REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10220/8/08_chapter 2.pdf · ground water or soil solution available for plant uptake, thus strongly influence

Chapter 2

REVIEW OF

LITERATURE

8

Plants grow well as long as they have optimum levels of nutrients, moisture,

light and temperature however, any significant deviation in their required optimum

“limit” is injurious, causes sickness in plants, leading to lower growth rate and

eventually parts of plant or whole plant dies (Agrios, 2005; Kumar et al., 2008; Larcher,

2003; Parvaiz and Satyawati, 2008). This damage to plants is caused by an array of

abiotic as well as biotic stress factors. Abiotic stress is due to unfavorable

environmental conditions caused by physical or chemical or mechanical factors whereas

biotic stress is imposed by living pathogenic microorganisms or insects (Fig. 2.1)

(Larcher, 2003; Hopkins and Hüner, 2004; Agrios, 2005). At a given time, plant may

face one or a combination of more than one of these stresses resulting in growth

retardation, reduced fresh weight and seed or fruit production (Salvatore et al., 2008).

STRESS

ABIOTIC

Physical

Drought

Gases

Light

Temperature

Radiations

Water Logging

Chemical

Heavy metals

Herbicides

Pesticides

Minerals

Pollutants

Salt

Mechanical

Wind

Solifluction

Burial

Snow

Cover

Ice sheets

BIOTIC

Plants

Allelopathy

Competition

Crowding

Parasitic

plants

Animals

Grazing

Trampling

Pathogens

Bacteria

Fungi

Insects

Nematodes

Protozoa

Viruses

Humaninterference

Electromagnetic

Radiations

Ionizing

Radiations

Fire

Soil Compaction

Agro-

Chemicals

Fig. 2.1. Abiotic and Biotic stress Factors (Modified after Larcher, 2003)

Review of Literature

9

A common consequence of most abiotic and biotic stresses is that they result, at

some stage of stress exposure, in cell signaling cascades and cellular responses, like

activation of stress proteins, up-regulation of antioxidant enzymes and antioxidants and

accumulation of compatible solutes (Triantaphylidès and Havaux, 2009). The

imposition of abiotic and biotic stresses can further intensify ‘reactive oxygen species’

(ROS) production as compared with ROS generated during normal metabolic processes

(Bhattacharjee, 2005; Mittler, 2006). Major ROS scavenging pathways include various

antioxidants and antioxidant enzymes (Dat et al., 2000; Mittler, 2002).

2.1 HEAVY METALS STRESS

The highly electronegative metals with a density greater than 5g cm-3 are termed

“heavy metals”. These metallic elements are intrinsic components of the environment.

Although the natural background of metal fraction in air, soil and plants is highly

variable, there are anomalous areas of high levels caused by man-made pollution due to

mining of metal rich ores, metal smelting industries (Foy et al., 1978). Heavy metal

concentration in atmosphere can be attributed to both natural and anthropogenic

sources. Human activities like mining, smelting, processing and manufacturing of metal

articles and using of substances containing metal contaminants are responsible for metal

accumulation. Industrial wastewaters and agricultural runoff are also potential source of

heavy metals in agricultural soils and water (Saint-Laurent et al., 2010; Alam et al.,

2011). When the capacity of the soil to retain heavy metals is reduced, they leach into

ground water or soil solution available for plant uptake, thus strongly influence their

speciation and hence bioavailability (Chary et al., 2008). In the top ten of the priority

list as provided by the American Agency for Toxic Substances and Disease Registry

(ATSDR-2007), mercury is at 3rd place among heavy metals like arsenic (1st place),

lead (2nd place), cadmium (7th place), nickel (53rd place) and chromium (65th place).

Industrial revolution and anthropogenic activities have aggravated the metal

pollution of biosphere and it has posed a serious threat to mankind by its incorporation

to the food chain resulting in the degradation of ecosystem (Chary et al., 2008). Being


10

persistent, these get bio-accumulated at alarming rates thereby disturbing ecosystem.

Some heavy metals are essential for most of the redox reactions which are fundamental

for cellular functions. However, their concentrations beyond tolerable limits lead to

production of reactive oxygen species (ROS). In young and healthy plant cells this

process is kept under control but unfavorable environmental conditions may lead to the

generation of oxidative stress. The ROS are highly toxic and can oxidize biological

macromolecules such as nucleic acids, proteins and lipids, thereby disturbing the

membrane permeability or ultimately leading to plant cell death (Schützendübel and

Polle, 2002; Sudo et al., 2008). Besides, mitogen activated protein Kinase (MAPK)-

driven phosphorylation cascades, regulatory post transcriptional modifications such as

protein oxidation and nitrosylation might also be involved in ROS-dependent cell death

(Breusegem and Dat, 2006).

