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CHAPTER - 1 INTRODUCTION

Chapter – 1 Introduction

1

WATER STRESS

“Millions of water drops without them no sound

No whispers of the living

Millions of water drops Treasured should they be

as belonging to all humanity”

− Morhardt Carmen Mencita Monoi Angel

Plants are frequently exposed to abiotic stresses such as drought, salinity, extreme

temperatures, chemical toxicity and oxidative stress. Each year, stresses on arable plants

in different parts of the world disrupt agriculture (Ludlow and Muchow, 1990; Hessine et

al., 2009). Water stress (drought) is the main abiotic factor limiting plant production in

several regions of the world, with crop growth and economic yield being severely

affected by water availability (Araus et al., 2002). Factors controlling stress conditions

alter the normal equilibrium, and lead to a series of morphological, physiological,

biochemical and molecular changes in plants, which adversely affect their growth and

productivity. The average yields from the major crop plants may reduce by more than

fifty percent owing to stresses (Bray et al., 2000; Wang et al., 2003). Plants require water

for photosynthesis, nutrient uptake and transportation as well as cooling (Farooq et al.,

2009b). Plants are sessile organisms and in contrast to most animals they are unable to

move when the environment becomes unfavorable. Accordingly, plants have to be able to

respond and adapt to the local environmental changes. Since water is essential for plant

survival, the ability to tolerate water stress is crucial.


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Water is the most copious compound in living organisms, comprising 80-90% of

the biomass of non-woody plants and more than 50% of the biomass of woody plants

(Kramer and Boyer, 1995). Water is the central molecule in all physiological processes of

plants by being the major medium for transporting metabolites and nutrients. Water is

one of the most important environmental factors which control plant productivity and

distribution of species. But, water is not consistently and lavishly available at all times

and places for supporting optimal growth of plants. Agriculture currently uses 75% of the

total global consumption of water (Molden, 2007), and in absolute terms consumptive

water use in agriculture will rise in coming decades (Falkenmark and Rockstr¨om, 2004).

With rising aridity and growing population, water will become an even scarce commodity

in the near future.

Plants experience water stress either when the water supply to their roots becomes

limiting or when the transpiration rate becomes intense (Jaleel et al., 2009a). Water stress

is primarily caused by the water deficit, i.e. drought or high soil salinity. In case of high

soil salinity and also in other conditions like flooding and low soil temperature, water

though exists in soil solution but plants cannot uptake it and such a situation commonly

known as ‘physiological drought’. Drought occurs in many parts of the world every year,

frequently experienced in the field grown plants under arid and semi-arid climates. Since

the dawn of agriculture, mild to severe drought has been one of the major production

limiting factors. Consequently, the ability of plants to withstand such stress is of immense

economic importance.


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Area under dryland agriculture in India:

Dryland agriculture means raising crops with rain water is spread in four

continents covering almost 48 countries. Worldwide, 6510 million hectares (mha) of land

is under rainfed agriculture of which approximately 60% are in developing countries.

India ranks first among the dryland agricultural countries in terms of both extent and

value of production. Out of every three hectares of cultivated land in India, nearly two

hectares are under rainfed agriculture (Singh, 2001).

India has about 91 million hectares of rainfed area which constitutes nearly 64%

of the total 142.1 million hectares of cultivated land. In such areas crop production

becomes relatively difficult as it mainly depends upon intensity and frequency of rainfall.

These areas get an annual rainfall between 400 mm to 1000 mm which is unevenly

distributed, highly uncertain and erratic. In certain areas the total annual rainfall does not

exceed 500 mm. The crop production, depending upon this rain, is technically called dry

land farming and areas are known as dry lands (Singh, 2001).

Importance of Dry farming in Indian Agriculture:

About 70% of rural population lives in dry farming areas and their livelihood

depend on success or failure of the mansoon. Insufficient water is the most serious

constraint to agricultural production. Dryland agriculture plays a distinct role in Indian

agriculture occupying 64% of cultivated area and supports 40% of human population and

60 % livestock population. Population growing at a rate greater than food production in

arid and semi arid regions, Hence tremendous efforts both in the development and

research fronts are essential to meet the food demand of human beings and live stock

food (Singh, 2001; Ram and Davari, 2010).


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The productivity of grains already showed a plateau in irrigated agriculture due to

problems related to nutrient exhaustion, salinity build up and raising water table.

Therefore, the challenges of the present millennium would be to produce more from

drylands while ensuring conservation of existing resources. Hence, new strategies would

have to be evolved which would make the fragile dryland ecosystems more productive as

well as sustainable.

Effect of water stress on whole plant system:

Water stress, as an abiotic stress, is multidimensional in nature, and it affects

plants at various levels of their organization (Yordanov et al., 2000; Chaves et al., 2003;

Wentworth et al., 2006). When the water availability is reduced, plants change the

biochemistry to be able to retain as much water as possible and take up whatever water

they can. During water stress plants produce and accumulate compatible solutes such as

sugars, polyols and amino acid to lower the osmotic potential in the cells to facilitate

water absorption and retention (Xiong and Zhu, 2002). Some of the compatible solutes

also contribute to maintaining the conformation of macromolecules by preventing

misfolding or denaturation (Xiong and Zhu, 2002). In fact, under prolonged drought,

many plants will dehydrate and die (Jaleel et al., 2009a). Water stress in plants reduces

the plant-cell’s water potential and turgor, as a result, cell enlargement decreases leading

to growth inhibition and reproductive failure (Mitra, 2001). At this stage, overproduction

of reactive oxygen species (ROS) aggravates the adverse influence (Ramakrishna and

Rao, 2012).


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Water stress not only affects plant water relations through the reduction of water

content, turgor and total water, it also affects stomatal closure, limits gaseous exchange,

reduces transpiration and arrests carbon assimilation rates. Negative effects on mineral

nutrition (uptake and transport of nutrients) and metabolism leads to a decrease in the leaf

area and alteration in assimilate partitioning among the organs. Alteration in plant cell

wall elasticity, disruption of homeostasis and ion distribution has been reported (Tuba et

al., 1996; Bray, 1997; Sarafis, 1998; Yordanov et al., 2003).

Synthesis of new proteins and mRNAs associated with the drought response is

another outcome of water stress on plants. Under the water stress cell expansion slows

down or ceases, and plant growth is retarded. However, water stress influences cell

enlargement more than cell division. Plant growth under drought stress is influenced by

altered photosynthesis, respiration, translocation, ion uptake, carbohydrates, nutrient

metabolism, and hormones (Xiong and Zhu, 2002; Jaleel et al., 2009b). Response to a

particular stress depends on the genetic and biochemical composition of plant species

(Dubey, 1994). Plants are unable to express their complete genetic potential, when

subjected to biotic and/or abiotic stresses (Ziegler, 1990). It is very clear that various

environmental stresses cause significant modifications in gene expression in plants. Such

variations may lead to accumulation or exhaustion of certain metabolites which probably

confer the plants to adapt to the stressful environment by physiological and biochemical

adjustments (Dubey, 1994).


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Water stress and plant growth:

Drought stress is a very important factor that adversely affects the seed

germination and establishment. It affects both elongation and expansion growth of plant

(Kusaka et al., 2005; Shao et al., 2008). The reduction in growth is common phenomenon

in plants under water stress conditions. (Heuer and Nadler, 1995; Specht et al., 2001; Wu

et al., 2008). Plant growth is achieved through cell division, cell elongation and cell

differentiation, and involves genetic, physiological, ecological and morphological events

and their complex interactions. The quality and quantity of plant growth depend on these

events, which are affected by water stress (Farooq et al., 2009b). Cell growth is one of the

most drought sensitive physiological processes due to the reduction in turgor pressure

(Taiz and Zeiger, 2006).The reduction in cell enlargement is highly turgor dependent

process and the cell turgor decreases with any dehydration in cell water potential (Levitt,

1980).

Root growth in soil can be limited by physical, chemical, and biological

properties of the soil. In terms of physical limitations to root growth, water stress (too

little water for root growth), hypoxia or anoxia (too little or no oxygen), and mechanical

impedance (soil that is too hard for roots to penetrate rapidly) are the major causes of

poor root system growth and development. Of these factors, there is a strong interaction

between the root growth and water content of soil. Roots are considered as an important

soil water deficity sensing organs in the plant (Wilkinson and Davies, 2002). Water stress

is known to manipulate various features of root such as root size, depth, length,

morphology, density and hydraulic conductance. The extent and pattern of root

development are closely related to the ability of the plant to absorb water and hence are a


7

great significance in drought tolerance. An increase in root growth with diminishing soil

moisture content has been reported in plants (Li et al., 2000; Jaleel et al., 2008c).

The allocation pattern that maximizes growth or water use efficiency depends on

the availability of water. Under conditions of mild water deficit the relative allocation of

biomass to roots usually increases (Hamblin et al., 1991; Gorai et al., 2010). The benefit

of higher allocation of biomass to the roots is an increased capacity for water uptake. The

costs of a larger root system are the costs of construction (possibly at the expense of

construction of photosynthetic tissue) and the increased respiratory losses associated with

its maintenance. So it can be hypothesized, that greater allocation of biomass to roots is

associated with benefits in terms of water uptake capacity and with costs in terms of

carbon. Not only the pattern of biomass allocation, but also differences in the rates of

uptake and loss of carbon and water of the different plant organs will contribute to

variation in growth and water use efficiency. For instance, a plant with a low leaf area but

a high rate of photosynthesis may assimilate as much as a plant with a high leaf area and

a low rate of photosynthesis. Likewise, rates of transpiration and water uptake need to be

considered to understand variation of water use efficiency.

Water stress reduced the height of the plants, and decreased shoot length in wheat

(Molnar et al., 2004) and in soybean (Zhang et al., 2004). In general shoot growth is

reduced more than root growth because more severe water deficits develop in the shoots

and probably continue longer. Therefore, the ratio root/shoot is generally increased by

water stress. Increase in root/shoot ratio may be an adaptive response to drought


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(Passioura, 1947). On the contrary, increased shoot/root ratio was reported in low land

rice cultivars under mild water stress by Cruz et al., (1986).

Water stress inhibits plant growth and development. Ahmad et al. (2007)

established dry matter reduction in wheat under water deficiency stress. Boutraa and

Sanders (2001) established that water deficit inhibited the relative growth rate by 25%.

The main reasons are changes in net assimilation rate (NAR) and in photosynthetic rate.

The same changes are reported in 24 wild species (Poorter and Remkes 1990), in ten

wheat cultivars (Van den Boogard et al., 1997), in durum wheat (Lutts et al. (2004) and in

young bean plants (Berova and Zlatev, 2002). Ramos et al., (1999) established that water

deficit inhibits accumulation in fresh plant mass in greater extent than dry biomass.

Relatively lower influence of drought on dry biomass than on fresh mass indicates a

disturbances in water relations. Ferrat and Lovatt (1999) established decrease of 14-27%

in dry biomass in young bean plants subjected to drought and significant increase in ratio

dry mass/fresh mass. It is considered that increased ratio dry mass/fresh mass is a stress

parameter at plant level (Baker, 1993; Augé et al., 2001).

From the above cited reports it is clear that the root growth and shoot growth of

plants were affected under water stress conditions.

Photosynthesis:

Water stress effects on photosynthesis have been well demonstrated (Gomes et al

2008; Farooq et al., 2009a). Plants resistance to water deficiency yields metabolic

changes along with functional and structural alterations of photosynthesizing apparatus.

Photosynthesis of higher plants decreases with the reduction in the relative water content


9

(RWC) and leaf water potential. Photosynthesis rate is a usual effect of water stress in

plants and has been attributed primarily to stomatal limitation and secondarily to

metabolic impairment. Water deficiency reduces the supply of carbon dioxide from the

environment due to the closure of stomata (Yokata et al., 2002). However, metabolic

impairment is the more complex phenomenon than the stomatal limitation though the

relative importance of stomatal or metabolic inhibitions is unclear (Anjum et al., 2003).

There are some co-factors, which decrease plants' photosynthesis under water

stress. Of them, changes in the pool of photosynthesizing pigments are important. Both

the chlorophyll a and b are prone to water stress (Farooq et al., 2009a). Carotenoids have

additional roles and help the plants to withstand adversaries of water stress. Changes in

the ratio of chlorophyll a and b and carotenoids have been reported under water stress

(Anjum et al., 2003). A reduction in chlorophyll content has been reported in drought

stressed soyabean (Zhang et al., 2004) and in rice (Reddy et al., 2007). Assimilation rates

in photosynthetic leaves decreases due to reduced photosynthetic metabolites and

enzymes activity, low carboxylation efficiency and inhibition of chloroplast activity at

low water potential (Wise et al., 1991). Among other co-factors of water stress the

damage of the photosynthetic apparatus through the production of ROS such as

superoxide and hydroxyl radicals, worth special mentions (Prochazkova et al., 2001;

Jaleel et al., 2009b).

The non stomatal limitation of photosynthesis may be attributed to reduced

corboxylation efficiency of RuBisCO (Wise et al., 1991). RuBisCO, the key enzyme for

carbon metabolism in leaves, acts as a carboxylase in the Calvin cycle and as an


10

oxygenase in the photorespiration which, however, the latter frequently is viewed as an

adverse process. RuBisCO is the most critical player influencing the physiology of plants

under water-stressed conditions. Under the conditions of water stress, it was noticed that

a rapid decrease in the amount of RubisCO as well as lower activity of the enzyme

(Karenchi et al., 1995).

Drought stress induced reduction in photosynthesis was reported in cowpea

(Souza et al., 2004), cotton (Massaci et al., 2008), grass (Xu et al., 2006), sugarcane

(Graça et al. 2010) and common beans (Franca et al., 2000). In the coconut palm, the

drought-induced photosynthetic reductions were initially attributable to limited CO2

diffusion from the atmosphere to the intercellular spaces as a result of stomatal closure

(Repellin et al., 1997). Non-stomatal factors have been shown to contribute to the

reduction in photosynthesis, both during a period of severe water deficit and during the

recovery phase after resuming irrigation (Gomes, 2006; Gomes et al., 2007).

Accumulation of compatible solutes:

When plants experience unfavorable environmental conditions associated with

high levels of drought, plant cells protect themselves by accumulating a variety of small

organic molecules that are collectively referred to as compatible solutes or

osmoprotectants (Tamura et al., 2001). Compatible solutes are highly soluble in water

and are also uniformly neutral with respect to the perturbation of cellular functions, even

at high concentrations (Yancy et al., 1982). The properties of compatible solutes allow

the maintenance of turgor pressure during water stress, which is an intrinsic feature of

major forms of abiotic stress. Compatible solutes are typically hydrophilic, which


11

suggests they could replace water at the surface of proteins and membranes, therefore

acting as osmoprotectants (Papageorgiou and Murata, 1995). In addition, compatible

solutes can serve as efficient scavengers of free oxygen radicals (ROS) produced by

water stress (Apel and Hirt, 2004).

A variety of organic solutes (eg. sugars, amino acids, amides, glycerol, proline

and glycinebetaine) accumulate in osmotically stressed plants in which proline appears to

be widely distributed osmolytes under stress conditions. (Delauney and Verma, 1993;

Yoshiba et al., 1997). In addition to acting as osmoprotectant, proline also serves as sink

for energy to regulate redox potentials (Aziz et al., 1999; Aktas et al., 2007), as a

reservoir of carbon and nitrogen for rapid recovery from the stress (Matysik, 2002), as a

scavenger of OH· radical and as a solute that protects macromolecules such as DNA and

proteins against denaturation (Kavi Kishore et al., 2005) and as an agent of detoxification

of NH3 produced during water stress (Blum and Ebercon, 1976) The accumulation of free

proline as a consequence of water stress has been observed in date palm (Djibril et al.,

2005), sorghum and chick pea (Dalvi et al., 2007), umbu tree (Silva et al., 2009), and

jatropha (Silva et al., 2010).

It is evident from the literature above cited, the increased levels of proline

contributes significantly to the turgor maintenance of cells and proline accumulation has

been considered as stress marker in several plant species under water stress conditions.

Nitrogen metabolism:

Nitrogen metabolism is the most important factor that influences plant growth and

performance. Disruption in metabolism is a crucial in plant injury under the water deficit


12

conditions. Nitrogen is a structural component of many organic compounds like amino

acids, nucleic acids, chlorophylls, proteins, hormones, vitamins and cytochromes. Some

studies showed the reduction of nitrate uptake and decrease in nitrate reductase activity

under water stress (Anuradha and Rao, 2003; Garg et al., 2005). Samia et al., (2009)

reported that reduction in growth of stressed maize plants is mainly related to the

decrease in nitrate uptake and its assimilation. Low level of nitrate in maize shoots

caused proportional decrease in nitrate reductase activity.

Although the nitrate reductase (NR) enzyme itself represents a very small

proportion of leaf protein (Calza et al. 1987), the activity of the enzyme plays a pivotal

role in the supply of nitrogen and the growth and productivity of plants (Srivastava,

1995). The activity of nitrate reductase, a key enzyme in nitrogen assimilation, is a

measure of the habitat-dependent nitrate utilisation of a plant (Larcher 1995). The

reduction in nitrate reductase activity under water stress has been known to inhibition of

protein synthesis (Morilla et al., 1973). The nitrate reductase activity can be modulated

rapidly in return to a water stress induced stomatal closure by a mechanism coupled to

net photosynthesis and this could be part of an integrated response of cell metabolism to

stomatal closure under stress conditions(Cornic et al., 1992). From the happening reports

it is clear that the nitrate reductase activity was altered in plants during water stress.

Water stress induced oxidative stress:

As regards 2.7 billion years before molecular oxygen was introduced in our

environment by the O2-evolving photosynthetic organisms and ROS have been the

uninvited companions of aerobic life (Halliwell, 2006). There are many studies that


13

report an increased ROS accumulation and oxidative stress under drought stress (Sgherri

and Navari-Izzo, 1995). When plants exposure to drought stress, ROS production is

enhanced through multiple ways. For instance, the limitation on CO2 fixation will reduce

NADP+ regeneration through the Calvin cycle, hence provoking an over reduction of the

photosynthetic electron transport chain. In fact, during photosynthesis and under drought

stress there is a higher leakage of electrons to O2 by the Mehler reaction. Also

photorespiratory pathway under drought stress is enhanced, especially when RuBP

oxygenation is maximal due to limitation on CO2 fixation. The predominance of

photorespiration on the oxidative load under drought stress has been recently put forward.

Photorespiration is likely to account for over 70% of total H2O2 production under drought

stress conditions (Noctor et al. 2002b). The ability of phototrophs to convert light into

biological energy is critical for life on Earth and therefore photosynthesizing organisms

are especially at the risk of oxidative damage, because of their bioenergetic lifestyle and

the abundance of the photosensitizers and polyunsaturated fatty acids (PUFA) in the

chloroplast envelope. On the other hand, electron transfer activities of mitochondria, and

oxidative metabolism in the peroxisomes represent the main sources of ROS (Sharma and

Dietz, 2009; Gill and Tuteja, 2010).

An inequality between ROS production and ROS scavenging leads to oxidative

stress resulting major physiological impairment. The ROS comprising O2- (superoxide

anion), H2O2 (hydrogen peroxide), 1O2 (singlet oxygen), HO2.- (perhydroxyl radical), OH-

(hydroxyl radical), ROOH (organic hydroperoxide), ROO- (peroxyl radical), and RO-

(excited carbonyl) are extremely reactive and toxic and causes damage to a variety of

macromolecules including proteins, lipids, carbohydrates, DNA which eventually results


14

in cell death (Mithöfer et al., 2004; Del Rio et al., 2006; Navrot et al., 2007). The most

popular ROS are singlet oxygen1O2, superoxide radical O2-, hydrogen peroxide H2O2 and

hydroxyl radical OH_ are produced as a result of spin inversion and one, two and three

electron transfers to dioxygen (O2) respectively (Figure 1).

Figure 1. Generation of different ROS by energy transfer (Gill and Tuteja, 2010)

Under physiological conditions .O2 - is not very reactive in opposition to the

biomolecules of the cell, and in aqueous solutions at neutral or slightly acidic pH

disproportionate to H2O2 and O2. H2O2 is comparatively stable and not very reactive,

electrically neutral ROS, however is very hazardous because it can pass through cellular

membranes and reaches cell compartments distant from the site of its formation (Mithöfer

et al., 2004; Michalak, 2006). H2O2 in the presence of .O2- can produce highly reactive

.OH- hydroxyl radicals. As a consequence the scavenging of H2O2 in cells is crucial to

avoid oxidative damage (Michalak, 2006). Overproduction of ROS leads to interaction

with the antioxidant system, disrupting the electron transport chain or disturbing the

metabolism of essential elements (Srivastava et al., 2004; Dong et al., 2006).

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Lipid peroxidation (LPO):

The peroxidation of membrane lipids is most damaging effects caused by ROS. In

the initiation step, hydroxyl radicals (OH-) and 1O2 oxygen can react with the methylene

groups of PUFA (polyunsaturated fatty acids) forming conjugated dienes, lipid peroxy

radicals and hydroperoxides (Heath and Packer, 1968; Montillet, 2005). In an aerobic

environment, oxygen will add to the fatty acid at the carbon-centered lipid radical to give

rise to a ROOˉ. Once initiated, ROOˉ can further propagate the peroxidation chain

reaction by abstracting a hydrogen atom from adjacent PUFA side chains. The resulting

lipid hydroperoxide can easily decompose into several reactive species including: lipid

alkoxyl radicals (ROˉ), aldehydes (malonyldialdehyde), alkanes, lipid epoxides (RO2˙),

and alcohols (Davies, 2001; Fam and Morrow, 2003). A single initiation event thus has

the potential to generate multiple peroxide molecules by a chain reaction. Increased

PUFA peroxidation decreases the fluidity of the membrane, increases leakiness and

causes secondary damage to membrane proteins (Moller et al., 2007). It has been reported

that water stress increased the LPO, membrane injury index, H2O2 and OH- production in

leaves of stressed Phalseolus vulgaris (Zlatev et al., 2006), in wheat plants (Simova-

Stoilova et al., 2010) and in five cherry tomato varieties (Sanchez-Rodriguez et al.,

2010).

Protein oxidation by ROS:

Protein oxidation is defined as covalent modification of a protein induced by ROS

or byproducts of oxidative stress. Protein carbonylation is widely used marker of protein

oxidation (Moller et al., 2007). The oxidation of certain protein amino acids, particularly

arginine, histidine, lysine, proline, threonine and tryptophan give free carbonyl groups


16

which may inhibit or alter their activities and increase vulnerability towards proteolytic

attack (Ghezzi and Bonetto, 2003). Whatever the location of ROS synthesis and action,

ROS are likely to target proteins that contain sulfur-containing amino acids and thiol

groups. Protein carbonylation may occur due to direct oxidation of aliphatic amino acids

to hydroxylated derivatives (e.g., threonine to aminoketobutyrate) and aromatic residues

to phenoxyl derivatives (e.g., tyrosine to dityrosine) by ROS, ultimately leading to

fragmentation of the peptide bond, cross-linking and increased susceptibility to

proteolysis (Shringarpure, and Davies, 2002; Gill and Tuteja, 2010). Proteins can be

damaged in oxidative conditions by their reactions with LPO products, such as 4-

hydroxy-2-nonenal and MDA generated from fatty acid degradation, which unlike free

radicals are long lived and therefore attack targets quite distant from their site of

production (Millar and Leaver, 2000, Cabiscol et al., 2000).

It has been found that various stresses lead to the carbonylation of proteins in

tissues. Carbonylation of storage proteins has been noted in dry Arabidopsis seeds but

carbonylation of a number of other proteins increased strongly during seed germination

(Job et al., 2005). Bartoli et al., (2004) found that protein carbonylation was higher in the

mitochondria than in chloroplasts and peroxisomes in wheat leaves which suggest that the

mitochondria are more susceptible to oxidative damage. A number of carbonylated

proteins in a soluble fraction from green rice leaf mitochondria have been identified

(Kristensen et al., 2004).


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Oxidative injure to DNA:

Though the plant genome is very stable but its DNA might get damaged due to

the exposure to biotic and abiotic stress factors which might damage the DNA, and

thereby exerts genotoxic stress (Tuteja et al., 2009). High levels of ROS can cause

damage to nucleic acids (Valko et al., 2006). It has been reported that hydroxyl radicals

(OH-) are the most reactive and cause damage to all components of the DNA molecule,

damaging both the purine and pyrimidine bases and also the deoxyribose sugar backbone

(adding or removing H atoms to/from DNA bases) and results in broad spectrum of DNA

lesions. The DNA lesions include DNA base modifications, inter- and intra-molecular

cross-linkage of DNA, DNA strand breaks, rearrangements and de-purination (Tuteja et

al., 2001; Hartwig and Schwerdtle, 2002). 1O2 primarily attacks guanine, and H2O2 and

O2- don’t react at all (Wiseman et al., 1996). DNA damage results in various

physiological effects, such as reduced protein synthesis, cell membrane destruction and

damage to photosynthetic proteins, and genomic instability which affects growth and

development of the whole organism (Britt et al., 1999; Cooke et al., 2003).

ROS scavenging antioxidant defense mechanism:

Exposure of plants to unfavourable environmental conditions increases the

production of ROS. To protect themselves against toxic oxygen intermediates, plant cells

and its organelles like chloroplast, mitochondria and peroxisomes employ antioxidant

defense systems. The accumulation of ROS also activates defense gene expression as part

of protective responses to both biotic and abiotic stimuli (Karpinski et al., 1999; Grant

and Loake, 2000; Fryer et al., 2003; Op den Camp et al., 2003).


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ROS detoxification reactions involve right balance between the formation and

detoxification of active oxygen species. As hydroxyl radicals (OH-) are most reactive to

be controlled easily, defense mechanisms are based on the elimination of its precursors

namely, O2-, H2O2. Removal of ROS and cellular homeostasis governed by antioxidant

systems, which includes the enzymatic as well as non-enzymatic components (Mittler et

al., 2004). The enzymatic antioxidants include enzymes such as superoxide dismutase

(SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) etc.

and a complex antioxidant system the ascorbate-glutathione cycle (AGC) and the

associated glutathione metabolism enzymes comprising γ-GCS, GST and GSH-PX.

(Anjum, 2010; Yadav, 2010)

Enzymatic antioxidants:

Superoxide dismutase (SOD)

Metalloenzyme SOD is the most effective intracellular enzymatic antioxidant

which is ubiquitous in all aerobic organisms and in all subcellular compartments prone to

ROS mediated oxidative stress. 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. The SOD removes O2- by catalyzing its dismutation, one O2

- being reduced to

H2O2 and another oxidized to O2. It removes O2- and hence decreases the risk of OH-

formation. H2O2 is potentially capable of reacting with O2 to form OH- (Quan et al.,

2008).

O2-+ O2

- +2 H+ 2 H2O + O2


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Catalase (CAT):

CAT is tetrameric heme containing enzyme with the potential to directly

dismutate H2O2 into H2O and O2. 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. CAT is important in the removal of H2O2 generated in peroxisomes by

oxidases involved in β-oxidation of fatty acids, photorespiration and purine catabolism

(Garg and Manchanda, 2009).

2 H2O2 2 H2O +O2

Peroxidase (POD):

Peroxidases constitute a class of haem-containing enzymes ubiquitously present

in prokaryotic and eukaryotic organisms (Welinder, 1992) which catalyze the

dehydrogenation of structurally diverse phenolic and endiolic substrates by H2O2 and are

thus often regarded as antioxidant enzymes, protecting cells from the destructive

influence of H2O2 and derived oxygen species (Prasad et al., 1994). In addition, these

enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2-) and

H2O2 by substrates such as NADH or dihydroxyfumarate (Chen Si-xue and Schopfer

Peter, 1999).

Donor + H2O2 Oxidized donor +2 H2O

Ascorbate peroxidase (APX):

APX is thought to play the most crucial role in scavenging ROS and protecting

cells in higher plants, algae, and other organisms. APX is involved in scavenging of H2O2

in water-water and AsA-GSH cycles and utilizes AsA as the electron donor. APX has a


20

higher affinity for H2O2 (mM range) than CAT and POD (mM range). Enhanced

expression of APX in plants has been demonstrated during different stress conditions

(Gill and Tuteja, 2010).

AA+ H2O2 DHA+2 H2O

Glutathione reductase (GR):

GR is a flavo-protein oxidoreductase, it is a potential enzyme of the AsA -GSH

cycle and plays an important role in defense system against ROS by supporting the

reduced status of GSH. GR catalyzes the reduction of GSH, a molecule involved in many

metabolic regulatory and antioxidative processes in plants where GR catalyses the

NADPH dependent reaction of disulphide bond of GSSG and is thus important for

maintaining the GSH pool. It is contained mostly in chloroplasts, also found in

mitochondria and cytosol (Edwards et al., 1990; Creissen et al., 1994).

NADPH+GSSG NADP +2GSH

MDHAR and DHAR:

Monodehydroascorbate reductase (MDHAR) is a flavin adenin dinucleotide

(FAD) containing enzyme. It is present in chloroplast and cytosol. MDHAR exhibits a

high specificity for monodehydro asorbate (MDHA) as the electron acceptor, preferring

NADH rather than NADPH as the electron donor. The reduced enzyme donates electrons

to MDHA, and producing two molecules of ascorbate (Asada, 1999). Dehydroascorbate

reductase (DHAR) regenerates AsA from the oxidized state of ascorbate (DHA) and

regulates the cellular AsA redox state which is crucial for tolerance to various abiotic

stresses leads to the production of ROS (Chen and Gallie, 2005).


21

MDHA + NADPH 2 AsA + NADP+

DHA + GSH AsA + GSSG

Glutathione peroxidase (GSH-PX):

GPX is present in cytosol, chloroplast, mitochondria and endoplasmic reticulum.

It catalyzes GSH dependent reduction of H2O2 and organic and lipid hydroperoxides, and

therefore help plant cells from oxidative stress (Noctor et al., 2002a). Leisinger et al.,

(2001) reported the upregulation of GSH-PX gene (Gpxh gene) in Chlamydomonas

reinhardtii following oxidative stress.

2GSH+PUFA-OOH GSSG+PUFA+2 H2O

Glutathione sulfo-transferases (GSTs):

GSTs detoxify endogenously produced electrophiles like 4-hydroxyalkenals, 4-

hydroxynonenals and base propenols the products of oxidative membrane damage and

DNA degradation respectively by conjugating glutathione to hydrophobic substrates

forming less reactive and more polar glutathione-s-conjugates (Haluskova et al., 2009).

Noctor et al., (2002a) also reported that GSTs have the potential to remove cytotoxic or

genotoxic compounds, which can react or damage the DNA, RNA and proteins in plants,

in response to biotic and abiotic stresses.

Non-enzymatic antioxidants:

Glutathione (GSH):

The tripeptide glutathione (GSH, γ-glutamylcysteinylglycine) is the most

abundant low molecular weight thiol in almost all eukaryotic cells as well as in proteo-

and cyanobacteria (Fahey and Sundquist, 1991; Masip et al., 2006). GSH is one of the


22

crucial metabolites in plants which is considered as most important, water soluble

intracellular defense against ROS induced oxidative damage. It occurs abundantly in

reduced form (GSH) in plant tissues and is localized in all cell compartments like cytosol,

endoplasmic reticulum, vacuole, mitochondria, chloroplasts, peroxisomes as well as in

apoplast (Ferreira et al., 2002; Del Rio et al., 2003). GSH plays a central role in several

physiological processes, including regulation of sulfate transport, signal transduction,

conjugation of metabolites, detoxification of xenobiotics (Alscher et al., 2002) and the

expression of stress-responsive genes (Sandalio et al., 1998). Glutathione functions as a

stress indicator occurring in two distinct redox forms i.e. GSH (reduced) and GSSG

(oxidised) promptly responding to oxidative stress and the balance between the GSH and

GSSG is a central component in maintaining cellular redox state (Foyer and Noctor,

2005). GSH is necessary to maintain the normal reduced state of cells so as to counteract

the inhibitory effects of ROS induced oxidative stress (Meyer, 2008). It is a potential

scavenger of 1O2, H2O2 (Briviba et al., 1997) and most dangerous ROS like OH- (Larson,

1988). The change in the ratio of its reduced (GSH) to oxidized (GSSG) form during the

degradation of H2O2 is important in certain redox signaling pathways (Shao et al., 2008;

Meyer, 2008; Anjum et al., 2012) GSH is particularly adequate electron donor or

acceptor in physiological reactions due to the chemical reactivity and relative stability in

water (Foyer and Noctor, 2005). Additionally, GSH plays a key role in the antioxidative

defense system by regenerating another potential water soluble antioxidant like AsA, via

the AsA-GSH cycle.

Glutathione is synthesized enzymatically from the free amino acids glutamate,

cysteine and glycine in two ATP- and Mg2+

-dependent steps. First gamma-


23

glutamylcysteine synthetase (γ-GCS) establishes a peptide bond between the amino group

of cysteine and the γ-carboxy group of glutamate, forming γ-glutamylcysteine (γ-GC). In

the second step, glutathione synthetase (GS) adds a glycine residue to the carboxy-

terminus of γ-GC, producing glutathione. While the γ-GCS reaction takes place in

plastids, GS is found in both plastids and cytosol (Yadav, 2010).

Ascorbate (AsA):

AsA is an excellent electron donor participating in various reactions of the plant

system even involved in photo protective xanthophylls cycle is water soluble ascorbate

(Horemans et al., 2000). It occurs in all plant tissues, usually being higher in

photosynthetic cells; about 30 to 40% of the total ascorbate is in the chloroplast and

stromal concentrations as high as 50 mM have been reported (Foyer and Noctor, 2005).

Ascorbic acid can also directly scavenge 1O2, O−2 and ·OH and acts as secondary

antioxidant by generating the lipophilic chloroplastic antioxidant α-tocopherol (vitamin

E) from the α-chromanoxyl radical thus providing membrane protection (Asada, 1994;

De Tullio et al., 1998; Shao et al., 2008). In chloroplast, AsA acts as a cofactor of

violaxantin de-epoxidase thus sustaining dissipation of excess excitation energy

(Smirnoff, 2000, 2001; Gill and Tuteja, 2010). In plants, mitochondrion play central role

in the metabolism of AsA. Plants mitochondria are not only synthesize AsA by └-

galactono-γ-lactone dehydrogenase but also take part in the regeneration of AsA from its

oxidised forms (Szarka et al., 2007). The regeneration of AsA is extremely important

because fully oxidized dehydroascorbic acid has a short half-life and would be lost unless

it is reduced back. AsA is considered as a most powerful ROS scavenger because of its

ability to donate electrons in a number of enzymatic and non-enzymatic reactions.


24

Figure 2. A diagramatic illustration of the antioxidant enzymes, AsA-GSH cycle and

glutathione metabolism.

Water stress

O2-

SODH2O2

H2O2

H2O + O2

CAT

H2O2

H2O GSH

GSSG

GSH-PX GR

NAD(P)H

NAD(P+)

H2O2

H2O

MDHA

AsA

APX MDHAR

NAD(P+)

NAD(P)H

O2- H2O

N.E.

AsA+DHA

AsA

GSSG

GSH NAD(P)H

NAD(P+)

DHAR GR

Non-enzymatic disproportion (N.E.)

HX+R-SS-GSH

GST RX *

Vacuole

ASCORBATE - GLUTATHIONE CYCLE

GLUTATHIONE METABOLISM

* (R - aliphatic, aromatic or hetero cyclic group, X - sulphate, nitrate or halide group)

Ascorbate-glutathione cycle (Figure 2), a major pathway that detoxifies H2O2 and

maintains cellular homeostasis in plant under stress conditions. Ascorbate peroxidase

(APX) use the ascorbic acid as reductant to reduce the H2O2 to water in the first step of

AsA-GSH cycle with concomitant generation of ascorbyl radical

(monodehydroascorbate) and oxidized form of ascorbate (dehydroascorbate) operates

both in chloroplast as well as the cytosol (Noctor and Foyer, 1998; Anjum, 2010). The

MDHA reduced to ASA by NAD(P)H-dependent mono-dehydroascorbate reductase

(MDHAR). However, monodehydroascorbate is a short lived radical and if not reduced

rapidly it disproportionates into ascorbate and dehydroascorbate. Dehydroascorbate is


25

reduced to ascorbate by dehydroascorbate reductase at the expense of GSH, yielding

oxidized glutathione (GSSG), which in turn reduced back to GSH by GR using NADPH

as electron donor (De Tullio et al., 1998). Therefore, operation of the AsA-GSH cycle not

only maintains the reduced active forms of GSH and ASA in cells on a suitable level, but

also participates in ROS detoxification (Kingston-Smith and Foyer, 2000; Ma and Cheng,

2003).

Carotenoids scavenge free radicals that are generated owing to excess excitation

energy from chlorophyll during photosynthesis (Arora et al., 2002). In plants, proline also

scavenges singlet oxygen and free radical induced damages and performs an important

role in protection of proteins against denaturation (Alia et al. 1991).

Polyethylene Glycol (PEG 6000) and Water stress:

Structural formula: HO (~CH2CH2O~) n H

Molecular weight: 6000 – 75000.

Properties: Clear, viscous liquids (M = 200 to 600) or waxiform (M = 1000 to 6000)

products. Solubility in water is inversely proportional to molecular mass. Liquid PEG is

colorless, almost odorless, and miscible with water. Waxiform PEGs (carbowaxes) are

soluble in water (50 to 73%). At a concentration of 1.0 g/1, they do not alter the color,

odor, or taste of water.

Osmotic solutions are often used to impose water stress reproducibly under in

vitro conditions (Kaya, et al., 2006). PEG widely used to induce water stress, is a non-

ionic water polymer, which is not expected to penetrate into plant tissue rapidly

http://en.wikipedia.org/wiki/Glutathione�

http://en.wikipedia.org/wiki/GSSG�


26

(Nepomuceno, et al., 1998). PEG molecules with a Mr ≥ 6000 cannot penetrate the cell

wall pores (Carpita, et al., 1979). Because PEG does not enter the apoplast, water is

withdrawn not only from the cell but also from the cell wall. Therefore, PEG solutions

mimic dry soil more closely than solutions of low–molecular weight range (Mr)

osmotica, the often used solute mannitol because mannitol has been shown to be taken

up by plant cells and cause specific toxic effects on growth which infiltrate the cell wall

with solute (Verslues, et al., 1998). It is reported that PEG induced significant water

stress in plants and not having any toxic effects (Emmerich et al., 1990).

Figure 3. The relationship between PEG 6000 concentration and osmatic potential.

(Williums and Shaykewich, 1969)


27

Brassinosteroids

Discovery of Brassinosteroids:

Brassinosteroids (BRs) are essential low molecular weight plant hormones that

are ubiquitous throughout the plant kingdom (Takatsuto, 1994). Mitchell et al., (1970)

first published in Nature describing the growth-promoting activity of Brassica napus L.

pollen extracts at very low concentrations. John W. Mitchell and his group initiated

screening pollen in search of new plant hormones at the USDA Agricultural Research

Center in Beltsville, Maryland, as pollen is a rich source of plant growth-regulating

substances. They screened nearly 60 species of plants and of which pollen crude extracts

from the alder tree (Alnus glutinosa L.) and rapeseed (Brassica napus L.) caused

maximum growth increase in the common bean (Phaseolus vulgaris L.) second internode

bioassay. The researchers proposed that this was due to a new class of steroidal

hormones, which they termed “brassins”. The application of brassins to field crops was

shown in some cases to enhance plant growth, crop yield and seed viability (Mitchell and

Gregory, 1972). To isolate the active component in brassins, 500 pounds of bee-collected

rapeseed pollen, was extracted and purified resulting in 10 mg of an active crystalline

material, and named it as ‘brassinolide’ (Grove et al., 1979). This was followed by the

discovery of the second steroidal hormone, castesterone, from the insect galls of chestnut

(Castanea crenata) in 1982 (Yokota et al., 1982). Since then numerous analogs have

been discovered and isolated from various plant species, of which approximately 60 are

fully characterized (Haubrick and Assmann, 2006). Japanese scientists also isolated a

compound from the leaves of Distylium racemosum, which they named Distylium factor

that had growth-promoting activities similar to Brassins in a rice lamina assay. Later it


28

was found that Brassins and Distylium factor were very similar chemically as well as

functionally, so both were subsequently classified as BRs (Yokota, 1999). All such

naturally-occurring steroidal compounds now constitute an independent class of plant

hormones, known as Brassinosteroids (BRs).

Chemical Structure:

BRs are a group of polyhydroxy lactones with a common 5α-cholestane skeleton,

with various hydroxyl substitutions and attached functional groups (Haubrick and

Assman, 2006). Their chemical structural variations come from the kind and position of

functionality in the A/B rings and the side chain. Only 3-oxygenated (22R, 23R)-5α-

cholestane-22-23-diols of plant origin, bearing alkyl or oxy substituents, conjugated or

not to sugars or fatty acids, and exhibiting the characteristic brassinolide activity. The

2α, 3α-dihydroxylation is necessary for biological activity (Zullo et al., 2002). The

classification of BRs as C27, C28 or C29 usually depends on the alkyl-substitution pattern

of the side chain (Yokota, 1997; Zullo and Adam, 2002)

Brassinolide (BL) was the first BR whose chemical structure was elucidated.

Spectroscopic methods, including X-ray crystallography were used to identify the

structure of BL isolated from Brassica napus pollen. BL was determined to be (22R,

23R, 24S)-2α, 3α, 22, 23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan-6-one

(Grove et al., 1979). Structurally, all BRs are composed of a steroid nucleus with four

rings, A, B, C and D and a side chain at C-17 (Figure 3a).


29

Figure 3a:

Chemical structure of plant steroid hormone, brassinolide, composed of a steroid nucleus

with four rings, A, B, C and D (adapted from Grove et al., 1979). The structural

requirements associated with optimal BR activity include: 1) presence of oxygen at C-6

in ring B; 2) presence of cis-glycols at C-2 and C-3 in ring A; 3) presence of hydroxyl

groups at C-22 and C-23; 4) presence of a trans A/B ring junction; and 5) presence of a

methyl or ethyl group at C-24 (Mandava, 1988).

Natural Occurrence of Brassinosteroids in the Plant Kingdom:

BRs have been detected throughout the plant kingdom in every species that has

been examined. To date, 54 BRs and five of its conjugates have been isolated from 60

plant species. These BRs have been detected in angiosperms, gymnosperms,

pteridophytes, bryophytes and an alga. Pollen grains and immature seeds are the richest

source of BRs with contents ranging from 1-100 ng/g of fresh weight, whereas the stems

and shoots have the lowest BR content ranging from 0.01-0.1 ng/g of fresh weight (Rao

et al., 2002; Bajguz and Tretyn, 2003).


30

Biochemical Analysis of Brassinosteroids:

BRs extraction and purification from vegetative tissue generally involves a

solvent-based partitioning followed by a chromatographic separation. The active fractions

containing BRs are tracked via one of the three bioassays described below. Further

analysis of the purified fractions can be performed using analytical techniques such as

gas chromatography-mass spectrometry (GC/MS), GC-MS selected ion monitoring

(SIM), fast atom bombardment-mass spectrometry (FAB), high pressure liquid

chromatography(HPLC), and liquid chromatography-mass spectrometry (LC/MS)

(Takasuto and Yokota, 1999).

Bioassays for Brassinosteroids:

Bioassays are very important studies of plant growth substances. The three main

bioassays that are utilized to study structure-activity relationships of BRs are:

Bean second-internode bioassay: In this assay, elongation in the bean second

internode length is measured four days after application of the test chemical.

Application of BL not only increases the internode length but also causes

swelling, curvature and splitting of the internodes (Thompson et al., 1981). This

assay played a critical role in isolation of BL containing fractions for its structural

analysis (Grove et al., 1979).

Rice-lamina inclination bioassay: This is the most commonly used bioassay to

isolate BRs. Rice explants composed of the leaf blade, leaf sheath and the lamina

joint are cut and floated on a solution of test chemical or distilled water (control).

This is a very sensitive bioassay as BRs cause lamina bending even at very low

concentrations.


31

Wheat leaf unrolling test: This bioassay was developed by Wada et al., (1985).

This is a relatively convenient bioassay, but is 10 times less sensitive than the

rice-lamina inclination test (Takatsuto and Yokota, 1999).

Physiological Role of Brassinosteroids:

Similar to other endogenous plant hormones like auxins, giberrellins, ethylene,

cytokinins and abscisic acid, BRs affect multiple physiological processes in plants. BRs

regulate various aspects of plant growth and development including, cell expansion and

organ formation, promotion of cell division, regulation of vascular differentiation,

promotion of senescence, regulation of fertility, modulation of biotic and abiotic stresses,

skotomorphogenesis and photomorphogenesis, timing of flowering, and regulation of

gene expression (Rao et al., 2002; Sasse, 2003; Ashraf et al., 2010). The identification

and characterization of BR-deficient and BR-insensitive mutants with pleiotropic

phenotypic defects further confirmed the physiological importance of BRs (Haubrick and

Assman, 2006).

Cell elongation:

A pronounced elongation of hypocotyls, epicotyls, and peduncles of dicotyledons,

as well as coleoptiles and mesocotyls of monocotyledons, is caused by the application of

brassinosteroids (Clouse, 1996). The elongation response caused by exogenous

application of BRs is mostly observed in young tissue, whereas mature tissue shows little

or no elongation (Sasse et al., 1992). The mechanism of BR-induced cell elongation is

similar to that of auxins, which involves the extrusion of protons via ATPases leading to

hyperpolarization of the cell membrane and acidification of the apoplast there by


32

activating the cell wall-modifying enzymes (Haubrick and Assmann, 2006). Treatment

with BRs promotes the cell elongation by regulating activity of vaculor H+- ATPases in

Azuki bean epicotyls and maize roots (Cerana et al., 1984). Cell walls consist of a

complex network of cellulose microfibrils tethered to hemicelluloses (xyloglucans). For

cell expansion to occur, these tethers need to be broken or loosened. It has been reported

that BRs regulate cell wall elongation by inducing a number of genes which encode a

number of cell wall modifying enzymes, including endo-1,4-beta-glucanases, xyloglucan

endo- transglycosylase/hydrolases (XTHs, formerly known as xyloglucan

endotransglycosylases or XETs), expansins, sucrose synthase and cellulose synthase in

soybean, Arabidopsis, tomato and rice indicating that all of these proteins may play an

important role in BR-mediated cell expansion ( Zurek et al., 1994; Sasse, 2003; Ashraf et

al., 2010). Application of 28-HBL restored the organization of microtubules and rescued

cell elongation in BR-deficient Arabidopsis mutants, boule or bull without activation of

tubulin gene expression (Catterou et al., 2001).

Cell division:

BRs have been shown to enhance cell division in combination with auxins and

cytokinins in cell cultures of Helianthus tuberosum L. and protoplast cultures of Chinese

cabbage (Clouse and Zurek, 1991; Clouse and Sasse, 1998). The transcript level of the

cell cycle regulatory enzyme, Cyclin-D3, involved in regulation of G1/S transitions in the

cell cycle, is up-regulated in Arabidopsis det2 (de-etiolated2) cell suspension cultures

treated with 24-epibrassinolide (Hu et al., 2000), which indicates a BRs play positive role

in controlling cell division. Brassinolide application promoted cell division in tobacco

BY-2 cells during the early phase of culture only in the absence of auxin (Miyazawa et


33

al., 2003). Microscopic examination of the BR-deficient cbb mutant showed that

dwarfism in the mutant was mainly a result of reduced cell size rather than number

(Kauschmann et al., 1996). Contradictory to BRs effect on cell division, anti-proliferation

activity of BR analogues, 28-homocastasterone and 24-epibrassinolide in breast cancer

and prostate cancer cells has been reported, indicating BRs may also have a role as cell

division inhibitors (Malikova et al., 2008).

Vascular Differentiation:

The evidence of BRs role in vascular differentiation has demonstrated in

Helianthus tuberosus explants and isolated mesophyll cells of Zinnia elegans, where low

concentrations of BR promote xylem differentiation (Fukuda, 1997; Fukuda, 2004). In

soyabean epicotyls BR splay a vital role in xylem formation (Zurek et al., 1994).

Subsequently, identification and characterization of a BR-deficient mutant, constitutive

photomorphogenesis and dwarfism (cpd) that had unequal cambium division established

the importance of endogenous BRs in regulating vascular differentiation in vivo

(Szekeres et al., 1996). Similarly a putative role of other signaling sterols has been

proposed in vascular differentiation as well (Clouse, 2002; Carland et al., 2002; Sasse,

2003). The interaction between BRs, auxins and cytokinin signaling pathways play a

critical role in the regulation of vascular development (Dettmer et al., 2009).

Reproduction:

It has been reported that male sterility in the BR-insensitive mutant,

brassinosteroid insensitive1 (bri1) and BR-deficient mutants such as dwarf 4 (dwf4) and

cpd demonstrate the importance of BRs in controlling fertility (Clouse, 1996; Choe et al.,


34

1999; Szekeres, et al., 1996). A number of additional studies have furthered our

understanding of BRs role in regulating fertility. Brassinolide application promoted

pollen tube elongation in vitro in Prunus avium at nanomolar concentrations (Hewitt et

al., 1985). Treatment with BL induced parthenogenetic production of haploid seeds in

Arabidopsis and Brassica juncea (Kitani, 1994). Although the BR-deficient mutant dwf5-

1 had fertility comparable to that of WT plants, its seeds required exogenous BR

treatment for full germination and seedling development (Choe et al., 2000). BR

signaling is also apparently involved in the induction of flowering. Two key proteins,

ELF6 and REF6, involved in regulating time of flowering, directly interact with BES1, a

transcription factor controlling BR-regulated gene expression (Yu et al., 2008). Ye et al.,

(2010) also showed that BES1 regulates a number of key genes involved in Arabidopsis

anther and pollen development.

Senescence:

Several studies also suggest a role for BRs also play crucial role in regulating

senescence (Rao et al., 2002). Application of BL enhanced senescence in Xanthum and

Rumex explants (Mandava, 1981). In contrast, in Arabidopsis BR mutants, senescence is

delayed by approximately 40 days (Clouse, 2002). Delayed senescence is correlated with

reduced fertility in BR mutants, e.g. the bri1 mutant has very delayed senescence,

whereas dwf 5-1 has normal senescence compared to WT (Choe et al., 2000). Application

of 28-homo-BL and 24-epi-BL to tomato fruit pericarp discs resulted in elevated levels of

lycopene and lowered chlorophyll levels and this accelerated ripening was associated

with increased ethylene production, which is a known ripening hormone in climacteric

fruits (Vardhini and Rao, 2002).


35

Environmental Stress:

BRs can induce the resistance to wide range of abiotic and biotic stresses such as

temperature, salt, drought and fungal infections (Rao et al., 2002; Krishna, 2003; Bajguz

and Hayat, 2009, Vardhini et al., 2010). BR treatment reduced chilling injury in rice,

maize and cucumber by enhancing chlorophyll synthesis, osmoregulation and membrane

stability in cells (He et al., 1991; Katsumi, 1991). Epibrassinolide treatment caused

thermo tolerance with increased heat shock protein synthesis and accumulation, as well

as increased expression of translational machinery in B. napus L. seedlings (Dhaubhadel

et al., 2002). BR-treatment also has an ameliorative effect on rice and barley plants

grown under high salt conditions (Hamada, 1986; Kulaeva et al., 1991). Treatment with

24-epi-BL increased seed germination and growth of Eucalyptus camaldulenesis seeds

under high salt conditions (Sasse et al., 1995).

BRs application increased relative water content (RWC), nitrate reductase

activity, chlorophyll content and photosynthesis during water stress, resulting in higher

leaf area, biomass, and grain yeild in wheat, (Sairam, 1994). BR-treated sorgum

(Sorghum vulgare) plants showed higher germination rates compared to non treated

control plants under osmotic stress (Vardhini and Rao, 2003), and 24-epi-BL treatment of

Arabidopsis thaliana and Brassica napus seedlings increased their survival rate under

drought conditions (Kagale et al., 2007). These studies also indicate that BR levels

increased under drought conditions (Catala et al., 2007). Foliar application of 24-EBR to

pepper (Capsicum annuum L.) plants grown under salt-stress significantly reduced the

inhibitory effects of the salt on shoot growth and leaf relative-water-content and had a

positive effect on root growth and chlorophyll fluorescence Houimli et al., (2008). BRs


36

induced tolerance in Oryza sativa L. under salinity stress (Anuradha and Rao, 2001,

2003). EBR supplemented tomato (Lycopersicon esculentum) seedlings significantly

alleviated water stress and increased the relative water content (RWC), net photosynthetic

rate (PN), and also increased the activities of antioxidant enzymes (catalase, ascorbate

peroxidase and superoxide dismutase) while it decreased the contents of H2O2 and MDA

(Yuan et al., 2010). Farooq et al., (2009a) also found that BRs application improved the

leaf water economy and CO2 assimilation, and enabled rice to withstand drought.

Exogenous application of 24-epibrassinolide and 28-homobrassinolide protected

the photosynthetic machinery and improved the leaf water potential in two tomato

cultivars (K-25 and Sarvodya) against cadmium induced oxidative stress. In addition,

BRs increased the carbonic anhydrase and nitrate reductase and proline levels in tomato

cultivars (Hasan et al., 2011). Kanwar et al., (2012) observed that supplementation of 24-

EBL to Brassica juncea subjected Ni toxicity, significantly improved the pool of

antioxidative enzymes (CAT, SOD, GR, MDHAR, DHAR and APX) and lowered the Ni

uptake which further manifested in improved plant growth and stress tolerance. BRs

enhanced activity of the antioxidative enzymes (CAT, POD and SOD) and proline

content in chickpea (Cicer arietinum) under cadmium stress (Hasan et al., 2008).

BRs increase plants’ resistance against a broad range of viral, fungal and bacterial

diseases (Khripach et al., 2000). It has found that BL-treated WT tobacco plants exhibited

enhanced resistance to the viral pathogen tobacco mosaic virus, the bacterial pathogen

Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp., and that

treatment of rice plants with BL increased their resistance towards rice blast and bacterial


37

blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae,

respectively (Nakashita et al., 2003). This BL-induced disease resistance (BDR) was

found to activate the innate immunity of higher plants, this is distinct from systemic

acquired resistance (SAR) and wound-inducible disease resistance, which require

salicylic acid (SA) biosynthesis and PR gene expression, respectively (Nakashita et al.,

2003). BRs treated cucumber increased its tolerance to both photo-oxidative and cold

stress, and increased resistance to cucumber mosaic virus. (Xia et al., 2009a).

BRs application reduces the toxicity of pesticides in plants by promoting pesticide

metabolism. Xia et al. (2009b) found that application of 24-epi-BL on cucumber

enhanced degradation of many fungicides and insecticides by regulating several pesticide

detoxification genes including P450 monooxygenases, glutathione-S-transferases and

UDP-glycosyltransferases. This study suggests the role of BRs as safeners, suitable for

reducing the risk of pesticide toxicity to humans and the environment.

Radish (Raphanus sativus L.) The experimental plant:

Radish (Raphanus sativus L.), belonging to the brassicaceae family, is an

important annual root vegetable. Radishes are good source of ascorbic acid, folic acid and

potassium, vitamin B6, riboflavin, magnesium, copper and calcium. It also a rich source

of two important medicinal compounds: glucosinolates and isothiocynates (Curtis, 2003;

Martı´nez-Villaluenga, 2008). It is easily grown under a wide range of agro ecological

regions. If adequate moisture is available it is a popular choice for cultivation, as they are

fairly easy to grow, rapidly maturing crop with many varieties able to reach maturity

within 60 days. Radish is considered as a model crop and extensively used in studies


38

related to stress (Kosta-Rick and Mannig, 1993). The advantage of radish and other

members of mustard family for stress studies are well established (Mathe-Gasper and

Anron, 2002).

The present study aimed at exploring the possibilities of mitigating the water

stress (as imposed by PEG 6000) employing brassinosteroids. The prime objectives of the

investigation are as follows.

Research Objectives:

a) To find out the effect of brassinosteroids on the germination and seedling growth

of radish under water stress.

b) To study the impact of brassinosteroids on the growth of radish plants subjected

to water stress.

c) To evaluate the influence of brassinosteroids on the changes in metabolite

contents of radish plants challenged by water stress.

d) To identify the quantitative changes as brought about by brassinosteroids in

certain compatible solutes which confer resistance to plants under water stress.

e) To decipher the protective role of brassinosteroids ‘if any’ in the structural and

functional impairment caused by drought stress.

f) To understand the changes in the activities of oxygen scavenging enzymes in

radish plants under water stress as influenced by the application of

brassinosteroids.

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