7

CHAPTER-2

REVIEW OF LITERATURE

In this chapter an attempt has been made to review the work done on the effects of abiotic

stresses on Andrographis paniculata. The general objective of the present study is to understand

the response of A. paniculata to various abiotic stresses (drought, salinity and heavy metal stress)

by evaluation of relative tolerance of plant in terms of morphological and physiological traits.

The review is limited to physiological responses and impact on secondary metabolites due to

abiotic stress. However, due to paucity of literature available on Andrographis paniculata,

studies on other plants have been incorporated in order to understand complexicities. The

literature is reviewed under following aspects:

2.1 Andrographis paniculata

2.2 Abiotic stress

2.3 Growth

2.4 Biomass and productivity

2.5 Physiological aspects

2.6 Nutrients

2.7 Plant antioxidant defense system

2.1 ANDROGRAPHIS PANICULATA

Andrographis paniculata Wall ex Nees (King of bitter/ Hempedu Bumi/ Kalmegh)

belonging to Acanthaceae family, is commonly used to cure many ailments and diseases (Kumar

et al., 2004). It is widely found and cultivated in tropical and subtropical Asia, South-east Asia

and India (WHO, 2002). It is a herbaceous annual, erect (1m height) with acutely quadrangular

branched stem (Fig. 2.1). Leaves are simple, opposite, lanceolate, glabrous, 2-12 cm long, 1-3 cm

wide; apex acute; margin entire, slightly undulate, upper leaves often bractiform; petiole short.

Inflorescence patent, terminal and axillary in panicle, 10-30 mm long; bract small; pedicel short.

Calyx 5-particle, small linear. Corolla tube narrow, about 6 mm long; limb longer than the tube,

bilabiate; upper lip oblong, white with a yellowish top; lower lip broadly cuneate, 3-lobed, white

with violet markings. Stamens 2, inserted in the throat and far exserted; anther basally bearded.

Superior ovary, 2-celled; style far exserted. Capsule erect, linear

wide, compressed, longitudinally furrowed on broad faces, acute at both ends, thinly glandular

hairy. Seeds small, subquadrate (

used medicinally, contains a large number of chemical constituents, m

diterpenoids, diterpene glycosides, flavonoids and flavonoids glycosides. Owing to its high

curing value and wild occurrence in diverse environments, it has been considered to be a

promising plant for marginal lands, new reclaimed

Every part of the plant

extensively used in Unani and Ayurvedic

(Srivastava et al., 2004). In the Unani system of medicine,

inflammatory, emollient, astringent, diuretic, emmenagogue, gastric and liver tonic,

carminative, antihelmintic and antipyretic. Due to its “blood purifying” activity it is

recommended for use in leprosy, gonorrhea, scabies

seasonal fevers (Srivastava et al

antiviral, choleretic, hypoglycemic, hypocholesterolemic, and adaptogenic effects (Bhatnagar

al., 1961).

Figure

celled; style far exserted. Capsule erect, linear-oblong, 1-2cm long and 2

, compressed, longitudinally furrowed on broad faces, acute at both ends, thinly glandular

hairy. Seeds small, subquadrate (WHO, 2002). A. paniculata is cultivated for its aerial parts,

used medicinally, contains a large number of chemical constituents, m

diterpenoids, diterpene glycosides, flavonoids and flavonoids glycosides. Owing to its high

curing value and wild occurrence in diverse environments, it has been considered to be a

promising plant for marginal lands, new reclaimed-soils and semi-arid regions.

has its therapeutic value, especially leaves (fr

Unani and Ayurvedic medicines and in various herba

In the Unani system of medicine, it is considered aperients, anti

inflammatory, emollient, astringent, diuretic, emmenagogue, gastric and liver tonic,

carminative, antihelmintic and antipyretic. Due to its “blood purifying” activity it is

recommended for use in leprosy, gonorrhea, scabies, boils, skin eruptions, a

al., 2004). The plant extracts exhibits antibacterial, antifungal,

antiviral, choleretic, hypoglycemic, hypocholesterolemic, and adaptogenic effects (Bhatnagar

Figure 2.1: Andrographis paniculata (Kalmegh)

8

2cm long and 2-5mm

, compressed, longitudinally furrowed on broad faces, acute at both ends, thinly glandular-

is cultivated for its aerial parts,

used medicinally, contains a large number of chemical constituents, mainly lactones,

diterpenoids, diterpene glycosides, flavonoids and flavonoids glycosides. Owing to its high

curing value and wild occurrence in diverse environments, it has been considered to be a

resh and dry) are

bal combinations

it is considered aperients, anti-

inflammatory, emollient, astringent, diuretic, emmenagogue, gastric and liver tonic,

carminative, antihelmintic and antipyretic. Due to its “blood purifying” activity it is

, boils, skin eruptions, and chronic and

s antibacterial, antifungal,

antiviral, choleretic, hypoglycemic, hypocholesterolemic, and adaptogenic effects (Bhatnagar et

9

The therapeutic activities in this plant are due to the andrographolide (AG) and its related

diterpene lactones such as andrographolic acid, andrographiside, neo-andrographolide (NAG)

and 14-deoxy-11,12-didehydroandrographolide (DDAG) (Srivastava et al., 2004) (Fig. 2.2). In

vitro studies showed AG activity against HIV virus (Calabrese et al., 2000). Neo-andrographolide

has also shown activity against malaria (Misra et al., 1992) and various liver disorders (Kapil et

al., 1993). Andrographolide is an important biological constituent present in aerial part of the

plant, which is a group of diterpene lactones belonging to ent-labdane class, present in both free

and glycosidic forms (Tang and Eisenbrand, 1992; Lim et al., 2012). Andrographolide (Fig. 2.2),

an extremely bitter substance, is colourless and neutral crystalline. It was first isolated by

Boorsma from different parts of the plant (Tang and Eisenbrand, 1992). In 1911, Gorter proved

that it is structurally a lactone and named it andrographolide (in the Chinese literature it is

sometimes cited as andrographis B). The chemical investigations of the compound and its

diterpenoid lactone nature, as well as its stereochemistry, conformation and crystal structure

were studied by means of infrared, X-ray diffraction, mass spectrometry and NMR analysis. Its

chemical formula is 3,14,15,18-tetrahydroxy-5,9 H,10-labda-8(20),12-dien-16-oic acid-lactone

(Fig. 2.2). Various epimers, geometric isomers, and rearrangement products of andrographolide

were isolated and structurally characterized (Matsuda et al., 1994; Pramanick et al., 2006).

Andrographolide is generally extracted with CHCl3/EtOH or acetone, and several methods are

described in the literature to determine it by titration with alkalis, TLC/UV spectrophotometry

and HPLC methods. Highest quality of andrographolide and related diterpenoids is in the mature

leaves (Sharma et al., 1992). The stem had 0.2%, seeds 0.13%; root 0.44%; and leaves 2.39% of

andrographolide. The andrographolide content varied due to region as well as with season. The

leaves contained more than 2% andrographolide before the plant blossoms; afterward the

contents decreased to less than 0.5% (Tang et al., 2012). The growth regulators ABA and GAs

treated plants showed increased contents of andrographolide when compared to control

(Anuradha et al., 2010). The pH modified the stability of andrographolide, and hydrolysis is

extremely slow below pH 7, but considerably faster on the alkaline side, producing some

structural changes. Andrographolide is sparingly soluble in water; soluble in acetone, methanol,

chloroform and ether. As a water soluble andrographolide derivative, the sodium bisulfite adduct

has been synthesized for medical use as an antipyretic agent.

10

Figure 2.2: Formulae of the main constituents

Preclinical properties include anti-retroviral (Reddy et al., 2005; Wiart et al., 2005),

antiproliferative and pro-apoptotic (Yang et al., 2010; Zhou et al., 2008), anti-diabetic (Yu et al.,

2008; Zhang et al., 2009), anti-angiogenic (Sheeja et al., 2007), anti-thrombotic (Tisoda et al.,

2006), anti-urothelial (Sheeja and Kuttan, 2006), anti-leishmaniasis (Sinha et al., 2000),

hepatoprotective (Handa and Sharma, 1990), protective activity against alcohol-induced hepatic

and renal toxicity (Singha et al., 2007), cardioprotective (Woo et al., 2008) and anti-

inflammatory (Bao et al., 2009; Hidalgo et al., 2005; Li et al., 2009; Parichatikanond et al.,

2010; Qin et al., 2006; Wang et al., 2004) characteristics.

11

2.2 ABIOTIC STRESS

Plants, because of their sessile nature, are the foremost organisms which always face

several environmental stresses such as extreme temperatures, drought, water logging, salinity,

and heavy metals, which severely affect productivity. Growth, yield, and quality of medicinal

and aromatic plants have been reported to be influenced by these environmental constraints.

Drought and salinity has the most effect on medicinal plants (Mohammadkhani and Heidari,

2008). Abiotic stresses (salinity, drought, heat/cold, light, heavy metals and other hostile

conditions) are responsible for oxidative stress and a common denominator in all these adverse

conditions is the overproduction of the reactive oxygen species (ROS) within different cellular

compartment of the plant cell (Pinheiro et al., 2004). ROS formation is initiated by the univalent

reduction of molecular oxygen using either one, two or three electrons generating superoxide,

hydrogen peroxide (H2O2) or hydroxyl radical (OH·), respectively or by the formation of singlet

oxygen (1O2) by transfer of excess excitation energy to O2 (Alscher et al., 2002; Collakova and

DellaPenna, 2003; Wu et al., 2008). ROS are divided in to two main classes consisting of non-

radical species (H2O2) or free radical forms (O2˙˙̄, OH·, OH2·). Accumulation of high

concentration of ROS is potentially detrimental to plant cells causing damage to valuable

biomolecules like DNA, proteins, lipids, chlorophyll, membrane etc. (Blokhina et al., 2003).

Generation of ROS has greater toxicity potential on biomolecules and membranes (Scandalios,

2005; Miller et al., 2007). It is also known that plants resist to the stress-induced production of

ROS by increasing component amounts of their defensive system (Foyer et al., 1994; Zabalza

et al., 2008). Plant cells are normally protected against such effect by a complex antioxidant

system as non-enzymatic and enzymatic antioxidants (Smirnoff, 1995; Ali et al., 2008). ROS are

now also considered as key regulatory molecules vital for cells, but they cause cellular damage

when produced in excess or when the antioxidant defense system is not properly functioning.

Excess ROS lead to plant death or considerable yield losses. Plants with the ability to scavange

and/or control the level of cellular ROS may be useful in future to withstand harsh environmental

conditions. The ability to tolerate abiotic stress by plants depends on several interacting

variables such as the plant growth stage, stress concentration and the duration of stress over

time.