Excessive accumulation of heavy metals in agricultural soils is a critical

environmental concern due to their potential adverse ecological effects. Such toxic

metals are considered as soil pollutants due to their widespread occurrence, and their

acute and chronic toxic effect on plants grown of such soils (Yadav, 2010). For

example, regulatory limit of cadmium (Cd) in agricultural soil is 100 mg/kg soil yet this

threshold is continuously exceeding because of several human activities (Salt et al.,

1995; Yadav, 2010). Plants exposed to high levels of Cd showed reduction in

photosynthesis, water uptake, and nutrient uptake and also visible symptoms of injury

reflected in terms of chlorosis, growth inhibition, browning of root tips, and finally

death (Yadav, 2010). Also, studies on nickel (Ni), which is a transition metal and found

in natural soils at trace concentrations except in ultramafic or serpentinic soils revealed

that Ni2+ concentration is increasing in certain areas by mining works, emission of

smelters, burning of coal and oil, sewage, phosphate fertilizers and pesticides (Gimeno-

García et al., 1996; Yadav, 2010). In contaminated soils, Ni2+ concentration may

enhance to 20-fold to 30-fold (200–26,000 mg/kg) higher than the overall range (10–

1000 mg/kg) found in natural soil (Izosimova, 2005; Yadav, 2010). Excess of Ni2+ in


11

soil resulted in various physiological alterations and diverse toxicity symptoms such as

chlorosis and necrosis in different plant species (Zornoza et al., 1999; Pandey and

Sharma, 2002; Yadav, 2010). Plants grown in high Ni2+ containing soil showed

impairment of nutrient balance and resulted in disorder of cell membrane functions.

Thus, in plants, heavy metals stress caused various morphological abnormalities,

altered the biochemical parameters thus resulting in oxidative damage due to the

excessive production of oxygen-, carbon-, sulfur- and nitrogen- radicals, that not only

originates from superoxide radical, hydrogen peroxide, and lipid peroxides but also

from chelates of amino-acids, peptides, and proteins complexed with the toxic metals

(Flora et al., 2008). One of the major mechanisms behind heavy metal toxicity has been

attributed to ROS induced oxidative stress (Flora et al., 2008). Redox-active metals,

such as iron (Fe), copper (Cu) and chromium (Cr), might undergo redox cycling

whereas redox-inactive metals, such as lead (Pb), cadmium (Cd), nickel (Ni), mercury

(Hg) etc. deplete cells' major antioxidants, particularly thiol-containing antioxidants and

enzymes. Either redox-active or redox-inactive metals might induce an increase in

production of ROS such as hydroxyl radicals, superoxide radicals or hydrogen peroxide

(Ercal et al., 2001). Generally, in plants ROS are continuously produced predominantly

in chloroplast, mitochondria and peroxisomes. However, this production and removal of

ROS is a balanced mechanism. But, a number of biotic and abiotic factors including

heavy metal stress are reported to disturb the production and scavenging of ROS (Flora

et al., 2008). The enhanced generation of ROS might overwhelm cells' intrinsic

antioxidant defenses, and result in a condition known as "oxidative stress" or "oxidative

burst" (Ercal et al., 2001). This oxidative damage can not only damage plant cells but

might also signal prime stresses to gene expression through activation of Ca2+ influx

and K+ efflux ion channels (Demidchik, 2010).

2.2 ANTIOXIDANT DEFENCE SYSTEM

Stresses induced accumulation of ROS leads to imbalance in pro-oxidative and

antioxidative defence system resulting in oxidative stress. The ROS capable of causing


12

oxidative damage include superoxide (O2·-), perhydroxyl radical (HO2·

-), hydrogen

peroxide (H2O2), hydroxy radical (OH-), alkoxy radical (RO·), peroxy radical (ROO·),

organic hydroperoxide (ROOH), singlet oxygen (1O2), excited carbonyl (RO·), etc.

(Arora et al., 2002; Bhattacharjee, 2005). Major ROS scavenging pathways include

various antioxidants and antioxidant enzymes (Dat et al., 2000; Mittler, 2002).

Antioxidants such as ascorbate, tocopherol and glutathione react directly or via enzyme

catalyis reactions with hydrogen peroxide (H2O2), hydroxyl radical (OH.) or O2- while

carotenes directly act as effective quenchers of reactive oxygen species (Shakirova,

2002; Nunez et al., 2004; Ozdemir et al., 2004).The pathway involved in antioxidant

defence system for removal of ROS is described in Fig. 2.2.

Glutathione (GSH) and cysteine are the two most important low molecular

weight biological thiols that have high affinity for toxic heavy meatls. Glutathione

reduced or GSH is a sulfur-containing tri-peptide thiol with the formula γ-glutamate-

cysteine-glycine and its synthesis is catalyzed by two ATP dependent enzymes γ-

glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2). Also, GSH is

used as a substrate for phytochelatin synthesis which catalyzes the synthesis of

phytochelatins and thus, it is very crucial for detoxification of heavy metals (Freeman et

al., 2004; Yadav et al., 2010). The phytochelatins (PCs) are small, heavy metal-binding,

cysteine-rich polypeptides with the general structure of (γ-Glu-Cys)nGly (n = 2–11) that

are present in plants, fungi and other organisms (Grill et al., 1985; Gekeler et al., 1988;

Piechalak et al., 2002; Yadav et al., 2010). PCs form complexes with toxic metal ions in

the cytosol and subsequently transported them into the vacuole (Salt and Rauser, 1995).

Hence, protect plants from the deleterious effect of heavy metals. Heavy metal ions

produce ROS and in chloroplasts SOD act as first line of defence dismutating

superoxide radical (O2•-) to H2O2 (Mittler, 2002; Sharma et al., 2011b). Consequently

CAT, APOX, POD or GPOX detoxify the H2O2 to H2O. The enzyme APOX detoxifies

H2O2 to H2O by oxidizing ascorbate into monodehydroascorbate (MDHA), which is

reverted back to ascorbate by MDHAR. Further, MDHA is converted into


13

dehydroascorbate (DHA) and DHAR is required for ascorbate regeneration

(Bhattacharjee, 2005). Besides, GR is requisite to maintain levels of reduced glutathione

(GSH) in cells (Mittler, 2002) as explained in Fig. 2.2.

Fig. 2.2. Heavy Metals induced ROS generation and Antioxidant Defence System

in plants. (Abbreviations: O2.-

: Superoxide radical; SOD: Superoxide dismutase; POD :

Guaiacol peroxidase; CAT: Catalase; GPOX: Glutathione peroxidase; APOX: Ascorbate

peroxidase; AA: Ascorbic acid; MDHA: Monodehydroascorbate; DHA:

Dehydroascorbate; MDHAR: Monodehydroascorbate reductase; DHAR:

Dehydroascorbate reductase; GSSG: Oxidized glutathione; GSH: Reduced glutathione;

GR: Glutathione reductase)

2.3 PHYTOHORMONES IN AMELIORATION OF OXIDATIVE STRESS

Apart from regulation of several antioxidant genes, the oxidative stress also

consequences in the evolution of new metabolic pathways, the accumulation of low


14

molecular weight metabolites, the synthesis of special proteins and changes in

phytohormone levels (Banu et al., 2009; Bari and Jones, 2009). Phytohormones include

naturally occurring growth promoting and growth retarding substances that strongly

influence, at micromolar (µM) to nanomolar (nM) concentrations, the growth,

development, differentiation of plant cells and organs (Hopkins and Hüner, 2004).

Burgeoning evidences indicate that phytohormones regulate each other’s synthesis and

are involved in plant defence pathways against stresses (Grsic et al., 1999; Yi et al.,

1999; Hansen et al., 2000; O’Neill and Ross 2002). A number of hormones such as

abscisic acid (ABA; terpenes), auxin (cyclopentane derivatives), brassinosteroids (BRs;

steroids), cytokinins (CKs; purine derivatives), ethylene (hydrocarbon), gibberellins

(Gas; terpenes), jasmonates (JAs), polyamines, salicylic acid (SA), strigolactones (a

novel class of phytohormones) and systemins (peptides) are produced in plants for their

growth and development including defence responses (Farrokhi et al., 2008; Gomez-

Roldan et al., 2008; Umehara et al., 2008; Santner and Estelle, 2009).

Several plant hormones like abscisic acid (ABA), ethylene, jasmonates and BRs

play a determinant role in implicating oxidative stress (Bari and Jones, 2009; Depuydt

and Hardtke, 2011). Ethylene, JA and SA play major role in biotic stress and ABA in

abiotic stress although little is known about the role of other phytohormones under

stress (Bray et al., 2000; Crozier et al., 2000; Hammond-Kosack and Jones, 2000; Ent

et al., 2009). Thus, under normal or stressed conditions, the inter- or intra-cellular roles

of one phytohormone on another phytohormone’s synthesis via downstream targets or

signalling mechanisms are collated. Such interactions result in the evolution of new

metabolic pathways, the accumulation of low molecular weight metabolites, and the

synthesis of special proteins as well as changes in levels of some special

phytohormones (Banu et al., 2009; Bari and Jones, 2009). Recent studies indicate that a

class of plant steroid hormones known as brassinosteroids (BRs) has been explored for

their promising role in reducing the effects of environmental stresses (Dhaubhadel et

al., 2002; Krishna, 2003; Kagale et al., 2007; Ali et al., 2008; Arora et al., 2008; Bari

and Jones, 2009). BRs are unique in their activities for not only regulating the diverse


15

physiological and morphogenetic responses in plants but also having a significant role

in amelioration of various biotic and abiotic stresses at nanomolar to micromolar

concentrations (Clouse, 1998; Krishna, 2003).

Recently, two important BRs namely 28-homobrassinolide (HBL) and 24-

epibrassinolide (EBL) at 10-6 to 10-11 M doses have been reported to combat salt and

heavy metals stress in B. juncea, R. sativus, T. aestivum and Z. mays (Bhardwaj et al.,

2007; Hayat et al., 2007; Sharma et al., 2007, 2010; Yusuf et al., 2010). Exogenous

application of BRs improved the antioxidant system by regulating the activities of

antioxidant enzymes (SOD, POD, APOX, CAT, GR, DHAR, MDHAR etc.) and

antioxidants (alkaloids, carotenoid flavonoids, prolines, ascorbate, tocopherol,

glutathione, reducing sugars, glycinebetaine, mannitol, sorbitol etc.) to provide

protection to the plants under stress conditions (Núñez et al., 2004; Özdemir et al.,

2004; Hayat et al., 2007; Sharma and Bhardwaj, 2007; Arora et al., 2008). Nascent

studies have revealed that apart from the role of BRs in biotic or abiotic stress

tolerance and they have prospective in phytoremediation (Barbafieri and Tassi,

2011; Sharma et al., 2010, 2011a, 2011b). Also, the BRs ability to regulate cell

membrane permeability and ion transport had revealed their implications in remediation

of heavy metals or radionuclide rich soils.

2.4 STEROID HORMONES IN PLANTS

For over 30 years, researchers’ have explored that steroid hormones are not only

active/effective in animals but are dynamic in the plants also (McLachlan, 1979; 2001;

Tilghman et al., 2010). These steroids are not only produced by animals, plants, fungi

and insects but are also derived from synthetic industrial pollutants, or pharmaceuticals

(Tilghman et al., 2010). Although steroid hormone-like activities could be mimicked by

environmental chemicals, but there is growing concern about the persistence of natural

and synthetic estrogens (Shore and Pruden, 2009). Of particular concern are estrone and

17β-estradiol that are excreted in large quantities by humans and animals and have been

shown to exert measurable effects on aquatic or terrestrial organisms’ at10 ng/L

(Hanselman et al., 2003; Hu et al., 2011). These lipophilic and low molecular weight


16

steroid hormones also referred as ‘mammalian sex hormones’, play a key role in

controlling the process of development and reproduction, and regulates the metabolism

of mineral and proteins in mammals (Kliewer et al., 1998; Janeczko and Skoczowski,

2005; Alon et al., 2007; Erdal and Dumlupinar, 2011; Hu et al., 2011). Recently these

animal steroid hormones were isolated from various plant species and their tissues and

organs like roots, leaves and flowers (Dogra and Kaur, 1994; Erdal et al., 2010a, 2010b;

Erdal and Dumlupinar, 2011).

Even though steroid hormones such as androgen, estrogen and progesterone are

naturally present in plants but the available knowledge is not sufficient to consider such

compounds as plant hormones (Geuns, 1978; Erdal and Dumlupinar, 2011). Since the

beginning of the 19th century when such hormones were reported first in plants, various

investigations have been carried out to depict their presence, importance and structural

elucidation or biosynthetic pathways in plants (Dohrn et al., 1926; Janeczko and

Skoczowski, 2005). In Plants, various steroidal sex hormones like estrone, testosterone,

progesterone, corticosteroids and several analogues of these compounds had been

reported (Janeczko and Skoczowski, 2005). Recently, their receptors and specific

binding sites have been studied to elucidate their mechanism of action in plants (Erdal

and Dumlupinar, 2011). These studies revealed the putative steroid-binding membrane

proteins and the presence or location of specific-binding sites for progesterone and

estradiol (Janeczko and Skoczowski, 2005; Yang et al., 2005; Lino et al., 2007;

Janeczko et al., 2008; Simersky et al., 2009; Erdal and Dumlupinar, 2011).

Animal steroid sex hormones were reported to control sex expression in many

plants and also act as feeding deterrents in protecting the plants from overgrazing by

insects, mammals and other animals. Exogenous applications of mammalian steroids on

plants have been reported to significantly affect the seed germination, seedling or plant

growth and development (Martínez-Honduvilla et al., 1976; Dogra and Thukral, 1991a,

1991b, 1994a and 1994b; Bhardwaj and Thukral, 2000a; Czerpak and Szamrez, 2003;

Szamrez and Czerpak, 2004). Treatments of estrone and testosterone had significantly

accelerated the meristem activity in the roots of Melandrium dioecium, Rumex acetosa


17

and Anthoxanthum aristatum (Helmkamp and Bonner, 1952) and enhanced the growth

in the seedlings of pea, pine, germinating wheat and winter wheat (Kopcewicz, 1969,

1970; Kopcewicz and Rogozinska, 1972; Dogra and Kaur, 1994; Janeczko, 2000).

Recently, certain evidences have been congregated to establish a phylogenetic tree

between plant and animal steroids for example presence of progesterone in a higher

vascular plant i.e., Juglans regia. Besides, evidence is gleaned from the characterization

of new plant steroids viz., 3-O-sulfated pregnenolones, sulfated H-5 cardenolide and

spirophanthigenin, a novel C-18 oxygenated spirocyclic derivative of strophanthidin

from Adonis aleppica. Such investigations highlight the cross-linkages between

mammals and higher plants based on the structural similarity of their metabolite pool

that can be interpreted as biochemical congruence in steroid metabolism (Pauli et al.,

2010). Effect of various animal steroid hormones in plants has been briefly discussed in

Table 2.1.

2.5 BRASSINOSTEROIDS

Brassinosteroids (BRs) are a group of phytohormones which have structures

similar to animal steroid hormones and are distributed throughout the plant kingdom

(Mandava, 1988; Clouse and Sasse, 1998; Bhardwaj et al., 2008; Sharma et al., 2010).

These steroidal plant hormones were discovered in 1970’s, when Mitchell et al. (1970)

reported that treatment of organic extracts of B. napus pollen had promoted the stem

elongation and cell division. The pollen extracts of around 60 different plant species

were tested after employing bean second internode bioassay (Mandava and Mitchell,

1971). The pollens of rape (B. napus L.) and alder tree (Alnus glutinosa L.) produced an

unexpected response, combining elongation (typical of gibberellins) with swelling and

curvature of the treated internode (Zullo and Adam, 2002). Thus, rape pollens were

supposed to contain a new group of lipidic plant hormones, named as ‘brassins’

(Mitchell et al., 1970). Further, the occurrence of an active fraction of the brassins

containing, mainly, glucosyl esters of fatty acids was reported by Mandava and Mitchell

(1972) and Mandava et al. (1973). Furthermore, in order to isolate the active brassin

compounds, about 250 kg of bee collected rape pollen were extracted with isopropanol


18

in batches of 25 kg (Mandava et al., 1978). In 1979, Grove et al. for the first time

isolated an active BR in a crystalline form, from the pollens of rape and named it as

brassinolide (BL). Its structure was elucidated by spectroscopic methods including X-

ray analysis and systematically designated as (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-

24-methyl-B-homo-7-oxa-5α-cholestan-6-one.

Testosterone 17 β-estradiol

(Mammalian Sex Steroid Hormone)

Ecdysone

(Insect Steroid Hormone)

Castasterone (CS) Brassinolide (BL)

(Plant Steroid Hormones)

Fig. 2.2. General Structures of Steroid Hormones

Due to its low concentration, the identification of BL took 10 years of dedicated

work on the part of United States Department of Agriculture (USDA) researchers at a

cost of over one million U.S. dollars (Mandava, 1988; Bishop and Koncz, 2002). The

general structures of BR is characterized by a carbon skeleton with four fused rings,

generally arranged in a 6-6-6-5 fashion and have 5-α cholestane structure similar to

animal or insect steroidal hormones (Fig. 2.2). Different BRs vary by the oxygen moiety

at C-3 position in combination with others at the C-2, C-6, C-22 or C-23 positions in BL


19

structure. Castasterone (CS) was isolated as 2nd BR at the University of Tokyo (Yokota

et al., 1982). According to Zullo and Adam (2002), BRs can be derived from the 5-α

cholestane carbon skeleton bearing the following structural characteristics:

i) Ring A mono- to trioxygenated, always oxygenated at carbon 3

ii) Ring B presenting a 6-oxo-7-oxalactone or a 6-keto function or saturation

iii) all-trans -junctions of rings A - D

iv) 22 α, 23 α -dihydroxylated, mostly alkylated at carbon 24, sometimes

methylated at carbon 25 and sometimes unsaturated between carbons 24 and 28.

These characteristics belonged to natural brassinosteroids comprised of 3-oxygenated

(20b)-5 α -cholestane-22 α, 23 α -diols of plant origin, bearing additional alkyl or oxy

substituents (Fig. 2.3).

Fig. 2.3. General Structure of Naturally Occuring Brassinosteroid (BR)

The most active BRs exhibit either a 6-oxo function (in CS) or a lactone

structure (in BL) at the B ring which in the latter case is 7-numbered. Mostly the natural

BRs are hydroxylated at the C-2, C-3, C-22 and C-23 positions, the latter two being in

R configuration. Structural variations other than the substitution patterns of rings A and

B lie in the different alkylations of C-24 (Altmann, 1999). The most active BRs include

the 7-oxalactone-type compounds like brassinolide (BL), 24-epibrassinolide (EBL)

and 28-homobrassinolide (HBL), while the corresponding 6-keto-type substances like


20

CS, 24-epicastasterone and ethylbrassinone possess comparatively reduced bio-activity

(Takasuto et al., 1983; Adam and Marquardt, 1986; Mandava, 1988; Marquardt and

Adam, 1991; Altmann, 1999).

2.5.1 Occurrence of Brassinosteroids in Plants

Till date, more than 70 analogues of BRs have been isolated from 66 plant

species (Table 2.2) which includes: 56 angiosperms (12 monocots and 44 dicots), 6

gymnosperms, 1 pteridophyte (Equisetum arvense), 1 bryophyte (Marchantia

polymorpha) and 2 algae (Chlorella vulgaris and Hydrodictyon reticulatum).

Apparently, there are a number of BRs and their conjugates in plants yet to be explored

(Bajguz and Tretyn, 2003). Four types of BRs viz., brassinolide, castasterone, 28-

homoteasterone and teasterone have been reported from the seeds of radish plants

(Raphanus sativus L.) (Schmidt et al., 1991, 1993b). The endogenous content of BRs

varies from tissue to tissue and with the age of the plant. Pollen and immature seeds are

found to have highest concentration of BRs (Khripach et al., 2000). These phyto-

steroidal hormones are ubiquitously distributed throughout the plant kingdom and play

essential role in modulating growth and differentiation (Clouse and Sasse, 1998). The

structural resemblance of BRs with ecdysteriods, have been widely used to delay insect

molting, as they compete with natural hormones for hormone receptor binding site

(Richter and Koolman, 1991).

Table 2.2. Occurrence and distribution of Brassinosteroids in Plants

DICOTS

S.No. Botanical Name Family Plant part Reference

1. Aegle marmelos Rutaceae Leaves Sondhi et al. (2008)

2. Alnus glutinosa Betulacae Pollen Plattner et al. (1986)

3. Apium graveolens Umbelliferae Seeds Schmidt et al. (1995)

4. Arabidopsis thaliana Brassicaceae Seedlings,

seeds, root-

callus

suspension

cultures,

siliques, shoots

Fujioka et al. (1996,

1998, 2000a);

Konstantinova et al.

(2001); Schmidt et

al. (1997)


21

5. Banksia grandis Proteaceae Pollen Takasuto (1994)

6. Beta vulgaris Chenopodiaceae Seeds Schmidt et al. (1994)

7. Brassica campestris var.

pekinensis

Brassicaceae Immature seeds

and sheaths

Abe et al. (1982,

1983); Arima et al.

(1984); Ikekawa and

Takasuto (1984)

8. Brassica napus Brassicaceae Pollen Grove et al. (1979);

Ikekawa et al. (1984)

9. Camellia sinensis Theaceae Leaves,

immature seeds

Gupta et al. (2004);

Bhardwaj et al.

(2007)

10. Cannabis sativa Cannabaceae Seeds Takasuto et al.

(1996b); Gamoh et

al. (1996)

11. Cassia tora Fabaceae Seeds Park et al. (1994a)

12. Castanea crenata Fagaceae Galls, shoots Abe et al. (1983);

Ikekawa et al.

(1984); Yokota et al.

(1982)

13. Catharanthus roseus Apocynaceae Crown gall

cells, culture

cells

Park et al. (1989);

Choi et al. (1993,

1997); Fujioka et al.

(2000b)

14. Centella asiatica Apiaceae Leaves Sondhi et al. (2010)

15. Cistus hirsutum Cistaceae Pollen Takasuto (1994)

16. Citrus sinenesis Rutaceae Pollen Motegi et al. (1994)

17. Citrus unshiu Rutaceae Pollen Abe et al. (1991);

Takasuto (1994)

18. Cucurbita moschata Cucurbitaceae Seeds Jang et al. (2000)

19. Daucus carota sativus Apiaceae Seeds Schmidt et al. (1998)

20. Diospyros kaki Ebenaceae Seeds Takasuto (1994)

21. Distylium racemosum Hammamelid-

aceae

Galls, leaves Abe et al. (1984);

Ikekawa et al. (1984)

22. Dolichos lablab Leguminosae Seeds Baba et al. (1983);

Yokota et al. (1982b,

1983b)

23. Echium plantagineum Boraginaceae Pollen Takasuto (1994)

24. Eriobotrya japonica Rosaceae Pollen, anthers Yasuta et al. (1995)

25. Eucalyptus calophylla Myrtaceae Pollen Takasuto (1994)


22

26. Eucalyptus marginata Myrtaceae Pollen Takasuto (1994)

27. Fagopyrum esculentum Polygonaceae Pollen Takasuto et al.

(1990b)

28. Gypsophilla perfoliata Caryophyllaceae Seeds Schmidt et al. (1996)

29. Helianthus annuus Asteraceae Pollen Takasuto et al.

(1989)

30. Lychnis viscaria Caryophyllaceae Seeds Friebe et al. (1999)

31. Lycopersicon esculentum Solanaceae Roots, shoots Yokota et al. (1997,

2001)

32. Nicotiana tabacum Solanaceae Cultured cells Park et al. (1994b)

33. Ornithopus sativus Fabaceae Seeds, shoots Schmidt et al.

(1993a); Spengler et

al. (1995)

34. Perilla frutescens Labiatae Seeds Park et al. (1993b,

1994a)

35. Pharbitis purpurea Convolvulaceae Seeds Suzuki et al. (1985)

36. Phaseolus vulgaris Fabaceae Seeds Kim et al. (1988;

2000); Yokota et al.

(1983c, 1987)

37. Pisum sativum Fabaceae Seeds, shoots Nomura et al.

(1997); Yokota et al.

(1996)

38. Psophocarpus

tetragonolobus

Fabaceae Seeds Takasuto (1994)

39. Raphanus sativus Brassicaceae Seeds Schmidt et al. (1991,

1993b)

40. Rheum rhabarbarum Polygonaceae Panicles Schmidt et al.

(1995a)

41. Robinia pseudo-acacia Fabaceae Pollen Abe et al. (1995)

42. Solidago altissima Asteraceae Stem Takasuto (1994)

43. Vicia faba Fabaceae Pollen, seeds Ikekawa et al.

(1988); Park et al.

(1988)

44. Zinnia elegans Asteraceae Cultured cells Yamamoto et al.

(2001)

MONOCOTS


1. Erythronium japonicum Liliaceae Pollen, anthers Yasuta et al. (1995)

2. Lilium elegans Araceae Pollen Susuki et al. (1994b)


23

3. Lilium longiflorum Araceae Anthers,

culture cells,

pollen

Abe et al. (1991,

1994); Asakawa et

al. (1994, 1996);

Soneo et al. (2000)

4. Lolium perenne Poaceae Pollen Taylor et al. (1993)

5. Oryza sativa Poaceae Bran, seeds,

shoots

Abe et al. (1984b,

1995a); Ikekawa and

Takasuto (1984),

Park et al. (1993c,

1994a)

6. Phalaris canariensis Poaceae Seeds Shimada et al.

(1996)

7. Phoenix dactylifera Arecaceaea Pollen Zaki et al. (1993)

8. Secale cereale Poaceae Seeds Schmidt et al.

(1995b)

9. Triticum aestivum Poaceae Grain Yokota et al. (1994)

10. Tulipa gesneriana Liliaceae Pollen Abe et al. (1991);

Takasuto (1994)

11. Typha latifolia Typhaceae Pollen Schneider et al.

(1983)

12. Zea mays Poaceae Pollen, seeds Gamoh et al.

(1990a); Park et al.

(1995)

GYMNOSPERMS


1. Cryptomeria japonica Taxodiaceae Pollen, anthers Takasuto (1994);

Yokota et al. (1998);

Watanabe et al.

(2000)

2. Cupressus arizonica Cupressaceae Pollen Griffiths et al. (1995)

3. Ginkgo biloba Gingkoaceae Seeds Takasuto et al.

(1996a)

4. Piceae sitchensis Pinaceae Shoots Yokota et al. (1985)

5. Pinus silvestris Pinaceae Cambial region Kim et al. (1983a)

6. Pinus thunbergii Pinaceae Pollen Yokota et al. (1983a)

PTERIDOPHYTE


1. Equisetum arvense Equisetaceae Strobilus Takasuto et al.

(1990a)


24

BRYOPHYTE


1. Marchantia polymorpha Marchantiaceae Whole bodies Kim et al. (2002)

ALGAE


1. Chlorella vulgaris Chlorophyceae

(Trebouxiophyc

eae)

Algal cultures Bazguj (2009)

2. Hydrodictyon reticulatum Hydrodictyaceae Green alga Yokota et al. (1987a)

2.5.2 Brassinosteroids in Growth and Development of Plants

Experimental evidences on higher plants revealed that BRs play a critical role in

a range of physiological and developmental processes such as growth, seed

germination, rhizogenesis, senescence, flowering, abscission, maturation, germination,

leaf bending and epinasty, root inhibition, induction of ethylene biosynthesis, proton-

pump activation, xylem differentiation, regulation of gene expression and vegetative

and reproductive developments (Cerena et al., 1983; Bhardwaj and Thukral, 2000;

Krishna, 2003; Sasse, 2003; Cao et al., 2005; Çağ et al., 2007). Due to their active

involvement in miscellaneous physiological activities in plants and their exogenous

applications have improved yield and quality of vegetables, fruits etc., BRs are now

regarded as hormones of the 21st century (Han et al., 1987; Ikekawa and Zhao, 1991;

Khripach et al., 2000; Ramraj et al., 1997; Hnilička et al., 2007; Štranc et al., 2008).

Besides this, BRs have been reported to affect the expression of several genes

involved in plant defence as well as biosynthesis of other hormones (Bari and Jones,

2009). Also, BRs can be implicated in plant stress protection as they are reported to

play a vital role in abiotic and biotic stress management (Nakashita et al., 2003;

Romanutti et al., 2007; Kagale et al., 2007; Arora et al., 2008; Ohri et al., 2008; Sharma

et al., 2010; Choudhary et al., 2010). Various reports of BRs in agriculture in terms of

enhanced plant growth and development, crop improvement and stress protection are

briefed below:


25

2.5.2.1 Brassinosteroids in Seed Dormancy Release and Germination

Seed germination is the process of the growth and development of the embryo

present in a seed into a seedling or a young plant capable of independent existence.

Among phytohormones, ABA suppresses seed germination, while BRs, ethylene and

gibberellin acid (GA) alleviate the ABA-induced inhibition of seed germination (Matilla

and Matilla-Vazquez, 2008; Linkies et al., 2009; Wang et al., 2011). Exogenous

application of most bioactive BRs i.e., BL, HBL and EBL had significantly promoted

seed germination in Eucalyptus camaldulensis, L. sativus, Arachis hypogea, B. juncea,

O. sativa, T. aestivum, Lycopersicum esculentum and Orabanchae minor (Sasse et al.,

1995; