular that of salinity and oxidative stress and seems

to be an important signal molecule by acting as a direct link between environmental

stress and key adaptive responses (Rennenberg 1982, May et al. 1998, Wingate et al

88

1998). Glutathione is the electron donor of glutathione peroxidase (EC 1.11.1.9),

which reduces H2O2 and organic and lipid hydroperoxides (Eshdat et al. 1997).

Increased glutathione content observed in the stressed and post stress recovered roots

and shoots of Lunishree, whereas a decreased GSH content was observed in case of

Begimbitchi roots and leaves (stress and post stress) may reflect, at least partially, its

increased demand as a substrate by enzymes participating in the detoxification of

membrane lipid peroxidation in Lunishree compared to Begunbilchi (Marrs 1996,

Gueta-Dahan et al. 1997, Khan and Panda 2002a, Khan and Panda 2003) or increased

glutathione redox state may serve as signal affecting the expression of defence genes

(Foyer et al. 1997). Changes in processes that regulate GSH concentration and / or

redox status are considered to be one of the important adaptive mechanisms of plant

exposed to stress conditions (Fadzilla et al. 1997, Alscher et al. 2002). Fadzilla et al.

(1997) reported severe oxidative damage with elevated concentrations of H2O2 and

reduced concentrations of GSH in rice plants exposed to salt stress. Srivalli et al.

(2003) reported no significant increase in GSH content in rice under water stress,

however the GSH content decreased after watering the plants i.e. post stress recovery.

It has been shown that salinity induced glutathione synthesis in Brassica napus (Ruiz

and Blumwald 2002). The increase in GR activity and in GSH contents as observed

by Tsai et al. (2004) in NaCl-treated rice roots suggests that GSH contents may be

regulated by its synthesis and regeneration. GSH can, directly or by means of

glutathione peroxidase react with ROS and lipid peroxides to GSSG (Noctor et al.

1998). Elevated GSH levels have been measured in plants exposed to NaCl

(Meneguzzo et al. 1999, Khan et al. 2002). However, there are other reports

indicating that GSH levels decreased in plants in response to NaCl stress (Hernandez

et al. 1999, 2000, Shalata et al. 2001). In addition, it has also been suggested that

variations in the oxidized / reduced ratio of glutathione can serve as a signal for the

modulation of ROS scavenging mechanisms and ROS signal transduction (Foyer et

al. 1997, Mittler 2002). GSH synthesis is enhanced in plants exposed to xenobiotics

and environmental stresses, such as in soybean nodules subjected to paraquat (Dalton

1992) or in Brassica napus plants under salt stress (Ruiz and Blumwald, 2002). The

latter attributed the increase of GSH synthesis to an enhancement of sulphur

assimilation and cysteine biosynthesis. Elevated GSH levels have been measured in

plants exposed to NaCl (Meneguzzo et al. 1999, Khan et al. 2002, Tsai et al. 2004).

However, there are reports that ASC and GSH levels decreased in plants in response

89

to NaCl stress (Hernandez et al. 1999, 2000a, 2000b, Shalata et al. 2001).

Furthermore osmotic and salt media stimulated the non-enzymatic antioxidants in the

leaves and stems of Mains domesUca explants, however, this increase was more

pronounced in the NaCl-treated leaves (Molassiotis et al. 2006).

In addition, it has also been suggested that variations in the oxidized / reduced ratio of

the ascorbate can serve as a signal for the modulation of ROS scavenging mechanisms

and ROS signal transduction (Foyer et al. 1997, Mittler 2002). The ascorbate-

glutathione cycle is an efficient way for plant cells to dispose of H2O2 in certain

cellular compartments where this metabolite is produced (Halliwell and Gutteridge

1989). This cycle makes use of the non-enzymic antioxidants ascorbate and

glutathione in a series of reactions catalyzed by four antioxidative enzymes and has

been demonstrated in chloroplasts, cytosol, and root nodule mitochondria (Foyer and

MuUineaux 1994). In peroxisomes and mitochondria purified from pea leaves, the

presence of all the enzymes of the ascorbate-glutathione cycle was reported (Jimenez

et al. 1997, 1998). The four enzymes, APX, MDHAR, DHAR, and GR, were present

in peroxisomes. Likewise, in intact peroxisomes and mitochondria, the presence of

AsA and GSH and their oxidized forms DHA and GSSG, respectively, was found by

HPLC analysis (Jimenez etal. 1997). The ascorbate-glutathione cycle of peroxisomes

was also affected by senescence. In dark-induced senescent leaves the peroxisomal

APX and MDHAR activities were notably decreased but DHAR was considerably

enhanced. In contrast, GR activity was not affected by senescence (Jimenez et al.

1997, 1998) to the peroxisomal antioxidants, whereas the ascorbate content was only

slightly increased by senescence, the total glutathione content augmented about 20

times. The predominant form of glutathione during senescence was GSSG, resuhing

in a 43-fold increase in the GSSG / GSH ratio in peroxisomes (Jimenez et al. 1998.