The impact of different types of stresses is reviewed herewith:

12

2.2.1 Salinity Stress

Salinity is a major abiotic stress affecting approximately 7% of the world’s total land

area resulting in billion dollar losses in crop production around the globe (Shabala and Cuin,

2007). With the use of chemical fertilizers and development of irrigation agriculture, salinity

land areas have increased year by year. Salt stress causes multifarious adverse effects in plants.

It is one of the major environmental stresses affecting the performance of many crop plants

(Yamaguchi and Blumwald, 2005). Soil salinity is detrimental to plant growth and productivity

as it causes nutritional imbalances by altering the uptake of nitrogen, phosphorus, potassium,

and calcium, and interferes with the cellular metabolism by causing ion toxicity and osmotic

perturbations. Massive changes in ionic and water balance cause molecular damage and arrest

growth (Jaleel et al., 2008a). Further, under field conditions, plants may experience several

stresses simultaneously. These stresses elicit composite responses which can be additive and

synergistic but rarely antagonistic. As a consequence of salinity stress-induced ion imbalance

and hyper-osmotic effects, the functioning of important cellular organelles like mitochondria and

chloroplast is adversely affected. Salinity stress decreased almost all of growth parameters in

Nigella sativa, some growth parameters and essential oil amount in Matricaria chamomilla

(Razmjoo et al., 2008) and essential oil yield in Melissa officinalis (Ozturk et al., 2004).

High concentrations of Na+ and Cl- ions impair biochemical as well as photochemical

processes of photosynthesis (Munns and Tester, 2008), cause stomatal closure and increase

mesophyll resistance for CO2 diffusion (Tuffers et al., 2001). However, damage on the

photosynthetic machinery may also occur due to non-stomatal limitation, i.e., decrease in

RuBisCO activity (Delfine et al., 1998), destruction of fine chloroplast structure (Barhoumi et

al., 2007), and damage to photosynthetic systems. Specific stress-related biochemical markers

have been identified in plants that serve as indicators to assess the extent of damage caused by

exposure to stress and also the ability of the affected plant to withstand the imposed stress.

Proline is one such biochemical marker which has been widely studied in plants and accumulate

exorbitantly in plants exposed to different abiotic stresses (Arora and Saradhi, 2002).

Accumulation of proline is one of the most widespread and consistent responses to salinity

stress. Proline is a compatible osmolyte (Taylor, 1996) that is reported to play diverse roles

under stress conditions as in stabilization of proteins, membranes and sub-cellular structures,

13

2free radical scavenging, and in protecting cellular functions (Bohnert et al., 1999; Matysik et

al., 2002). Production of reactive oxygen species (ROS) is one of the deleterious effects of

salinity stress. When molecular O2 undergoes reduction, it gives rise to ROS such as superoxide

(O-), hydrogen peroxide (H2O2) and the hydroxyl radical (OH•). Singlet oxygen (1O2), which

may arise due to the reaction of O2 with excited chlorophyll, is also considered as one of the

potential ROS (Ashraf, 2009). These ROS are highly reactive because they can interact with a

number of cellular molecules and metabolites thereby leading to a number of destructive

processes causing cellular damage. Plants possess antioxidant metabolites, enzymes and

non- enzymes to a variable extent, which have the ability to detoxify ROS (Ashraf, 2009).

Antioxidants have been considered as beneficial for enhancing plant stand and mitigating the

effects of biotic and abiotic stresses (Singh et al., 2010). However, in spite of having a potential

antioxidant defense system, plants do suffer extensive damage on exposure to salinity stress.

This indicates that the plant’s defense system has a limited capacity to detoxify reactive oxygen

species produced as a result of exposure to stress. There is evidence to suggest that the

alleviation of oxidative damage and increased resistance to salinity stress is correlated with an

efficient antioxidant defense system (Acar et al., 2001; Shigeoka et al., 2002).

2.2.2 Water Stress

The amount of water available to plants is important, since water accounts for 80-90% of

the fresh weight of most herbaceous plant structures and over 50% of the fresh weight of woody

plants (Kramer and Boyer, 1995). Certainly, most land plants are exposed to short or long term

water stress at some times in their life cycle and have tended to develop some adaptive

mechanisms for adapting to changing environmental conditions. Water stress may range from

moderate, and of short duration, to extremely severe and prolonged summer drought that has

strongly influenced evolution and plant life. Water deficit is a major abiotic factor affecting

global crop yield (Manavalan et al., 2009). In arid and semiarid regions, plants are often exposed

to water deficit stress, also known as drought stress. It seems the worldwide losses in crop yields

from water deficit probably exceed the cumulative loss of all other stresses (Kramer, 1983).

Field experiments related to water stress has been difficult to handle due to significant

environmental or drought interactions with other abiotic stresses (Rauf, 2008). An alternative

approach is to induce water stress through polyethylene glycol (PEG) solutions (Nepomuceno et

14

al., 1998; Kulkarni and Deshpande, 2007; Rajendran et al., 2011; Zhang et al., 2011; Patade et

al., 2012; Khodarahmpour, 2013). Polyethylene glycol molecules with a molecular weight C

6,000 (PEG 6000) are inert, non-ionic and cell impermeable. Therefore, they are frequently used

to simulate osmotic stress, as this solution interferes with the roots to absorb water due to

reduction of osmotic potential (Dodd and Donovan, 1999; Bhargava and Paranjpe, 2004; Van

den Berg and Zeng, 2006; Radhouane, 2007; Sidari et al., 2008). To understand drought stress

tolerance in plants different morphological, physiological and biochemical parameters were

investigated by many researchers under drought stress induced by PEG (Li and Wang, 2001;

Kocheva and Georgiev, 2003; Gui-fang, 2008; Patade et al., 2012; Ye et al., 2013;

Khodarahmpour, 2013; Uzilday et al., 2014).

Water deficit has been known to induce a sequence of morphological, biochemical and

molecular alterations that negatively affect plant growth and productivity (Wu et al., 2008;

Efeoglu et al., 2009). Roots are the place where plants first encounter water stress, it is likely that

roots may be able to sense and respond to the stress condition (Xiong et al., 2006;

Khodarahmpour, 2013). It plays an important role in water stress tolerance by reduction in leaf

expansion and promotion of root growth (Achten et al., 2010). Root length at seedling stage

provides a fair estimate about the root growth in field (Ali and Ashraf, 2011; Rajendran et al.,

2011). Reduction in shoot length perhaps is due to less water absorption and decrease in external

osmotic potential created by PEG (Kaydan and Yagmur, 2008). Drought has drastically affected

fresh and dry shoot and root weight in some cultivars of sorghum, wheat, maize and sunflower

(Bibi et al., 2012). Drought stress can cause primarily the formation of reactive oxygen species

(ROS) and the alteration of water potential within the plant (Sanchez-Rodriguez et al., 2010).

The ROS is potentially harmful to cell membranes, resulting in oxidative degradation of

membrane lipids (lipid peroxidation). Malondialdehyde (MDA) is one of the end breakdown

products of lipid peroxidation and can be used as an indicator of in vivo lipid peroxidation. To

protect against oxidative stress, plants have developed different scavenging mechanisms to

control the level of ROS by an interacting network of antioxidant enzymes such as superoxide

dismutase (SOD) and catalase (CAT) (Xue and Liu, 2008). The SOD converts superoxide to

H2O2 and O2. H2O2 is then scavenged by CAT and a variety of peroxidases through the oxidation

of co-substrates such as phenolics or other antioxidants (Basu et al., 2010). As studied earlier,

accumulation of proline is one of plant adaptive strategies to environmental stresses, particularly

15

low water stress. Proline is also closely related with plant drought stress, in which free proline

can significantly accumulate in crops and other plants (Hare and Cress, 1997; Ain-Lhout et al.,

2001; Kim et al., 2008; Lee et al., 2009). As an osmoprotectant in plants subjected to drought

conditions, proline can accumulate to high concentrations in plant cells without disrupting

cellular structure or metabolism. Accumulation of proline is believed to play an important role in

osmotic adjustment, detoxification of ROS and membrane integrity in plants under stress

conditions (Smirnoff and Cumbes, 1989; Hare and Cress, 1997; Matysik et al., 2002). Ghane et

al. (2012) reported higher accumulation of proline, glycine betaine and total soluble sugars and

lower damage to membrane lipids under increased water deficit in niger (Guizotia abyssinica).

2.2.3 Heavy Metal Stress In Relation To Cadmium

Environmental pollution due to heavy metals became extensive as mining and industrial

activities increased. Heavy metal pollutants, derived from a growing number of diverse

anthropogenic sources i.e., industrial effluents and wastes, urban runoff, sewage treatment plants,

agricultural fungicide runoff, domestic garbage dumps, and mining operations, have

progressively affected more and more different ecosystems (Macfarlane and Burchett, 2001).

Increased contents of heavy metals, especially non-essential metals like Cd, in soil affect

physiological processes in plants. Cadmium (Cd) is a potential environmental phytotoxicant. The

main sources of its pollution on agricultural soils are the use of phosphate fertilizers, dispersal of

sewage sludge, mining and atmospheric deposition of industrial emission (Pinto et al., 2004;

Kuriakose and Prasad, 2008). In living organisms, Cd accumulation results into cytotoxic,

mutagenic and carcinogenic effect (Kuriakose and Prasad, 2008). In plants, Cd is known to

cause physiological and morphological effects such as stunted growth, chlorosis and decreased

reproducibility. Cadmium adversely affects germination of seeds, plant growth and metabolism.

Siddiqui et al. (2009) reported that Cd has inhibitory effect on seed germination of pea (Pisum

satiivum) plant. Leaf chlorosis, leaf and root necrosis, general decrease in growth are the main

symptoms of Cd toxicity in plants (Hernandez and Cooke, 1997). Earlier studies have shown

that Cd toxicity in rice plants leads to inhibition in seedling vigour, causes stunted growth of the

plants, decreases the activities of many key hydrolytic enzymes and induces synthesis of proline

and certain novel proteins (Shah and Dubey, 1998). Cd interacts with proteins and nucleic acids,

as a result affects enzyme activities and causes alteration in membrane permeability (Sanita di

16

Toppi and Gabbrielli, 1999) leading to the loss of membrane function (Khan et al., 2007).

Cd causes significant decrease in fresh and dry weight, length of root and shoot, protein,

chlorophyll, carotenoids and sugar and increase in starch content in leaves of Phyllanthus amarus

(Rai et al., 2005). Cadmium can alter the uptake of minerals by plants through its effects on the

availability of the minerals from the soil (Moreno et al., 2001). In general, Cd has been shown to

interfere with the uptake, transport and use of several elements (Ca, Mg, P and K) and water by

plants (Das et al., 1997). Besides, the other plant systems which were studied to investigate

Cd stress are: Allium sativum (Liu et al., 2007), Hordeum vulgare (Finkemeier et al., 2003),

Cannabis sativa (Linger et al., 2005), Colocassia esculentum (Patel et al., 2005), Triticum

aestivum (Khan et al., 2007), Arabidopsis thaliana (Smeets et al., 2008), Lactuca sativa

(Cornu et al., 2008), Nicotiana tabacum (Wang et al., 2008), Solanum lycopersicon

(Chamseddine et al., 2008), Sorghum bicolor (Kuriakose and Prasad, 2008), Glycine max

(Luan et al., 2008; Shamsi et al., 2008), Vigna mungo (Molina et al., 2008), Zea mays

(Kumar et al., 2008), etc.

At the cellular level, the possible underlying mechanisms may be the bindings of Cd to

sulfhydryl and carbonyl groups or the replacement of essential cofactors which can finally

lead to oxidative stress (Smeets et al., 2008). The plant cell membranes are generally

considered as the primary site of injury and destabilization of membrane is attributed to lipid

peroxidation (Singh et al., 2006). Cd stress is known to increase MDA production in pea

(Metwally et al., 2005), rice (Shah et al., 2001), Thlaspi caerulescens, Brassica juncea (metal

accumulator) and Nicotiana tabacum (Wang et al., 2008). Increase in lipid peroxidation has also

been reported in maize (Kumar et al., 2008). On the other hand, Cd accumulation can also

induce changes in antioxidative systems of a cell (Balestrasse et al., 2006); e.g., in maize (Zea

mays), Cd enhanced antioxidative defence system with increased lipid peroxidation and

hydrogen peroxide (H2O2) accumulation (Kumar et al., 2008). Over accumulation of superoxide

anion in Cd-treated plants has been demonstrated by Rodriguez-Serrano et al. (2006). On

the other hand, Brassica juncea, a metal accumulator, develop superior antioxidative defense

system (CAT, POX, SOD, APX, GPX) to adapt to the oxidative stress (due to ROS generation)

induced by Cd toxicity (Wang et al., 2008). Namjooyan et al. (2012) reported elevated

antioxidant (GSH and α-tocopherol) and enzymatic antioxidant (SOD, APX, and GR) levels in

Carthamus tinctorius callus and found this activity more important and promising to the higher

17

Cd tolerance. Furthermore, substantial increases were observed in antioxidant enzymes, such as

catalase (CAT), glutathione S-transferase (GST) and peroxidase (POD), in Cd-stressed plants in

comparison with control in leaves and roots of Raphanus sativus (El-Beltagi et al., 2010). In

Phyllanthus amarus, the therapeutically active compounds-phyllanthin and hypophyllanthin,

enhanced at certain levels of Cd (Rai et al., 2005). This may be due to abiotic stress of metals

which often induce the synthesis and accumulation of the secondary metabolites (Mithofer et al.,

2004; Rai et al., 2004). There are many examples available where plants synthesize and

accumulate secondary metabolites upon treatment with heavy metals (Mithofer et al., 2004). For

example, Cd stress in Lactuca sativa and in Lupinus albus increases the level of aspargine in root

exudates (Sanita di Toppi and Gabbrielli, 1999) and lubimin and 3-OH- lubimin in Datura

stramonium (Mithofer et al., 2004).

2.3 GROWTH

Plant growth and productivity is adversely affected due to abiotic stresses. Plants are

frequently exposed to many stress conditions, such as low temperature, salt, drought, flooding,

heat, oxidative stress, and heavy metal toxicity (Mahajan and Tuteja, 2005). Growth is an

important tool for assessing crop productivity in various crops (Li and Wang, 2003).Growth is

one of the most stress-sensitive physiological processes due to the reduction of turgor pressure.

Cell expansion occurs when turgor pressure is greater than the cell wall yield threshold. Water

stress greatly suppresses cell expansion and cell growth due to the low turgor pressure

(Karthikeyan et al., 2007; Jaleel et al., 2007). Li et al. (2008) demonstrated in pearl millet that

osmotic regulation enabled to maintain cell turgor for survival and to assist plant growth under

severe drought conditions. The reduction in plant height is associated with the decline in the cell

enlargement and more leaf senescence, as reported in Abelmoschus esculentus under water stress

(Sankar et al., 2007).

The impact of water stress on seed germination and morphological parameters are reviewed

herewith:

2.3.1 Seed Germination

Seed germination and early seedling growth are considered the most critical phases for

18

establishment of any species. Germination of seed is strongly influenced by various abiotic

stresses like water-deficit, salinity, heavy metals, temperature and light requirement and these

factors often show significant interaction in their effects on germination. Salinity impairs seed

germination, reduces nodule formation, retards plant development and reduces crop yield

(Greenway and Munns, 1980). Seed germination was negatively affected by increased

concentrations of salinity in Atriplex patula and Plantago psyllium (Ungar, 1996; Mohammadi et

al., 2013). Jamil et al. (2006) reported that salt treatments strongly affect seed germination in

sugar beat, cabbage, amaranth and pak-choi. Seed germination speed decreased when the salinity

level was raised in Canola cultivars (Bybordi et al., 2010). But, on the other hand, saline stress

(NaCl 1g/l) stimulated the seed germination in jojoba (Berrichi et al., 2010).

Polyethylene glycol (PEG) induced water-deficit stress caused decreased percent seed

germination in Niger (Guizotia abyssinica), which is due to decreased water potential (Ghane et

al., 2012). PEG also affected seed germination by lowering its water potential in soybean

cultivars and Anabasis aphylla (Sakthivelu et al., 2008; Soleimani et al., 2011). Okcu et al.

(2005) reported a decrease in seed germination by increase in water-deficit stress induced by

PEG. In peanut (Arachis hypogea), cadmium stress do not inhibit seed germination even in the

presence of high concentration of Cd2+ (500µM) (Titov et al., 1995; Rascio et al., 2008), while in

Phaseolus vulgaris seed germination was reduced by 10 % (Bahmani et al., 2012) under

cadmium stress. Chen et al. (2003) reported a decrease in seed germination in carrot and radish

with an increase in cadmium concentration. Bhattacharya and Puri (2012) reported a decrease in

seed germination of Dioscorea deltoidea with an increase in concentration of polyethylene

glycol, NaCl and Cd; the degree of suppression was maximum in case of NaCl.

2.3.2 Stem Length

Abiotic stresses cause a reduction in stem length. Alla et al. (2011) reported a significant

decrease in the shoot length of Atriplex halimus at 300 and 550 mM NaCl by about 15 and 27%,

respectively; nevertheless, 50mM had no effect. Length of shoot decreased in response to salinity

in two wild species of potato i.e., Solanum stoloniferum and Solanum bulbosus (Daneshmand et

al., 2010). Under salinity stress the two species of Pistacia exhibited differences in terms of stem

length. In P. atlantica stem length was not affected by salt, however, P. vera underwent a

19

significant decrease at 60 and 80mM NaCl in terms of stem length (Chelli-chaabouni et al.,

2010).

Stem length decreased in Albizzia seedlings under drought stress (Patel and Golakia,

1988). Similar results were observed in Eucalyptus microtheca seedlings (Marron et al., 2002)

and Populus species (Nicholas, 1998). There was a significant reduction in shoot height in

Populus cathayana under water deficit (Nautiyal et al., 2002). In Glycine max, the stem length

decreased under water-deficit, but this decrease was not significant when compared to

wellwatered control plants (Manavalan, 2009). In peanut (Arachis hypogea), cadmium stress

reduced the shoot length at high concentration of 500µM (Shan et al., 2012). Reduction in

shoot length was also observed in Phaseolus vulgaris and Oryza sativa in all concentrations of

Cd (Bahmani et al., 2012; Panda et al., 2011). Bhattacharya and Puri (2012) reported that stem

length of Dioscorea deltoidea was suppressed by three abiotic stresses namely PEG, NaCl as

well as Cd.

2.3.3 Root Length

Roots are often reported to play a key role in the salt tolerance of plants for they represent

the first organ to control the uptake and translocation of nutrients and salt throughout the plant.

Root characteristics, especially root length density, and the number of thick roots, are important

for a plant to have comparatively well-established aboveground parts by exploiting the available

water, as in rice (Manivannan et al., 2007a). The accumulation of Na+ in the roots is an adaptive

response used by several woody species to avoid its toxicity in the shoots (Picchioni et al., 1990;

Gucci and Tattini, 1997). Enhancement in salinity decreased root length for all Brassica napus

cultivars and also in Seuvium portolacastrum (Bybordi, 2010; Slama et al., 2008), however, an

increase in root length with increasing concentration of NaCl (0-200mM) was observed in

Kochia prostrata (Karimi et al., 2005).

Drought avoidance due to a profound root system that enhances the ability of a plant to

capture water is a fundamental adaptation mechanism to drought (Liu et al., 2005). More severe

drought stress suggests that the dynamics of root growth under drought conditions might be a

key factor to the understanding of the contribution of roots to drought avoidance (Moran et

al., 1994). Water stress reduces the biomass of fibrous roots in Avocado cultivars (Nicholas,

20

1998) and in pearl millet (Manivannan et al., 2007; Lawlor and Cornic, 2002). The growth rate

of wheat and maize roots was found decreasing under moderate and high water-deficit stress

(Noctor and Foyer, 1998). However, the development of the root system increases water

uptake and maintains the right osmotic pressure through higher proline levels (Munns et al.,

1979; Munns and Weir, 1981; Noctor et al., 2002). The root growth was not significantly

reduced under water deficits in Zea mays and Triticum aestivum (Rao et al., 1993). Roots

became shorter and thicker and the whole root system became more dense and compact due

to cadmium stress in Miscanthus sinensis (Scebba et al., 2006). Reduction in root length was

reported at high concentrations of Cd in Arachis hypogea seedlings and in carrot and radish

(Shan et al., 2012; Chen et al., 2003) and reduction by 83.9% in Phaseolus vulgaris

(Bahmani et al., 2012). Reduction in root length was reported in Dioscorea deltoidea with

increasing concentration of stresses namely PEG, NaCl and Cd (Bhattacharya and Puri, 2012).

2.3.4 Leaf Area

Reduction in leaf area by salinity, water and heavy-metal stress is an important cause

of reduced crop yield through reduction in photosynthesis. Salt stress had reducing effect on

leaf area of seedlings. Leaf area reduction under salinity stress can be considered an avoidance

mechanism which minimizes water losses (Shaheen and Hood-Nowotny, 2005; Ruiz-Sanchez et

al., 2000). Sucre and Suarez (2011) opined that in Ipomoea pes-caprae the concentration of salt

reduced leaf area proportionately. In Oryza sativa the leaf areas in salt treated seedlings (0, 25,

50, 100 and 200mM) decreased by 79%, 40.7%, 28.7% and 3% compared to untreated seedlings

(Amirjani, 2010). Salinity stress also reduced the leaf area in Sesuvium portulacastrum (Slama

et al., 2008). Bybordi (2010) reported a decrease in leaf area as the concentration of NaCl

increased (0, 75, 150, 200, 250mM) but it increased at higher concentration of 300mM NaCl.

Leaf-area plasticity is important to maintain the control of water use in crops. The total

leaf area per plant decreased significantly in Eragrostis curvula, Oryza sativa, Abelmoschus

esculentum and Asteriscus maritimus due to water deficit (Rucker et al., 1995; Sadras et al.,

1993; Passioura, 1977; Rose et al., 1993; Shubhra and Goswami, 2003). In Sorghum, leaf area

reduced significantly under water stress. This reduction occurred before stomatal conductance

decreased in the remaining viable leaf area (Babu and Rao, 1983; Correia et al., 2001;

21

Meenakshi et al., 2005). The reduction in leaf area under water stress may be associated with

the decline in the cell enlargement and more leaf senescence in Abelmoschus esculentum

(Sankar et al., 2007). Water-deficit stress mostly reduced leaf growth and in turn the leaf area

in many species of plants, like Populus, Ziziphus, etc. (Suther and Patel, 1992; Tahir et al.,

2003; Thakur and Kaur, 2001; Thakur and Sood, 2005).

Increase in cadmium concentration decreased leaf area in dill (Anethum graveolens),

radish (Raphanus sativus) and five cultivars of Indian mustard (Brassica juncea) (Aghaz et

al., 2013; El-Beltagi et al., 2010; Gill et al., 2011). Nikolic et al. (2008) observed that in

hybrid poplar (Populus nigra × maximowitzii × P. nigra var. Italica) the leaf area decreased

from 1259 to 527 at 10-5 to 10-4M Cd concentration. Munoz et al. (2008) observed that a dose

of 10 μM cadmium increased soybean (Glycine max) leaf area, while a dose of 40 μM

inhibited it.

2.4 BIOMASS AND PRODUCTIVITY

2.4.1 Fresh and Dry Weight

A decrease in total dry matter (biomass) is generally due to the decrease in plant growth,

photosynthesis and canopy structure under abiotic stress conditions. Amirjani (2010) reported

that both fresh and dry weights of rice culms with leaves reduced with increasing salinity.

Biomass reduced by 89.7%, 84.6%, 69.6% and 42.2% when grown with 25, 50, 100 and 200mM

salt, respectively, compared to untreated plants. Joshi et al. (2011) reported that shoot fresh and dry

weight of four varieties of Brassica juncea decreased progressively with an increase in NaCl

concentration. The root dry weight under mild and severe water stress decreased in Beta

vulgaris (Pan et al., 2002). Nearly one third of biomass decreased in Ziziphus rotundifolia under

drought conditions (Tsialtas et al., 2001). Progressive drought resulted in a significant reduction

in early allocation of dry matter and decreased biomass in Populus davidiana (Zhang et al.,

2004a). Plant productivity under drought stress is strongly related to the processes of dry matter

partitioning and temporal biomass distribution in Populus cathayana (Willekens et al., 1997).

Regulated deficit irrigation and partial root drying caused a significant reduction in shoot biomass

when compared to control in common bean plants (Yancy et al., 1982). Many studies revealed

that biomass is dependent upon Cd concentration. Cd is toxic at concentrations more than 5–

22

10μg Cd g-1 leaf dry weight except for Cd-hyperaccumulators which can tolerate 100μg Cd g-1

leaf dry weight (Reeves and Baker, 2000; Broadley et al., 2001; Verbruggen et al., 2009). Scebba

et al. (2006) reported no visible symptoms of Cd toxicity in shoots and rhizomes of Miscanthus

sinensis grown in presence of Cd but dry biomass was affected by Cd concentrations.

2.4.2 Relative Water Content (RWC)

Relative water content (RWC) of leaves is an important character, which is

directly related to soil water content (Sarker et al., 1999). The maintenance of favorable plant

water status is an essential strategy for plant tolerance to stresses that result in cellular water-

deficit and loss of turgor pressure. RWC is the major tool for assessing changes in plant water

relations for studying plant responses to stress and subsequent relation to stress tolerance. RWC

has been used as a trait for screening different environmental stresses, where plants that maintain

higher RWC tend to be more tolerant to stress compared to plants that cannot efficiently control

leaf water status. It has been observed that the species which are better adapted to dry

environment have higher relative water content (RWC) at given water potential (Jarvis and

Jarvis, 1963).

One of the early symptoms of salinity stress in plant tissue is the decrease of relative

water content (RWC). This reduction of RWC in stressed plants may be associated with a

decrease in plant vigor and was observed in many plant species (Halder and Burrage 2003).

Relative water content in the leaves of plants grown under salinity stress decreased significantly

in two sugar beet cultivars and three rice cultivars compared to those grown in non-saline soil

(Farkhondeh et al., 2012; Pattanagul and Thitisaksakul, 2008). However, differences between

cultivars were very small. Cicek and Cakirlar (2002) also observed a decrease in RWC after salt

stress in two maize cultivars, but this reduction was visible only at the -0.5 MPa. Salinity level

caused reduction in relative water contents (RWC) of leaves. It reduced from 71% in untreated

plants to 67%, 64%, 60% and 58% in plants treated with 25, 50, 100 and 200mM salt,

respectively, in Oryza sativa (Amirjani, 2010). Salt stress induced a reduction in the relative

water content of the leaves, which indicates a loss of turgor that resulted in limited water

availability for cell extension process (Katerji et al., 1997).

23

Merah (2001) opined that relative water content is an important indicator of water stress

in leaves. Stress exposed plants immediately lower down relative water content of their leaves,

the decrease in leaf water potential and osmotic potential is also reported (Grover et al., 2011).

Sorghum bicolor suffers a smaller decrease in relative water content per unit change in leaf water

potential than cotton (Ackerson and Krieg, 1977) and Zea mays (Levitt, 1980). It has also been

observed that un-irrigated plants show a much smaller change in relative water content per

change in water potential (Leviit, 1980). Coyne et al. (1982) studied correlation of relative water

content (RWC) with tissue elasticity and concluded that low tissue elasticity contribute to

drought resistance by maintaining higher relative water content at zero turgor potential. Klar

(1984) studied RWC in the leaves of Triticum aestivum under controlled environment and

suggested as drought resistance indicator.

It has been reported that relative water content is an indicator of water status and through

its relation to cell volume; it reflects the balance between water supply to the leaf and

transpiration. Relative water content is used as the selection criterion for drought tolerance in

many plants (Sinclair and Ludlow, 1985; Matin et al., 1989; Ritchie et al., 1990). Khan et al.

(2009) evaluated four Triticum aestivum cultivars for drought resistance and found that cultivars

with smallest decrease in relative water contents produced higher dry weight and grain yield per

plant and were taller compared to other cultivars. There was also a positive correlation between

yield and relative water contents (Khan et al., 2009). Cadmium also interacts with the water

balance of the plant. Cadmium generally decreases the water stress tolerance of plants, causing

low turgor at higher leaf water potentials and relative water content. Aghaz et al. (2013) reported

that relative water content in the leaf of untreated Anethum graveolen showed highest amount.

Reduced RWC due to heavy metals, especially cadmium, is because of reduced uptake of water

by roots (Alsokari and Aldesuquy, 2011). Aksoy and Dinler (2012) viewed that relative water

content decreased under combined effect of Cd and salt and Cd treatment alone as compared to

control group.

2.5 PHYSIOLOGICAL ASPECTS

2.5.1 Membrane Stability

Under environmental stresses plant membranes are subject to changes often associated

with increase in permeability and loss of integrity (Blokhina et al., 2003). Therefore, the ability

24

of a cell membrane to control the rate of ion movement in and out of cells is used as a test of

damage to a great range of tissue.

Salt condition under irrigation water affects physiological process negatively including

water relations and gas exchange attributes (Maeda and Nakazawa, 2008), nutritional imbalance

(Yang et al., 2008), and disturbing the stability of membranes (Dogan et al., 2010). An excessive

amount of toxic ions (Na+ and Cl-) in plant tissues unstable the cellular membranes by displacing

K+ and Ca2+ (Grattan and Grieve, 1992) and affect their permeability. These variations in cell

membrane permeability due to the toxic ions i.e. Na+ and Cl-, results in the disturbances in

various physiological processes (Kao et al., 2006; Sayed, 2003). Membrane stability index (MSI)

decreased under salt stress in pea genotypes at all NaCl treatments, but maximum reduction was

under 75 mM (Shahid et al., 2012). Since membranes damage increased with an increase in

salinity level, hence MSI is considered as a significant tool for evaluating the salt tolerance

potential. Salt stress also caused considerable membrane injuries to the leaves of tested varieties

of cotton and membrane stability index (MSI) were negatively influenced by salinity (Saleh,

2013).

Biological membranes are the first target of many abiotic stresses. It is generally accepted

that the maintenance of integrity and stability of membranes under water stress is a major

component of drought tolerance in plants (Bajji et al., 2002). Cell membrane stability, reciprocal

to cell membrane injury, is a physiological index widely used for the evaluation of drought

tolerance (Premachandra et al., 1991). Tolerance to drought seems to be due to increase in cell

membrane stability under water deficit conditions (Premachandra et al., 1991). Cell-membrane

stability is considered to be one of the major selection indices of drought tolerance in cereals

(Zhang et al., 2004). The water stress induced decrease in membrane stability indicates the extent

of lipid peroxidation caused by active oxygen species (Dhindsa et al., 1981; Pastori and Trippi,

1992; Baisak et al., 1994; Menconi et al., 1995). Lower lipid peroxidation and higher membrane

stability (lower ion leaching) have also been reported in tolerant genotypes of Triticum aestivum

(Kraus et al., 1995) and Zea mays (Pastori and Trippi, 1992). Sairam and Saxena (2000) found

decrease in the membrane stability index in leaves with increasing age as well as under water

stress. They opined that accumulation of H2O2 under stress also led to enhance potential for

production of hydroxyl radicals, which leads to lipid peroxidation and membrane deterioration.

The extent of water stress induced damage to membranes is negatively correlated with the

25

capacity for increasing activities of SOD and CAT in drought tolerant and drought-sensitive

mosses (Dhindsa and Matowe, 1981) and jute (Chowdhury and Choudhuri, 1985).

Cadmium has been shown to damage cell membrane, induce oxidative stress, and

enhance the ionic-leakage in plants (Sharma et al., 2012). Cell membrane injury is associated

with the production of ROS, which also leads to the production of MDA (Hossain et al., 2012).

CdCl2 treatment decreased membrane stability index in Vigna radiata seedlings and it decreased

by 8% to 25% with 25 to 100 µM CdCl2 (Verma et al., 2012). In Helianthus annuus Cd toxicity

was linked to free radical processes in membrane components leading to alterations in membrane

stability and increasing their permeability (Moradkhani et al., 2012).

2.5.2 Carbohydrate

Carbohydrates play a central role in plant metabolism and regulate plant growth and

development. Sucrose and its derivatives are the major transport forms of assimilated carbon in

plants. For long distances in most plants sucrose is transported from source to sink organs via the

phloem. The transport of sugars is important as sugar acts as signalling substance in plants.

Among all organic compounds, soluble carbohydrates represent about 50% of the total

osmotically active organic solutes (Ashraf and Harris, 2004). The accumulation of soluble

carbohydrates in plants has been widely reported as a response to salinity or drought, despite a

significant decrease in net CO2 assimilation rate (Murakeozy et al., 2003). Carbohydrate

composition of seedlings got altered due to salinity, as reported by Amirjani (2010). There are

reports that salinity increase carbohydrates in some plant species (Lacerda et al., 2003; Silva et

al., 2003) or decrease in others (Agastian et al., 2000). Silva et al. (2008) reported that the

carbohydrate content in leaves of Spondias tuberosa plant showed a small but significant increase

(18 %) in plants grown above 50 mM NaCl (P< 0.01). In stems, carbohydrates increased by 40 %

in 25 mM NaCl treated plants. In roots, a reduction of 32 % in the soluble carbohydrate was

observed at salt levels of 50, 75, and 100 mM NaCl. In Phaseolus vulgaris (L.), Vassey and

Sharkey (1989) observed a similar decline in partitioning of carbohydrates to starch but not to

sucrose. Dhanapackiam and Ilyas (2010) reported that the total carbohydrate in the leaves was

much higher than in the root of the NaCl (10, 20, 30, 40 and 50 mM) treated Sesbania

grandiflora seedlings.

26

Many researchers viewed that the carbohydrates in the leaves of various water stressed

plants alters and may act as a metabolic signal in response to drought (Akinci and Losel, 2009;

Akinci and Losel, 2010; Chaves et al., 2003; Koch, 1996). Among the major effects in water

stressed plants is the accumulation of sugars and a number of other organic solutes (Kameli and

Losel, 1995). Munns et al. (1979) and Quick et al. (1992) showed that sugars contribute to

osmotic adjustment in wheat leaves. Many studies support this conjecture that soluble sugars

accumulate in leaves during water stress and might contribute to osmoregulation (Quick et al.,

1992; Jones et al., 1980; Munns and Weir, 1981; Ackerson, 1981; Kameli and Losel, 1996).

Total carbohydrates increase was reported in cotton (Timpa et al., 1986) and total soluble sugar

increase in durum wheat (Kameli and Losel, 1996) and nodulated alfalfa (Irigoyen et al., 1992).

Soluble carbohydrate contents in plants decreased with increasing concentration of heavy

metals (Bhardwaj, 2009). The presence of excessive amounts of Cd in soil commonly elicits

many stress symptoms in plants, such as disturbances in carbohydrate metabolism (Moya et al.,

1993). The carbohydrate content decreased in the plant body of Salvinia natans, with the time of

exposure and concentration of cadmium. This may be attributed to the formation of complexes of

carbohydrate with Cd molecules which prevents the enzymatic degradation by changing the

conformation of the carbohydrate (Mohan and Hosetti, 2006). Vijayarengan (2012) observed

that excess of cadmium disturbed carbohydrate metabolism in the leaves of rice as carbohydrate

content decreased with the corresponding increase in cadmium concentration.

2.5.3 Amino Acids

Amino acids have traditionally been considered as precursors and constituents of

proteins. Many amino acids act as precursors of other nitrogen containing compounds, e.g.,

nucleic acids. The amino acids have several roles in plants, for example, they act as osmolytes in

response to abiotic stress, detoxify heavy metals, regulate ion transport, stomatal opening, affect

synthesis and activity of enzymes, gene expression and redox homeostasis (Rai, 2002). DeCosta

and Huang (2006) reported that amino acids are one of the major solutes responsible for osmotic

adjustment. Amino acids (alanine, arginine, glycine, serine, leucine, and valine, together with the

imino acid, proline and the non-protein amino acids, citrulline and ornithine) have also been

reported to accumulate in plants subjected to salt stress (Mansour, 2000). Total free amino acids

27

in the leaves have been reported to be higher in salt tolerant than in salt sensitive lines of

Helianthus annuus (Ashraf and Tufail, 1995), Carthamus tinctorius (Ashraf and Fatima, 1995),

Eruca sativa (Ashraf, 1994) and Lens culinaris (Hurkman et al., 1991). Rao et al. (2009)

reported an increase in total free amino acid content in leaves of five cultivars of Emblica

officinalis with increasing salinity.

Some reports are available where accumulation of other free amino acids under drought

has been shown, e.g., asparagines, alanine and gamma-amino butyric acid in Phaseolus mungo

(Rai and Bapat, 1977), arginine in Cryptomeria (Mori et al., 1971), aspartic acid, glutamic acid

and glutamine in Gossypium (Hanower and Brzozowska, 1975), asparagines, aspartic acid, serine

and glycine in Zea mays (Slukhai and Shvedova, 1972; Thakur and Rai, 1982), aspartic acid and

alanine in Iris (Paulin, 1972), proline, ornithrine, arginine, and glutamic acid in detached Oryza

sativa leaves (Yang et al., 2000). Stewart et al., (1980) found an accumulation of amino acids in

the presence of water deficit, leading to a dynamic adjustment of N metabolism. Navari-Izzo et

al. (1990) suggested that an increase in free amino acids contribute to the tolerance of the plant

to water deficit through an increase in osmotic potential, or as a reserve of N, principally for the

synthesis of specific enzymes. Under water stress, a decrease in proteins could reflect either

diminished synthesis or increased breakdown, leading to higher levels of free amino acids

(Navari-Izzo et al., 1990). Since the effects of drought depend on species, tissue and age, as well

as the nature, duration and degree of the stress, it is not surprising that marked differences have

been found in the amino acid pattern for stress conditions (Hanson and Hitz, 1982). Once water

deficit occurs, the level of free amino acids in sorghum plants increased from 32 to 39mM under

moderate stress and from 29 to 45mM under severe stress (Jones and Turner, 1980).

Total free amino acid with an increasing concentration of heavy metals increased in

plants. In Phaseolus vulgaris, for different concentrations of Cd i.e. 1.5,2 and 2.5g/kg soil, amino

acid content increased by 46.77%, 52.30% and 54.08%, respectively (Bhardwaj et al., 2009).

Vijayarengan (2012) reported that amino acid content of rice leaf decreased with an increase in

cadmium level in the soil. Amino acid content was 3.0 per 50mg kg-1 soil in Cd treated plants

compared to 5.7 in untreated plants. Total amino acid content increased progressively in leaves

and roots of Lycopersicon esculentum when treated with Cd and amino acid content increase was

higher in roots (Chaffei et al., 2004).

28

2.5.4 Protein

Abiotic stresses usually cause protein dysfunction. Maintaining proteins in their

functional conformations and preventing the aggregation of non-native proteins are particularly

important for cell survival under stress. Abiotic stress (e.g. drought, heat, hypoxia, heavy metal

pollution) strongly affect senescence and the degradation of chloroplast proteins (Feller et al.,

2008). In general, such a stress causes an early or accelerated senescence (Wingler and Roitsch,

2008). Reactive oxidative species (ROS), the partially reduced or activated derivatives of

oxygen, are highly reactive and toxic and can lead to cell death by causing damage to proteins,

lipids, carbohydrates and DNA (Mittler et al., 2004). Upon encountering oxidative stress,

proteins are oxidized by highly reactive and oxidative species, and these damaged, oxidized

proteins need to be degraded rapidly and effectively.

Proteins that accumulate in plants under saline conditions may provide a storage form of

nitrogen that is reutilized later and may play a role in osmotic adjustment (Singh et al., 1987). A

higher content of soluble proteins has been observed in salt tolerant cultivars of Hordeum

vulgare, Helianthus annuus, Eleusine coracana and Oryza sativa (Ashraf and Harris, 2004).

Agastian et al. (2000) have reported that soluble protein increases at low salinity and decreases at

high salinity in mulberry cultivars. The results of certain studies (Sultana et al., 2002; Tort and

Turkyilmaz, 2004; Chen et al., 2007; Kapoor and Srivastava, 2010; El-Beltagi et al., 2013)

demonstrate a decrease or increase, in protein content in plants treated with different salt

concentrations. Ashraf and Fatima (1995) found that salt tolerant and salt sensitive accessions of

safflower did not differ significantly in leaf soluble proteins. The soluble protein contents

decreased with increasing salinity in rice (Amirjani, 2010).

Proteins synthesized in response to drought stress are called dehydrins (dehydration

induced) and belong to the group II late embryogenesis abundant (LEA) proteins (Close and

Chandler, 1990). The dehydrin family of proteins accumulates in a wide range of plant species

under dehydration stress. Drought stress induced changes in protein synthesis in maize (Bewley

et al., 1997). The accumulation of dehydrin like proteins was detected in the roots and leaves of

drought-stressed plants, which could protect plants from further dehydration damage

(Mohammadkhani and Heidari, 2008). As result of plant exposure to water stress during growth,

alterations in protein expression, accumulation and synthesis have been observed in many plant

29

species (Chen and Tabaeizadeh, 1992). Both quantitative and qualitative changes to proteins are

known to occur during water stress (Riccardi et al., 1998). Ghorbanli et al. (2013) reported

that leaf total protein content decreased in severe drought and increased under mild drought

stress treatments compared with control in two tomato cultivars.

Protein content was also greatly affected by cadmium stress treatments. The reduction in

the amount of protein could be due to decrease in protein synthesis or an increase in the rate of

protein degradation (Balestrasse et al., 2003). Bavi et al. (2011) reported that protein content in

pea roots reduced significantly in the presence of high cadmium concentrations. In roots of

soybean plants cadmium treatments 50 and 200μM had caused an increase in the rates of

protease activity. However in nodule, higher concentrations of cadmium (200μM) decreased both

protease activity and total protein content (Balestrasse et al., 2003). This reflects the toxic effects

of supra cadmium concentrations on protein synthesis machinery which results in both a

decrease in protease activity and the content of other proteins. Possibly, by reacting with the SH-

groups cadmium may result in protein denaturation (Fuhrer, 1982). Heavy metals are known to

promote protein denaturation and to increase the hydrolytic activities of proteases, RNAase and

DNAase enzymes (Gadd and Griffith, 1978).

2.5.5 Proline

Amino acid proline is known to occur widely in higher plants and normally accumulates

in large quantities in response to environmental stresses (Rains, 1989; Ashraf, 1994; Rhodes et

al., 1999; Ozturk and Demir, 2002; Hsu et al., 2003). In plants, the precursor for proline

biosynthesis is l -glutamic acid. Two enzymes, pyrroline -5-carboxylate synthetase (P5CS) and

pyrroline -5-carboxylate reductase (P5CR), play major roles in proline biosynthetic pathway

(Delauney and Verma, 1993). In addition to its role as an osmolyte for osmotic adjustment,

proline contributes to stabilizing sub-cellular structures (e.g. membranes and proteins),

scavenging free radicals, and buffering cellular redox potential under stress conditions. It may

also function as a protein compatible hydrotrope (Srinivas and Balasubramanian, 1995),

alleviating cytoplasmic acidosis, and maintaining appropriate NADP/NADPH ratios compatible

with metabolism (Hare and Cress, 1997). Also, rapid breakdown of proline upon relief of stress

may provide sufficient reducing agents that support mitochondrial oxidative phosphorylation and

30

generation of ATP for recovery from stress and repairing of stress-induced damages (Hare and

Cress, 1997; Hare et al., 1998).

Accumulation of proline under stress in many plant species has been correlated with

stress tolerance, and its concentration has been shown to be generally higher in stress-tolerant

than in stress-sensitive plants. For example, while in salt-tolerant Medicago sativa, proline

concentration in the roots rapidly doubled under salt stress, in salt-sensitive plants the response

was slow (Fougere et al., 1991; Petrusa and Winicov, 1997). Similarly, salt-tolerant ecotypes of

Agrostis stolonifera accumulated more proline in response to salinity than salt-sensitive ecotypes

(Ahmad et al., 1981). Besides positive effects of proline on improving plant salt tolerance at the

organismal level, considerable improvement in salt tolerance has been observed at the cellular

level. For example, in vitro studies with brown mustard (Brassica juncea) indicated that salt-

adapted calli had higher accumulation of free proline compared with non-stressed calli (Madan et

al., 1995). Proline accumulation also occurs in plants subjected to drought stress. For example, in

Oryza sativa plants subjected to water deficit, the concentration of proline increased in the leaves

(Hsu et al., 2003). This drought-induced accumulation of proline was related to increased

contents of the precursors for proline biosynthesis, including glutamic acid, ornithine and

arginine. In Triticum aestivum, an assessment of the effects of drought stress on proline

accumulation in a drought-tolerant and a drought-sensitive cultivar revealed that the rate of

proline accumulation and utilization was significantly higher in the drought-tolerant cultivar

(Nayyar and Walia, 2003). Zhang et al. (2011) reported higher levels of free proline

accumulation in cv. Xiuyan than cv. Yuline under water-deficit stress induced by PEG 6000

suggesting that the free proline also contributes to the better protection against reduced oxidative

damage in Jerusalem artichoke. Significantly higher accumulation of proline under increased

waterdeficit stress (i.e., at 80 and 60% FC) conditions in cultivar IGPN 2004 of niger (Guizotia

abyssinica) suggest its more tolerance capacity to water deficit in comparison to other cultivars

of Hyoscyamus niger (Ghane et al., 2012).

Proline accumulation in plants is a response to heavy metals to maintain the osmotic

balance in the cells of plant, which can be used as a physiological parameter for the tolerance

response of plant. Heavy metal stress stimulates free proline accumulation in some plant species

(Schat et al., 2002; Pandey and Sharma, 2002; Zengin and Munzuroglu, 2005). Free proline

31

increases plant tolerance to stress by osmoregulation, stabilizing protein synthesis, and protecting

enzymes against denaturation (Kuznetsov and Shevyakova, 1997). Singh and Gautam (2013)

suggested that the accumulation of proline in plants protect the cell membrane and proton pump

against damage. Zhou et al. (2010) studied that the concentration of proline in the leaves and

roots of Iris pseudacorus increased significantly under 25 mg L-1 Cd and 500 mg L-1 Pb.

Balestrasse et al. (2005) found that proline concentration increased significantly in nodules

and roots of soybean plants under Cd treatments.

2.6 NUTRIENTS

Plant growth depends on the supply of inorganic nutrients, which vary in time and space.

To cope with such fluctuating environments, plants have developed high levels of plasticity both

at the individual level and within species and ecotypes. Nevertheless, extreme nutrient conditions

cause deficiency or toxicity to a varying extent for different plant species. Excessive amount of

soluble salts in the root environment cause osmotic stress, which may result in disturbance of the

plant water relations, in the uptake and utilization of essential nutrients, and also in toxic ion

accumulation. As a result of these changes, the activities of various enzymes and the plant

metabolism are affected (Munns, 2002; Lacerda et al., 2003). The interaction of salts with

mineral nutrients may result in considerable nutrient imbalances and deficiencies (McCue and

Hanson, 1990). Ionic imbalance occurs in the cells due to excessive accumulation of Na+ and

Cl– and reduces uptake of other mineral nutrients, such as K+, Ca2+, and Mn2+ (Karimi et al.,

2013).

Salt stress decreases the calcium/sodium ratio in the root zone, which affects membrane

properties, due to displacement of membrane-associated Ca2+ by Na+, leading to dissolution of

membrane integrity and selectivity (Cramer et al., 1995; Kinraide, 1998). The increased levels of

Na+ inside the cells change enzyme activity resulting in cell metabolic alteration;

disturbance in K+ uptake and partitioning in the cells and throughout the plant that may

even affect stomatal opening, thus diminishing the ability of the plant to grow. High sodium to

potassium ratio due to accumulation of high amounts of sodium ions inactivates enzymes and

affects metabolic processes in plants (Booth and Beardall, 1991). Potassium (K) plays an

important role in survival of plants under environmental stress conditions. Potassium is essential

for many physiological processes, such as photosynthesis, translocation of photosynthates into

32

sink organs, maintenance of turgescence, activation of enzymes, and reducing excess uptake of

ions such as Na and Fe in saline and flooded soils (Marschner, 1995; Mengel and Kirkby, 2001).

Environmental stress factors that enhance the requirement for K also cause oxidative damage to

cells by inducing formation of ROS, especially during photosynthesis (Bowler et. al., 1992;

Elstner and Osswald, 1994; Foyer et al., 1994).

The reason for the enhanced need of K by plants suffering from environmental stresses

appears to be related to the fact that K is required for maintenance of photosynthetic CO2

fixation. For example, drought stress is associated with stomatal closure and thereby with

decreased CO2 fixation. Decrease in photosynthesis caused by drought stress is particularly high

in plants supplied with low K, and are minimal when K is sufficient (Gupta et al., 1989).

Alleviation of detrimental effects of drought stress, especially on photosynthesis, by sufficient K

supply has also been shown in legumes (Sangakkara et al., 2000). Under water-deficit conditions,

K nutrition increases crop tolerance to water stress by utilizing the soil moisture more efficiently

than in K-deficient plants. Potassium maintains the osmotic potential and turgor of the cells

(Lindhauer, 1995) and regulates the stomatal functioning under water stress conditions (Kant and

Kafkafi, 2002). It enhances photosynthetic rate, plant growth and yield under stress conditions

(Egila et al., 2001). The protective role of K in plants suffering from drought stress by

maintenance of a high pH in stroma and against the photooxidative damage to chloroplasts was

also reported by Cakmak and Romheld (1997). Cadmium accumulates in the roots and decreases

the water uptake by roots (Barcelo and Poschenrieder, 1990; Vozary et al., 1997). However,

cadmium accumulates also in the leaves (Di Cagno et al., 1999). The content of Na and K in

leaves of pea plant diminished significantly by Cd treatment (Sandalio et al., 2001). Na and K

also showed a significant reduction in Cd treated barley plants (Smykalova and Zamecnikova,

2003).

2.7 PLANT ANTIOXIDANT DEFENSE SYSTEMS

Exposure of plants to unfavorable environmental conditions can increase the production

of reactive oxygen species e.g., 1O2, O2•-, H2O2 and OH•. These ROS are highly reactive because

they can interact with a number of cellular molecules and metabolites thereby leading to a

number of destructive processes causing cellular damage. During the course of evolution, plants

have developed several strategies to defend themselves against reactive oxygen species

33

generated under environmental stresses (Foyer et al., 1994; Inze and Montagu, 1995).

Antioxidants have been touted as beneficial for enhancing plant stand and mitigating the effects

of biotic and abiotic stresses (Singh et al., 2012). Antioxidant activity, possessed by numerous

vitamins, secondary metabolites and other phytochemicals, serve as protection against the

damaging effects of highly reactive molecules known as the radicals (McDermott, 2000; Eraslan

et al., 2007; Hong-Bo et al., 2008). Plant cells are normally protected by two main antioxidant

systems as enzymatic which includes SOD, CAT, APX, MDHAR, DHAR and GR (Table 2.1;

Fig. 2.3) and non-enzymatic antioxidants such as GSH, AA, carotenoids and tocopherols

(Smirnoff, 1995; Ali et al., 2008). Without these defense mechanisms, plants could not efficiently

convert solar energy to chemical one.

2.7.1 Enzymatic Antioxidants

Enzymatic antioxidants include enzymes capable of removing, neutralizing, or

scavenging oxy-intermediates. They comprise superoxide dismutase (SOD), ascorbate

peroxidase (APX) and glutathione reductase (GR), which are believed to scavenge H2O2 in

chloroplast and mitochondria. The other enzymatic anti-oxidants, catalase (CAT) and peroxidase

(POD), are capable of removing H2O2, neutralizing or scavenging free radicals and

oxyintermediates (Karpinski and Muhlenbock, 2007).

Table 2.1: Major ROS scavenging antioxidant enzymes

Enzymatic antioxidants Enzyme Code Reaction catalyzed

Superoxide dismutase (SOD)

Catalase (CAT)

Ascorbate peroxidase (APX)

Guaicol peroxidase (GPX)

Monodehydroascorbate reductase (MDHAR)

Dehydroascobate reductase (DHAR)

Glutatione reductase

EC 1.15.1.1

EC 1.11.1.6

EC 1.11.1.11

EC 1.11.1.7

EC 1.6.5.4

EC 1.8.5.1

EC 1.6.4.2

O2. + O2

. + 2H+ → 2H2O2 + O2

H2O2 → H2O + 1/2 O2

H2O2 + AA → 2H2O + DHA

H2O2 + GSH → H2O + GSSG

MDHA +NAD(P)H → AA + NAD(P)+

DHA + 2GSH → AA + GSSG

GSSG+NAD(P)H→2GSH+ NAD(P)+

34

Abiotic Stresses

O2-

H2O

H2O2

½ O2 + H2OOH·

H2O + GSSG

SOD APX

Fenton reaction

AA

MDHAR

NADP

NADPHMDHA

DHAR

DHA

Figure 2.3: ROS and antioxidants defense mechanism

Superoxide dismutase (SOD) is a metal-containing enzyme that catalyzes the dismutation

of superoxide radical to oxygen and H2O2. It is well established that various environmental

stresses often lead to the increased generation of ROS where SOD has been proposed to be

important in plant stress tolerance and provide the first line of defense against the toxic effects of

elevated levels of ROS. Dismutation to H2O2 by SOD initiates subsequent formation of hydroxyl

radical by the Fenton reaction (Foyer et al., 1994) (Fig. 2.3). Oxidative damage alleviation was

reported in salt stressed Catharanthus plants through the action of SOD in all parts (Jaleel et

al., 2007, 2008b) indicating that there might be an efficient active oxygen scavenging system.

SOD activity in potato increases at lower salt level in salt-tolerant cultivars. However, at higher

salt levels, SOD activity is reduced in all cultivars (Chen et al., 2011).

Under drought stress, enhanced SOD activity was found in pea (Moran et al., 1994) and

tobacco (Van Rensburg et al., 1994) and SOD activity decreased in sunflower seedlings

(Quartacci and Navaril, 1992); unaffected activity of SOD in maize (Luna et al., 1985); while in

35

wheat, SOD activity increased or remained unchanged in the early phase of drought but

decreased with prolonged water stress (Zhang and Kirkham, 1995). Under cadmium stress SOD

activity showed no clear effects in Miscnathus sinensis. It enhanced in Carthamus tinctorius at

lower concentration of Cd but declined at higher concentration of Cd (Scebba et al., 2006;

Namjooyan et al., 2012). However, Cd induced an increase in SOD activity at all concentrations

in Arachis hypogaea and poplar (Dinakar et al., 2008; Nikolic at al., 2008).

Catalase (CAT) is tetrameric heme containing enzymes that catalyze the dismutation of

H2O2 into water and oxygen (Table 2.1) and is indispensable for ROS detoxification during stress

conditions (Garg and Manchanda, 2009). CAT has one of the highest turnover rates for all

enzymes: one molecule of CAT can convert ≈ 6 million molecules of H2O2 to H2O and O2 per

minute. Eyidogan and Oz (2005) reported a significant increase in CAT activity in Cicer

arietinum leaves under salt treatment. Similarly, increase in CAT activity in C. arietinum roots

following salinity stress was noted by Kukreja et al. (2005). Srivastava et al. (2005) reported a

decrease in CAT activity in Anabaena doliolum under NaCl and Cu2+ stress. Simova-Stoilova et

al. (2010) reported increased CAT activity in wheat under drought stress but it was higher

especially in sensitive varieties. In another study, Sharma and Dubey (2005) reported a decrease

in CAT activity in rice seedlings following drought stress. It has also been reported that high

light conditions increased the CAT acivity in Picea asperata under drought stress (Yang et al.,

2008). Pan et al. (2006) studied the combined effect of salt and drought stress and found that it

decreased the CAT activity in Glycyrrhiza uralensis seedlings. The variable response of CAT

activity has been observed under heavy metal stress. Its activity declines in Glycine max

(Balestrasse et al., 2001); Phragmites australis (Iannelli et al., 2002); Capsicum annum (Leon et

al., 2002) and Arabidopsis thaliana (Cho and Seo, 2005) whereas its activity increased in

Brassica juncea (Mobin and Khan, 2007), Triticum aestivum (Khan et al., 2007) and Cicer

arietinum (Hasan et al., 2008) under Cd stress.

Peroxidases (POD) constitute a wide variety of heme containing enzymes involved in

many physiological processes in plants. It is involved in responses to biotic and abiotic stresses,

auxin catabolism, cell wall lignification and degradation (Vicuna, 2005). They also provide

downstream signaling molecules for other transduction pathways (Hoang, 2010). Peroxidases are

also involved in the scavenging of Reactive Oxygen Species (ROS). Peroxidases comprise a

36

large family of related proteins that catalyze the conversion of hydrogen peroxide to water in the

presence of an electron acceptor. However, peroxidases can also be a source of hydrogen

peroxide (H2O2) (Apel and Hirt, 2004; Vicuna, 2005). Water deficit stress, salinity stress and

cadmium stress increased the POD activity in Glycine max, Vigna unguiculata and Brassica

juncea (Pujari and Chanda, 2002; Mobin and Khan, 2007; Zhang et al., 2008).

Ascorbate peroxidase (APX) is one of the most important antioxidant enzymes that play

an essential role in scavenging ROS and protecting cells in higher plants, algae, euglena and

other organisms. APX is involved in scavenging of H2O2 in water-water and ascorbate–

glutathione cycles and utilizes ascorbate as the electron donor (Table 2.1). Different isoforms

of APX are active in chloroplasts, cytosol and microsomes. APX is more specific and use

ascorbic acid as electron donor, but to a lesser extent, can also use guaiacol or other substrates

(Mehlhorn et al., 1996; Jaleel et al., 2007). Ascorbate peroxidase utilizes H2O2 to oxidize

ascorbic acid to monodehydroascorbate (MDHA) radical, which disproportionate to DHA non-

enzymatically. monodehydroascorbate reductase (MDHAR) regenerate AA at the expense of

nicotinamide adenine dinucleotide phosphate (NADPH) and dehydroascorbate reductase

(DHAR) regenerate AA utilizing glutathione (GSH) to form oxidized glutathione (GSSG) (Fig.

2.3). Enhanced expression of ascorbate peroxidase (APX) in plants has been demonstrated

during different stress conditions. In different plant species, APX increases in response to a

variety of biotic and abiotic stresses (Asada, 1999). Enhanced activity of APX was also found in

salt stressed Anabaena doliolum (Srivastva et al., 2005). Significant increase in APX activity

was noted under water stress in three cultivars of Phaseolus vulgaris (Zlatev et al., 2006) and

Picea asperata (Yang et al., 2008). Sharma and Dubey (2005) found that mild drought stressed

plants had higher chloroplastic-APX activity than control grown plants but the activity declined

at the higher level of drought stress. It has been noted that overexpression of APX in Nicotiana

tabacum chloroplasts enhanced plant tolerance to salt and water deficit (Badawi et al., 2004).

Increased leaf APX activity under Cd stress has been reported in Ceratophyllum demersum

(Arvind and Prasad, 2003), Brassica juncea (Mobin and Khan, 2007) and Triticum aestivum

(Khan et al., 2007).

37

GPXs (GPX) are a large family of diverse isozymes that use GSH to reduce H2O2 and

organic and lipid hydroperoxides and therefore help plant cells from oxidative stress (Noctor et

al., 2002) These reductions are catalysed by glutathione peroxidases (GPOXs):

R-OOH + 2GSH → ROH + H2O + GSSG

GPX is capable of membrane lipid peroxidation repair and is generally considered to be the

main line of enzymatic defense against oxidative membrane damage (Kuhn and Borchert,

2002). de Azevedo Neto et al., (2006) investigated responses of salinity stress to maize and

observed an increase with time in GPX activities. Stress increases GPX activity in cultivars of

Capsicum annum (Leon et al., 2002) but decreases in roots and cause no significant change in

the leaves of Cd exposed Pisum sativum plants (Dixit et al., 2001). Glutathione peroxidase

(GPX) activity in leaves of Phragmites australis decreased in Cd2+ plants compared to control

plants (Iannelli et al., 2002).

The plant glutathione transferases, formerly known as glutathione S-transferases (GST,

EC 2.5.1.18), are a large and diverse group of enzymes which catalyse the conjugation of

electrophilic xenobiotic substrates with the tripeptide glutathione (GSH; γ-glu-cys-gly). Plant

GSTs are known to function in lignin biosynthesis, degradation of indole-3-acetic acid,

herbicide detoxification, hormone homeostasis, vacuolar sequestration of anthocyanin, tyrosine

metabolism, hydroxyperoxide detoxification, regulation of apoptosis and in plant responses to

biotic and abiotic stresses (Asada, 1992; Dixon et al., 2010). Noctor et al. (2002) reported that

GSTs have the potential to remove cytotoxic or genotoxic compounds, which can react or

damage the DNA, RNA and proteins. In fact, GSTs can reduce peroxides with the help of GSH

and produce scavengers of cytotoxic and genotoxic compounds. In an experiment drought

tolerant (M35-1) and drought sensitive sorghum (SPV-839) varieties were subjected to 150 mM

NaCl for 72 h. M35-1 exhibited efficient H2O2 scavenging mechanism with significantly higher

activities of GST and CAT (Jogeswar et al., 2006). An increased GST activity was found in

leaves and roots of Cd-exposed Pisum sativum plants (Dixit et al., 2001) and in roots of Oryza

sativa and Phragmites australis plants (Iannelli et al., 2002; Moons, 2003). Gapinska et al.

(2008) noted increased GST activity in Lycopersicon esculentum roots under salinity stress. It

has also been found that GST overexpression also enhance plant tolerance to various abiotic

38

stresses. Overexpression of GST/GPX in transgenic tobacco seedlings provides increased GSH-

dependent peroxide scavenging and alterations in GSH and ASH metabolism that lead to

reduced oxidative damage (Roxas et al., 2000).

2.7.2 Non-Enzymatic Antioxidants

Ascorbic acid (AA) is one of the most abundant, extensively studied antioxidant and has

been detected in majority of plant cell types, organelle and apoplast (Smirnoff, 2000;

Giovannoni, 2007). It is usually being higher in photosynthetic cells and meristems (and some

fruits). Its concentration is reported to be highest in mature leaves with fully developed

chloroplast and highest chlorophyll. It has been reported that ascorbic acid mostly remains

available in reduced form and chloroplast under normal physiological conditions (Smirnoff,

2000). Ascorbic acid is a water soluble antioxidant which acts to prevent or in minimizing the

damage caused by ROS in plants (Smirnoff, 2005; Athar et al., 2008). AA is linked to cell

growth, being involved in the cell cycle and other mechanisms of plant cell growth and division

as well as acting as a co-factor for many enzymes (Lee and Kader, 2000). AA has effects on

many physiological processes including the regulation of growth, differentiation and metabolism

in plants (Mehlhorn et al., 1996). AA influences many enzyme activities and minimizes the

damage caused by oxidative process through synergic function with other antioxidants (Asada

1999; Foyer et al. 1994). AA scavenges many types of free radicals affecting many enzyme

activities and it is also required for regeneration of α-tocopherol (Smirnoff, 2000). In the

ascorbate–glutathione cycle, two molecules of AA are utilized by APX to reduce H2O2 to

water with con- comitant generation of monodehydroascorbate (MDHA). MDHA is a radical

with a short lifetime and can disproportionate into dehydroascorbate (DHA) and AA. The

electron donor is usually NADPH and catalyzed by MDHA reductase (MDHAR) or ferredoxin in

water–water cycle in the chloroplasts (Asada, 1999). In plant cells, the most important reducing

substrate for H2O2 removal is AA. H2O2 removal by ascorbate is also important in the apoplast

(Blokhina et al., 2003). A direct protective role for AA has also been demonstrated in rice,

where partial protection against damage caused by a release from flooding conditions was

provided by the prior addition of AA (Chen and Gallie, 2004). Yang et al. (2008) reported that

high light condition and drought significantly increased the ascorbic acid content in Picea

asperata seedlings. Demirevska-Kepova et al. (2006) reported that the content of oxidized

39

ascorbate increased during Cd exposure in Hordeum vulgare plants. Nicotiana tabacum and

Populus × Canescens plants have higher foliar ascorbic acid contents and improved tolerance to

oxidative stress (Aono et al., 1993). Contrarily, a decrease in the AA in the roots and nodules of

Glycine max under Cd stress has also been observed (Balestrasse, 2001).

Tocopherols (vitamin E) are considered as a major lipophilic antioxidant synthesized by

all plants in the thylakoid membrane of chloroplasts, where they play both antioxidant and non-

antioxidant functions. Out of four isomers of tocopherols (α-, β-, γ-, δ-) found in plants, α –

tocopherol has the highest antioxidative activity due to presence of three methyl groups in its

molecular structure (Kamal-Eldin and Appelqvist, 1996) (Fig. 2.3). It is synthesized from γ-

tocopherol in chloroplasts by γ-tocopherolmethyltransferase. α-Tocopherols interact with the

polyunsaturated acyl groups of lipids, stabilize membranes and scavenge and quench various

ROS and lipid soluble byproducts of oxidative stress (Wang and Quinn, 2000). Singlet oxygen

quenching by tocopherols is highly efficient, and it is estimated that a single α-tocopherol

molecule can neutralize up to 120 singlet oxygen molecules in vitro before being degraded.

Because of their chromanol ring structure, tocopherols are capable of donating a single electron

to form the resonance stabilized tocopheroxyl radical (Yamaguchi et al., 2001). α-tocopherol

was consumed predominantly as radical scavenging antioxidant against the lipid peroxidation

as observed in the Glycine max membrane system (Clement et al., 2002). α-tocopherol levels

increase in photosynthetic plant tissues in response to a variety of abiotic stresses (Munne-

Bosch and Algere, 2002). Synthesis of low-molecular-weight antioxidants, such as α-tocopherol,

has been reported in drought-stressed plants (Munne-Bosch and Algere, 2002). Increase in

tocopherol during water stress in plants has also been reported by many workers (Wu et al.,

2008; Shao et al., 2007). Srivastava et al. (2005) reported a general induction in α-tocopherol

content in Anabaena doliolum under NaCl and Cu2+ stress.

Chlorophyll content is one of the major factors affecting photosynthetic capacity. Total

chlorophyll content is considered as a parameter reflecting salt tolerance (Srivastava et al., 1988;

Jaleel et al., 2008c). An increase in leaf chlorophyll content due to salinity was found in rice by

Asch et al. (2000), who suggested that the increased chlorophyll content reflected the nitrogen

sources available to the plant and not the physiological stress experienced by the leaf. Severe

drought stress also inhibits the photosynthesis of plants by causing changes in chlorophyll

40

content, by affecting cholorophyll components and by damaging the photosynthetic apparatus

(Iturbe-Ormaetxe et al., 1998). Ommen et al. (1999) reported that leaf chlorophyll content

decreases as a result of drought stress. Drought stress caused a large decline in the chlorophyll a

content, the chlorophyll b content, and the total chlorophyll content in all sunflower varieties

investigated (Manivannan et al., 2007b). The decrease in chlorophyll under drought stress is

mainly the result of damage to chloroplasts caused by active oxygen species (Smirnoff, 1995).

Nyachiro et al. (2001) described a significant decrease of chlorophyll a and b caused by water

deficit in six Triticum aestivum cultivars. A reason for decrease in chlorophyll content as affected

by water deficit due to production of reactive oxygen species (ROS), such as O2- and H2O2,

which can lead to lipid peroxidation and consequently, chlorophyll destruction (Foyer et al.,

1994). Also, with decreasing chlorophyll content due to the changing green color of the leaf into

yellow, the reflectance of the incident radiation is increased (Schlemmer et al., 2005).

Carotenoids are pigments that are found in all plants and microorganisms. In all

photosynthetic organisms, the carotenoids, ẞ-carotene and zeaxanthin, act as protactive “antenna

pigments” for photosynthesis, gathering wavelengths of light that are not absorbed by

chlorophylls (Vichnevetskaia and Roy, 1999). Carotenoids also have a protective function

against oxidative damage as they dissipate excess excitation energy as heat or by scavenging

ROS and suppressing lipid peroxidation. First, they absorb light at wavelength between 400 and

550 nm and transfer it to the Chlorophyll (an accessory light- harvesting role) (Siefermann-

Harms, 1987). Second, they protect the photosynthetic apparatus by quenching a triplet

sensitizer (Chl3), 1O2 and other harmful free radicals which are naturally formed during

photosynthesis (an antioxidant function) (Havaux et al., 2000; Collins, 2001). Third, they are

important for the PS I assembly and the stability of light harvesting complex proteins as well as

thylakoid membrane stabilization (a structural role) (Niyogi et al., 2001). Singlet oxygen is very

powerfully quenched by ẞ-carotene, allowing a relatively low concentration of ẞ-carotene to

effectively protect membrane lipids from reactions of O2 leading to peroxidation (Larson, 1988).

Rai et al. (2006), Singh et al. (2008) and Ekmekci et al. (2008) reported decreased carotenoid

contents in Phyllanthus amarus, Vigna mungo and Zea mays cultivars with increasing Cd

concentration. Carotenoid content of Hordeum vulgare seedlings decreased under Cd-stress

(Demirevska-Kepova et al., 2006).

41

All plants produce an amazing diversity of secondary metabolites. One of the most

important groups of these metabolites are phenolics compounds. Phenolics are characterized by

at least one aromatic ring (C6) bearing one or more hydroxyl groups. Antioxidant action of

phenolics compounds is due to their ability to eliminate radical species and to function as metal

chelators (Ksouri et al. 2008). Phenolics possess hydroxyl and carboxyl groups, able to bind

particularly iron and copper (Jung et al., 2003). The roots of many plants exposed to heavy

metals excude high levels of phenolics (Winkel-Shirley, 2002). It has been suggested that

peroxidase could act as an efficient H2O2 scavenging system in plant vacuoles in the presence

of phenolics and reduced ascorbate (Zancani and Nagy, 2000). Tannin-rich tea plants, which are

tolerant to Mn excess, are protected by the direct chelation of Mn. Direct chelation, or binding to

polyphenols, was observed with methanol extracts of rhizome polyphenols from Nympheae for

Cr, Pb and Hg (Lavid et al., 2001). Phenolic antioxidants inhibit lipid peroxidation by trapping

the lipid alkoxyl radical. This activity depends on the structure of the molecules, and the number

and position of the hydroxyl group in the molecules (Milic et al., 1998). Santiago et al. (2000)

hypothesize a cycle where H2O2 is scavenged by phenolics through a peroxidase; phenolics

are oxidized to phenoxyl radicals which can be reduced by AA. This cycle can occur in both the

apoplast and in the vacuole, where phenolics are particularly concentrated.

Flavonoids are among the most bioactive plant secondary metabolites and are commonly

found in leaves, floral parts, and pollens. They are also abundant in woody parts such as stems

and bark. Flavonoids usually accumulate in the plant vacuole as glycosides, but they also occur

as exudates on the surface of leaves and other aerial plant parts. Flavonoids are suggested to

have many functions like flowers, fruits, and seed pigmentation, protection against UV light;

defence against phyto- pathogens (pathogenic microorganisms, insects, animals); role in plant

fertility and germination of pollen and acting as signal molecules in plant-microbe

interactions (Olsen et al., 2010). It has been recognized that several classes of flavonoids show

antioxidant activity toward a variety of easily oxidizable compounds, such as ASH and a-

tocopherol (Hernandez et al., 2009). Flavonoids serve as ROS scavengers by locating and

neutralizing radicals before they damage the cell thus important for plants under adverse

environmental condition (Lovdal et al., 2010). Flavonoids function by virtue of the number and

arrangement of their hydroxyl groups attachment to ring structures. Their ability to act as

antioxidants depends on the reduction potentials of their radicals and accessibility of the radicals.

42

It has been found that there is considerable increase in flavonoid levels following biotic and

abiotic stresses, such as wounding, drought, metal toxicity and nutrient deprivation (Winkel-

Shirley, 2002). Production of flavanoids in response to UV-B, cold and drought were reported

(Kilian et al., 2007).

The peroxidation of lipids (primarily the phospholipids of cell membranes) is considered

as the most damaging process known to occur in every living organism. It involves oxidative

degradation of polyunsaturated fatty acids caused by ROS – is responsible for degradation of

membrane lipids resulting in cell damage and formation of many toxic products. Membrane

damage is sometimes taken as a single parameter to determine the level of lipid destruction under

various stresses. Now, it has been recognized that during lipid peroxidation, products are formed

from polyunsaturated precursors that include small hydrocarbon fragments such as ketones,

malondialdehyde (MDA) and compounds related to them (Garg and Manchanda, 2009). Some of

these compounds react with thiobarbituric acid (TBA) to form coloured products called

thiobarbituric acid reactive substances (TBARS) (Heath and Packer, 1968). Lipid peroxidation,

in both cellular and organelle membranes, takes place when above threshold ROS levels

are reached, thereby not only directly affecting normal cellular functioning, but also aggravating

the oxidative stress through production of lipid-derived radicals (Montillet et al., 2005).

It has been noted that plants exposed to various abiotic stresses exhibit an increase in

lipid peroxidation due to the generation of ROS. Treatment with Cd significantly increased the

accumulation of lipid peroxides in different plants (Mobin and Khan, 2007; Noriega et al., 2007;

Ammar et al., 2007; Razinger et al., 2008; Wang and Song, 2009; Singh et al., 2008). Khan and

Panda (2008) studied the cultivar response of Oryza sativa under salt stress and found that it

increased the lipid peroxidation in both cultivars of rice. Kukreja et al. (2005) noted marked

increase in lipid peroxidation in Cicer arietinum roots under salinity stress. It has also been

reported that water stress increased the lipid peroxidation, membrane injury index, H2O2 and OH

production in leaves of stressed Phaleolus vulgaris plants (Zlatev et al., 2006). Simova-Stoilova

et al. (2010) reported that the weakening of membrane integrity and oxidative damage to lipids

were more pronounced in the sensitive varieties under field drought conditions in wheat plants.

Thus, it is evident that abiotic stress factors influence growth and physiology in plants.

Oxidative stress induced by abiotic stress triggers signaling pathways that affect production of

43

specific plant metabolites. In particular, reactive oxygen species (ROS), generated during abiotic

stress, may cause lipid peroxidation that stimulates formation of highly active signaling

compounds capable of triggering production of bioactive compounds (secondary metabolites)

that enhances the medicinal value of the plant. Further research is needed to clarify the

mechanism by which heavy metals induce responses that result in enhanced secondary

metabolite production.