Jimenez et al. (1997) interpreted that the ascorbate-glutathione cycle gains in

importance for the elimination of H2O2 in peroxisomes as senescence proceeds and

catalase disappears.

Reduced ascorbate is regenerated from either MDHA (through the Mehler-peroxidase

cycle) (Miyake and Asada 1992) by the NADPH-dependent enzyme

monodehydroascorbate reductase (MR) (Buettner and Jurkiewicz 1996), or by

dehydroascorbate reductase (DR) through the ascorbate-glutathione (ASC-GSH)

90

cycle or Asada-Halliwell Pathway (Asada 1994). Asada (1984) concluded that DR

would participate only in ascorbate regeneration when MR activity is limited by the

availability of NAD(P)H. The change in dehydroascorbate (DHA) / ascorbate (AsA)

ratio, an important indicator of the redox status of the cell, is one of the first signs of

oxidative stress (Lechno et al. 1997, Meneguzzo et al. 1998, 1999). While several

authors suggest that MR activity is important in the regeneration of foliar ascorbate in

tomato plants under salt stress (Mittova et al. 2000). Gorgocena et al. (1995) suggest

that DR is the enzyme responsible for AsA regeneration in the pea-nodule cytosol

under water stress and that this activity is critical for nodule functionality (Dalton et

al. 1993). Elevated AsA and GSH levels have been measured in plants exposed to

NaCl (Meneguzzo et al. 1999, Khan et al. 2002, Tsaio et al. 2004). However, there

are other reports indicating that AsA and GSH levels decreased in plants in response

to NaCl stress (Hernandez et al. 1999, 2000a, 2000b, Shalata et al. 2001). Tsaia et al.

(2004) showed that both AsA and DHA levels increased in NaCl stress in rice roots. It

appears that the increase in AsA levels in rice roots treated with NaCl depends on the

rates of its synthesis as well as on the rates of its regeneration. In addition to AFX

activity, ascorbate oxidase catalyzes the oxidation of AsA to DHA. The definitive role

of ascorbate oxidase is not clear, although it has been suggested that the enzyme may

participate in a redox system involving AsA (Weis 1975). It is mostly likely that NaCl

caused an increase in ascorbate oxidase activity in rice roots which in turn resulted in

an increase in DHA level. Tsaia et al. (2004) showed that CI" is not involved in the

NaCl-induced H2O2, AsA and DHA contents, as well as the NaCl induced in APX and

GR activities, and the NaCl-enhanced expression of OsAPX and OsGR, and CI' rather

than Na* is required for the NaCl-induced GSH and GSSG contents. How the CI'

contributes to the GSH and GSSG production remains to be investigated. When

growing in saline soils, roots also encounter osmotic stress resuhing fi"om salt

concentration in the soil that results in lowered water potential and consequent loss of

cell turgor in roots. Furthermore, it has been shown that ABA accumulates in plants

under salt stress (Montero et al. 1997, 1998), Thus, it is of great interest to know the

relative importance of osmotic stress and endogenous ABA in NaCl-induced

antioxidant systems in the roots of rice seedlings.

91

Effect of NaCl-salinity stress on proline content in plants

Amino-acids such as proline, asparagines, and a-aminobutyric acid, can play an

important role in the osmotic adjustment of the plant under saline conditions (Gilbert

et al. 1998). Plants grown under salinity accumulate compatible solutes, such as

sorbitol, mannitol and proline (Xiong and Zhu 2002), These substances, referred as

osmolytes, were originally thought to function as osmotic buffers lowering the

cellular osmotic potential to sustain water absorption from saline solutions and to

restore intracellular ion homeostasis (Zhu 2001). It has been demonstrated that these

substances exhibit also ROS scavenging properties although the underlying

mechanism is not clear (Xiong and Zhu 2002). Accumulation of proline primarily due

to de novo synthesis. It has been suggested that proline protects plant tissues against

osmotic stress because it is an osmosolute, a source of nitrogen compounds, a

protectant for enzymes and cellular structures (Stewart and Lee 1974, Le-Rudulier et

al. 1984, Serrano and Gaxiola 1994), and a scavenger for hydroxyl radicals (Smironff

and Cumbes 1989). In addition to acting as an osmoprotectant, proline also serves as a

sink for energy to regulate redox potentials (Blum and Ebercon 1976, Saradhi and

Saradhi 1991), as a hydroxyl radical scavenger (Smirnoff and Cumbes 1989), as a

solute that protects macromolecules against denaturation (Schobert and Lauchli

1990), and as a means of reducing the acidity in the cell (Venekamp et al. 1996). Over

expression of a bacterial gene allowing mannitol synthesis has been demonstrated to

confer salinity tolerance in transgenic plants (Tarczynski et al. 1993). Therefore, it is

likely that over-production of proline, one of the natural osmolytes (Yancey et al.

1982) may enable crop plants to tolerate water stress and thereby salt stress.

Proline accumulation is higher in the leaves of salt sensitive rather than salt tolerant

tomato genotypes (Alian et al. 2000). The greater accumulation of proline may resuh

from an increased endogenous production from a decreased metabolism or from a

direct effect of salinity on the uptake and translocation of proline (Heuer 2003).

Demiral and Tukan (2005) reported higher proline content in rice culitvar ER 28 than

Pokkali under NaCl stress with no change in the ratio. In function, proline is often

regarded as an compatible osmolite associated with the salt-resistance mechanisms

(Samaras et al. 1994) and can also help lower the cell's osmotic potential (Gilbert et

al. 1998). Parida et al. (2002) have reported the long-term effect of NaCl in

92

hydroponic culture in this species, which tolerated salinity levels up to 400 mmol/L.

Their results showed that acclimatization to high salt concentration enhanced the

accumulation of free amino acids and polyphenols. Thus, NaCl stress resulted in an

accumulation of osmolytes as an adaptive measure in this species. Rapid

accumulation of free proline is a typical response to salt stress. When expose to

drought or a high salt in the soil (both leading to water stress), many plants

accumulate high amount of proline, in some cases several times the sum of all the

other amino acids (Mansour 2000). Dash and Panda (2001) reported that salt

treatments triggered an accumulation of proline for salt-treated seeds of Phaseoltis

mimgo. Molassiotis et al. (2006) presented data on proline accumulation pattern

indicating that mannitol and salt treatments triggered an accumulation of proline in

the leaves and stems of explants of Mains domestica. Of particular interest in the

reported investigation is the finding that proline content was not increased in the

leaves and stems of MM 106 exposed to sorbitol as reported previously in rice by Al-

Khayri and Al-Bahrany (2002). There are reports that constitutive production of

proline could confer osmotolerance in transgenic tobacco plants. However, the lack of

correlation between proline level and salt tolerance in certain plant species has also

led to the conclusion that proline accumulation is merely a consequence of stress and

does not lead to sah tolerance (Hanson and Nelson 1978). Exogenous proline has been

reported to protect plants under stress. It improved the tolerance of somatic embryos

of celery {Apinm graveolens L. cv. SB 12) to partial dehydration (Saranga et al.

1992). Nanjo et al. (1999) using transgenic Arabidopsis plants with reduced

pyrroline-5-carboxylase synthase (P5CS) activity, which is the rate limiting enzyme

in the proline synthesis from glutamate, demonstrated that these plants were unable to

grow on saline media and that feeding with L-proline, but not D-proline, could

increase their ability to withstand the salt. They came to the conclusion that, in

addition to various known roles of proline, it was also involved in the synthesis of key

proteins that are necessary for stress responses. However, Iyer and Caplan (1998)

showed that a proline metabolite such as pyrroline-5-carboxylate, but not proline

itself, at 1 mM concentrations is able to induce stress-regulated genes such as

dehydrins and sail. Okuma et al. (2000) found that exogenous proline improved the

growth of salt-stressed tobacco cell cultures and the improvement was attributed to

the role of proline as an osmoprotectant for enzymes and membranes against salt

inhibition rather than as a compatible solute. Khedr et al. (2003) studying with

93

Pancratium marUimum found that exogenous proline induces the expression of salt-

stress-responsive proteins by acting as a component of signal transduction pathways

that regulates stress responsive genes in addition to its previous described

osmoprotective roles, thereby improving the tolerance to salt stress.

Effect of NaCI-salinity stress on the enzymic antioxidants in plants

Plants are naturally subjected to the noxious effects of ROS (Noctor and Foyer 1998),

thus evolving highly efficient mechanisms to counteract the attack on cellular

components and processes causing impairment of electron transport in chloroplasts

and mitochondria (Jimenez et al. 1997, 1998, Meneguzzo ei al. 1998), membrane

peroxidation (Quartacci et al. 1995, Navari-Izzo et al. 1996), protein denaturation,

and DNA damage (Conte et al. 1996). In plant cells ROS detoxification is controlled

by superoxide dismutases (SOD, EC 1.15.1.1), catalases (CAT, EC 1.11.1.6), and the

Asada-Halliwell scavenging cycle. This cycle, found in different cellular organelles

and the cytosol (Jimenez er al. 1997, 1998, Hamilton and Heckathom 2001), involves

the oxidation and re-reduction of ascorbate and glutathione mainly by the activities of

ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2)

(Noctor and Foyer 1998, Asada 1999). Although several authors have observed

variations in antioxidative defences in plants grown in nutrient solutions

supplemented with NaCl (Hernandez et al. 2000, Meneguzzo et al. 1998, 1999, 2000,

Silveira et al. 2001). It is well known in glycophytic as well halophytic species that

the exposure to NaCl imposes oxidative stress due to changes in the osmotic and ionic

environment of the cell (Allakhverdiev et al. 2000, Hasegawa et al. 2000). In turn,

this oxidative stress ahers the levels of antioxidants as well as antioxidative enzymes

that serve as amelioration of the defense system in plants (Gossett et al. 1994).

Production and removal of ROS must be strictly controlled as such plants have

developed a complex array of non-enzymatic and enzymatic detoxification

mechanisms. Nevertheless, the equilibrium between production and scavenging of

ROS may be disrupted by a number of environmental constraints such as water,

salinity and heavy metal stresses, pathogens, herbicides, high irradiance or high

temperatures leading to oxidative stress situations and thus cell damage (Apel and

Hirt 2004). There is also a great deal of evidence to support the suggestion that

oxidative stress resuhs in increases in enzymatic and non-enzymatic systems

associated with the ROS scavenging cycle (Okpodu et al. 1996, Apel and Hirt 2004).

94

Studies have shown that salt tolerance may be improved if the free radicals formed

during the accompanying activated oxygen damage are scavenged by an enhanced

antioxidative defense system (Alscher et al. 2002, Shigeoka et al. 2002). There is

good evidence that the alleviation of oxidative damage and increased resistance to

salinity and other environmental stresses is often correlated with an efficient

antioxidative system (Scandalios 1993, Hasegawa ei al. 2000, Acar et al. 2001, Sato

et al. 2001, Bor et al. 2003, Demiral and Turkan 2005). Dionisio-Sese and Tobita

(1998) studied the activities of SOD and POX enzymes under NaCl stress in the

leaves of four cultivars of rice exhibiting different sensitivities to NaCl. Their results

have indicated that salt tolerance capacity of salt-tolerant species is closely related

with the maintenance of specific activity of antioxidative enzymes studied (Dionisio-

Sese and Tobita 1998).

Effect of NaCi-salinity stress on protein content in plants

Salt stress responses observed were a reduction in protein content (Navari-Izzo et al.

1990, Gilbert et al. 1998). The effect of salt stress on protein content depends on the

concentration of NaCl. At lower levels of NaCl, there was an increase in the protein

content, but higher concentrations caused it to decline in both shoot and root. This

suggests that the initial response to salt / water stress involves increased protein

synthesis that is prevented when the stress becomes too severe (Khedr et al. 2003).

Other salt stress responses observed were a reduction in protein content, in agreement

with Gilbert <?/a/. (1998).

Effect of NaCl-salinity stress on superoxide dismutase (SOD) activity

Superoxide (O2") is atoxic by-product of oxidative metabolism. Thus, the dismutation

of superoxide into H2O2 and O2 by SOD is an important step in protecting the cell.

The family of metalloenzymes known as SODs (EC 1.15.1.1) play an important role

in protecting cells against the toxic effects of O2" produced in different cellular loci

(Fridovich 1986, Halliwell and Gutteridge 1989, Asada 1999, Foyer and Noctor 2000,

Kwon et al. 2002). Superoxide radicals generated by oxidative metabolisms are

dismutated into H2O2 and O2 by superoxide dismutase (SOD). SOD is classified

according to their metal co-factor like Mn-SOD, Fe-SOD, and Cu/Zn-SOD (Beyer

and Fridovich 1987) and occurs in various cellular organelles (del Rio et al., 1983,

Slooten et al 1995, Miszalski et al. 1998). SODs are distributed in different cell

95

compartments, mainly chloroplasts, cytosol, mitochondria (Fridovich 1986, Halliwell

and Gutteridge 1989, del Ryo ei al. 1992), and peroxisomes (del Ryo et al. 1992). The

occurrence of SODs in isolated plant peroxisomes has been reported in at least seven

different plant species (del Ryo ei al. 1992), and in four of these plants the presence of

SOD has been confirmed by immunoelectron microscopy (Corpas et al. 1998). From

studies performed with different plant species, it is known that chloroplasts are

equipped with Fe-SOD and / or with a specific isoform of CuZn-SOD (Van Camp et

al. 1996, Kurepa et al. 1997, Asada 1999). The chloroplastic CuZn-SOD is associated

with the thylakoid membrane, whereas Fe-SOD is in the chloroplastic stroma (Asada

1999). The role of the different SOD classes seems to be rather specific. However,

results on the over expression of different SODs in plants in oxidative stress tolerance

are conflicting. Artificial overproduction of Fe-SOD in chloroplasts and Mn-SOD

enhanced the oxidative stress tolerance in transgenic tobacco plants, whereas

overproduction of Mn-SOD in chloroplasts did not increase oxidative stress tolerance

(Van Camp et al. 1996, Kwon et al. 2002). There is pertinent information in the

literature indicating that Fe-SOD is of major importance for superoxide radical

scavenging. A Fe-SOD-lacking mutant of Synechococctis is sensitive to

photooxidative stress leading to inactivation of PSI (Herbert et al. 1992, Samson et al.

1994, Thomas et al. 1998). A comparison of transgenic tobacco plants overproducing

Fe-SOD or Mn-SOD in the chloroplast indicates that Fe-SOD provides better

protection against oxidative stress than Mn-SOD (Van Camp et al. 1996). While Fe-

SOD seems to be located in the chloroplast stroma, chloroplastic CuZn-SOD is

attached to the thylakoid membranes (Asada 1999). Conversely, the responses of the

Fe-SOD isoforms were very complex and indicate a differential regulation of isoform

activity, which indeed suggests increased superoxide production in the chloroplast

under NaCl treatment and under osmotic stress. The activity of one of them, Fe-SOD

I, was only increased by NaCl treatment and not by osmotic stress. On the other hand,

activities of Fe-SOD II and Fe-SOD UI, were down-regulated in the absence of K^

. and in the presence of Na* and were up-regulated under osmotic stress. The

mitochondrion is another important source of superoxide radicals. In this organelle,

Mn-SOD is scavenging superoxide radicals produced under conditions of malfunction

of mitochondrial electron transport (Bowler and Inze 1992). Lee et al. (2001)

observed that NaCl treatment induced a significant increase of SOD activity of rice

leaves, however, the activity was not affected in rice roots was affected by NaCl. A

96

significant increase of SOD activity occurred in pea leaves after short-term NaCl

stress (Hernandez and Almansa 2002). Tsai et al. (2004) observed that NaCl had no

effect on the activity of SOD and isozymes of SOD in rice roots.

Alterations in SOD activity against oxidative processes have been observed in many

plants, underscoring the critical role of these enzymes (Slooten et al. 1995, Van Camp

et al. 1996, Goel and Sheoran 2003, Sairam et al. 2003/4). Since it is very difficult to

measure superoxide production in vivo, generally activity changes of SOD are used as

an indicator of changes in superoxide production. The resuhs showed a decreased

SOD activity in the stressed roots of Luuishree and Begimbiichi during NaCl

treatment, may result from an increased inactivation by H2O2 (Scandalios 1993)

thereby lowering the dismutation and enabling the plant to resist the potential

oxidative damage caused by NaCl salinity exposure (Hernandez et al. 1994, Foyer et

al. 1997, Shalata and Tal 1998, Hernandez et al. 2000, Dash and Panda 2001, Khan

and Panda 2002a, Panda and Khan 2003). Molassiotis et al. (2006) showed SOD

activity to increase under osmotic and saline conditions whereas an additional Mn-

SOD isoform was detected in the NaCl-treated leaves. Enhancement of SOD activity

by salinity has been observed by Lee et al. (2001). Although SOD functions as the

first line of defense against oxidation at the membrane boundaries, its end product is

the toxic H2O2 (Mittler 2002). An excessive accumulation of superoxide due to

reduced activity of SOD under flooding stress was shown also (Yan et al. 1998).

Tsaia et al. (2004) observed that NaCl had no effect on the activity of SOD and

isoenzymes of SOD in rice roots. Demiral and Tukan (2005) reported that under NaCl

stress, rice culitvar Pokkali and IR 28 showed no change SOD activity, however

higher ratio of activity was reported in Pokkali than in IR 28. Srivalli et al. (2003)

reported that the rice showed slightly increased SOD increased during water stress,

however the SOD activity decreased after watering i.e. post stress recovery.

Effect of NaCI-salinity stress on catalase (CAT) and peroxidase activities

Catalase (CAT) is known to dismutate H2O2 into H2O and O2. Catalase is involved in

scavenging hydrogen peroxide that is produced during photorespiration and P-

oxidation of lipids in the peroxisomes under normal conditions and accumulated

under stressful conditions. Alterations in CAT activitiy against oxidative processes

97

have been observed in many plants, underscoring the critical role of these enzymes

(Slooten el al. 1995, Van Camp et al. 1996, Goel and Sheoran 2003, Sairam et al.

2003/4). The induction of catalase activity under water stress is well documented and

a positive relationship has been found between its up-regulation and stress tolerance

(Hernandez et al. 2000, Hamilton and Heckathorn 2001, Shalata et al. 2001,

Ushimaru et al. 2001). Hydrogen peroxide produced during salt stress can easily

permeate membranes and can be removed by catalase (CAT) or by peroxidase (POX)

(Meloni et al. 2003). Although decreased CAT activity was observed in the results of

the Liinishree and Begimbitchi stressed roots, increased CAT activity was observed in

the post stress recovered roots showing higher capacity for the decomposition of H2O2

generated during the post recovery NaCl stress period (Foyer et al. 1997, Jimenez et

al. 1997). Decreased CAT activity in stressed roots of both the cultivars, might have

promoted H2O2 accumulation, which could result in a Haber-Weiss reaction to form

hydroxy) radicals. Since OH radicals are known to damage biological membranes and

react with most compounds present in biological systems, they might have hastened

lipid peroxidation and membrane damage in the stressed roots. The results obtained in

this study are in accordance with those of Sairam et al. (2002) who reported

enhancement in CAT activity in both sah sensitive and salt-tolerant cultivars of

wheat. Gueta-Dahan et al. (1997) also reported similar observation. Vaidyanathan et

al. (2003) reported enhancement in CAT activity in salt-tolerant rice cultivar. Savoure

et al. (1999) found that NaCl stimulated catalase activity through activation of the

Cat2 and Cat3 genes. Fadzilla et al. (1997) reported that NaCl had no effect on CAT

activity on rice shoots. On the other hand, decrease in CAT by NaCl has been shown

in rice leaves (Dionisio-Sese and Tobita 1998, Lee et al. 2001). Tsai et al. (2004)

reported that NaCl does not influence the activity and isozymes of CAT in rice roots.

The down-regulation of catalase by salt stress may indicate that the plant is not able to

maintain protection against active oxygen under sah-stress particularly at high salt

concentrations. The decrease in CAT activity measured under severe water stress has

been reported (Baisak et al. 1994, Sgherri and Navari-Izzo 1995, Schwanz et al. 1996,

Khedr et al. 2003). Such a decrease may be due to some stress-induced damage to the

enzyme (Khedr et al. 2003). In Nicotiana plumbagimfolia, Savoure et al. (1999)

found that NaCl-stimulated catalase activity through activation of the Cat2 and Cat3

genes. Fadzilla et al. (1997) reported that NaCl had no effect on CAT activity in rice

98

shoots. On the other hand, decrease in CAT by NaCl has been shown in rice leaves

(Dionisio-Sese and Tobita 1998, Lee et al. 2001). Tsai et al. (2004) observed that

NaCl does not influence the activity and isoenzymes of CAT in rice roots. Tsai et al.

(2004) suggested that, if anything, CAT and SOD play a less important role in

scavenging ROS in roots of rice seedlings under NaCl stress conditions. The decrease

in catalase would lead to an increase in hydrogen peroxide levels, although this

accumulation in tissues could also be due to the action of SOD, since this metabolite

is a product of O2" dismutation (Becana et al. 1986). Despite its considerable

reputation, catalase is not a primary means of antioxidant defences in the cytosol of

nodule tissues because of its very high Km for H2O2 and its restricted location in

peroxisomes (Dahon 1995). Srivalli et al (2003) reported that the CAT activity

significantly increased during water stress with similar results of increased CAT

activity even after removal of water stress, i.e. watering. Demiral and Tukan (2005)

reported that the CAT activity increased under NaCl stress in rice cuhivar Pokkali,

whereas the CAT activity decreased in rice cuhivar IR 28.

Peroxidases (POXs) are involved not only in scavenging of H2O2 produced in

chloroplasts but also in growth and developmental processes (Dionisio-Sese and

Tobita 1998). POX is among the enzymes that scavenge H2O2 produced through

dismutation of O2 catalyzed by SOD. POX activity significantly increased in the

stressed roots of Ltmishree and mild increase in Begimhitchi roots showing better

detoxification of H2O2 produced during NaCl stress or H2O2 generated by SOD.

Increased POX activity has also been reported in tomato (Shalata and Tal 1998), rice

cultivars (Dionisio-Sese and Tobita 1998, Panda and Khan 2003) and sugar beet (Bor

et al. 2003). Increased POX activity in Lunishree and Begunbitchi roots may be

attributed to increased activity of POX encoding genes or increased activation of

already existing enzymes as suggested by Dionisio-Sese and Tobita (1998). Mittal and

Dubey (1991), Demiral and Turkan (2005) compared two sets of rice cuhivars

differing in saU tolerance to determine a possible correlation between peroxidase

activity and the degree of salt tolerance in rice; they found a negative correlation

between peroxidase activity and salt tolerance of rice cuhivars. They showed that the

POX decreased in cultivar Pokkali whereas increased in cultivar IR 28. Their resuUs

are not consistent with ours in that with increased salinity, there was a significant

increase in peroxidase activity in roots of Lunishree with a mild increase in roots of

99

Begwibitchi. Lin and Kao (2001) demonstrated that reduction of root growth with

increasing NaCl concentrations was correlated with an increase in ionically bound cell

wall POX activity. There are reports of increased POX activity in rice and other crops

on exposure to various environmental stresses (Gilham and Dodge 1986, Scandalios

1993, Gosset et al. 1994, Foyer et al. 1997, Jimenez et al. 1997, Oidaira ei al. 2000,

Meloni et al. 2003).

Like catalase, peroxidase is involved in neutralizing hydrogen peroxide, but at the

expanse of another substrate being oxidized such as ascorbate. Peroxidase is reported

to be enhanced by water stress and this was positively correlated with water stress

tolerance (Hernandez et al. 2000, Sairam and Saxena 2000, Lin and Kao 2001a,

Hamilton and Heckathom 2001, Shalata et al. 2001, Ushimaru et al. 2001). Increased

POX activity has also been reported in tomato (Shalata and Tal 1998), rice cultivars

(Dionisio-Sese et al. 1998, Panda and Khan 2003) and sugar beet (Bor et al. 2003).

The consistent increase in peroxidase activity in the shoots even during severe salt-

stress and its differential regulation show that peroxidase may be more stable or more

important for stress tolerance than catalase. This is probably dictated by the wide

range of metabolic processes in which peroxidase are known to be involved, such as

lignin biosynthesis and formation of isodityrosine bridges that are believed to

crosslink structural protein molecules, in addition to antioxidant activity (Khedr et al.

2003). Molassiotis et al. (2006) showed that POD activity increased due to osmotica

and salts whereas additional POD isoforms were detected in the explants of Malus

domestica exposed to salinity. The enhancement of POD activity by salinity has also

been observed in the rice leaves (Lee et al. 2001) and callus cultures of Stiaeda

midiflora (Cherian and Reddy 2003). Lee e/ al. (2001) pointed out that the induction

of specific POD isoforms by salinity occurs under CAT deactivation. Molassiotis et

al. (2006) studying with Malus domestica explants confirmed the inability of CAT to

cope with salinity since CAT activity depressed in the saline-treated leaves.

Effect of NaCI-salinity stress on ascorbate peroxidase (AFX) activity

Ascorbate peroxidases (APXs) play a key role in the removal of H2O2 in the

chloroplast and cytosol (Noctor and Foyer 1998), and changes in the activity of these

enzymes are strictly correlated with plant tolerance to oxidative stresses (Meneguzzo

et al. 1998, 1999, Lee et al. 2001, Sudhakar et al. 2001). The role of APX and GR in

100

the H2O2 scavenging in plant cells has been well established in the ascorbate-

glutathione cycle (Bowler et al. 1992, Asada 1994, 1999). Lee et al. (2001) showed

that NaCl stress resulted in a higher activity of APX in rice leaves but not in rice

roots. In shoot cultures of rice, activity of APX was similar whether the shoots were

grown in the presence or absence of NaCl (Fadzilla ei al. 1997). Gene expression in

response to environmental stress is usually studied at the level of steady-state mRNA

abundance because this gives a more precise estimate of antioxidant gene activation

than enzyme activity (Tsai et al. 2004). Expression of APX and GR genes have been

reported to be enhanced in plants by NaCl treatment (Kaminaka et al. 1998, Savoure

et al. 1999, Kawasaki et al. 2001). However, Lopez et al. (1996) demonstrated that

APX activity, rather than mRNA level, was enhanced in NaCl-stressed Raphauus

sativus plants. Hernandez et al. (2000) found that transcript levels for cytosolic APX

were strongly induced in the NaCl-tolerant pea variety but not in the NaCl-sensitive

pea variety. Lee et al. (2001) showed that NaCl stress resulted in a higher activity of

APX in rice leaves and not in rice roots. Hernandez and AJmansa (2002)

demonstrated that APX activity did not change in pea leaves during short-term NaCl

stress. Tsaia et al. (2004) that NaCl and Na-gluconate treatments resulted in an

enhancement of the expression of the OsAPX in the roots of rice seedlings. They

revealed that OsAPX gene expression is up-regulated by NaCl in rice leaves. APX

activity remained uniform under water stress in rice with similar resuhs in recovery

period (Srivalli et al 2003). APX activity increased slightly in rice cultivar Pokkali no

change in rice cultivar IR28 under NaCl stress (Demiral and Tukan 2005).

Dehydroascorbate reducatse (DHAR) and glutathione reducatse (GR) were found in

the soluble fraction of peroxisomes, whereas APX was bound to the external side of

the peroxisomal membrane. These results agree with findings of an APX isoenzyme

in membranes of pumpkin and cotton peroxisomes (Yamasaki et al. 1995). MDHAR

was also localized in the peroxisomal membranes. It has been proposed that the trans-

membrane protein MDHAR can oxidize NADH on the matrix side of the peroxisomal

membrane and transfer the reducing equivalents as electrons to the acceptor

monodehydroascorbate on the cytosolic side of the membrane. In this process,

molecular O2 could also act as an electron acceptor, with the concomitant formation

of O2" (Lopez et al. 1996). The evidence of the presence of APX and MDHAR in leaf

peroxisomal membranes suggests a dual complementary function in peroxisomal

101

metabolism of these membrane bound antioxidant enzymes. The first function could

be to reoxidize endogenous NADH to maintain a constant supply of NAD^ for

peroxisomal metabolism, an idea that was originally proposed for the membrane-

bound NADH dehydrogenase of glyoxysomes from castor bean endosperm (Fang ei

al. 1998). A second function of the membrane antioxidant enzymes could be to

protect against H2O2 leaking from peroxisomes. H2O2 can easily permeate the

peroxisomal membrane, and an important advantage of the presence of APX in the

membrane would be the degradation of leaking H2O2, as well as the H2O2 that is being

continuously formed by disproportionation of the O2" generated in the NADH-

dependent electron transport system of the peroxisomal membrane (del Ryo et al.

1992, Lopez 1996). This membrane scavenging of H2O2 could prevent an increase in

the cytosolic H2O2 concentration during normal metabolism and under certain plant-

stress situations, when the level of H2O2 produced in peroxisomes can be substantially

enhanced (del Ryo et al. 1992).

Effect of NaCl-salinity stress on glutathione reducatse (GR) activity

Glutathione reductase (GR) also plays a key role in oxidative stress by converting the

oxidised glutathione (GSSG) to reduced glutathione (GSH) and maintaining a high

GSH / GSSG ratio (Fadzilla et al. 1997, Irishimovitch and Shapira 2000). Increased

GR activity in leaves of sugar beet plant have been reported, may be closely related

with sah tolerance capacity of these plants (Bor et al. 2003), However, in our study,

GR activity decreased in the roots of both cultivars, this decline was slightly greater in

Begmihitchi than in Lunishree. Constitutive levels of GR were higher in Lunishree

than in Begioihilchi. Consistent with our results, Shalata and Tal (1998) observed a

decrease in GR activity in leaves of tomato cultivars under sah stress. Since decreased

GR activity enhances stress sensitivity (Aono et al. 1995), more severe decrease in

GR activity in roots of Begiwbitchi than in roots of Lunishree may result in more

sensitive structural arrangement in roots of Begmihitchi against NaCl stress. A lower

GR activity in the stressed roots of Limishee and Begimbitchi could be due to the

tendency of the plant to acclimate or inactivation of the enzyme losing its ability to

maintain a higher GSH / GSSG ratio or the plants are unable to exhibit a more active

ascorbate-glutathione cycle (Shalata and Tal 1998, Loginni et al. 1999, Mittova et al.

2000, Molina et al. 2002). GR increased in rice during water stress, however, GR

activity decreased after watering (Srivalli et al. 2003). GR activity decreased under

102

NaCl stress in both cultivars Pokkali and IR 28, however with a lesser decline in

Pokkali than in 1R28 (Demiral and Turkan 2005).

There was an early increase in GR activity in NaCl-exposed shoot cultures of rice

(Fadzilla et al. 1997). Increased GR activity facilitates improved stress tolerance and

has the ability to alter the redox poise of improved components of electron transport

chain (Tyystjarvi et al. 1999). Hernandez and Almansa (2002) demonstrated that GR

activity increased in pea leaves during short-term NaCl stress. Tsai et al. (2004)

showed that both the activities and isoenzymes of GR are enhanced by NaCl in the

roots of rice seedlings. In addition a statistical coupling of decline in APX, MR, and

GR by salt stress was detected. This suggests that GR plays a major role in the

regeneration of ascorbate by the AsA-GSH cycle, acting as a key detoxifying enzyme

under stress conditions. While several authors suggest that MR activity is important in

the regeneration of foliar ascorbate in tomato plants under salt stress (Mittova et al.

2000). In their study with Bnigtaera pan'ijlora, Parida et al. (2002) have analyzed the

gradient effect of salt treatment on accumulation of antioxidants as well as

antioxidative enzymes in hydroponic culture of B. pan'iflora. Their resuhs provide

information on the salt induced specific changes in the isoforms of the major

antioxidative enzymes. Salt tolerance is often correlated with a more efficient

antioxidative system (Cakmak and Marschner 1992, Bor et al. 2003). Since NaCl-

induced enzyme activity indicates a specific role in coping with the stress (Gueta-

Dahan et al. 1997), constitutive and / or induced activities of SOD, CAT, APX, and

GR further suggest improved tolerance to salt stress. Expression of GR have been

reported to be enhanced in plants by NaCl treatment (Kamanika et al. 1998, Savoure

et al. 1999, Kawasaki et al. 2001). Hernandez et al. (2000) found that transcript levels

for cytosolic GR were strongly induced in the NaCl-tolerant pea variety. Tsai et al.

(2004) that NaCl and Na-gluconate treatments resulted in an enhancement of the

expression of the OsGR in the roots office seedlings. They revealed that OsGR gene

expression is up-regulated by NaCl in rice leaves. There was an early increase in GR

activity in NaCl-exposed shoot cultures of rice (Fadzilla et al. 1997).

103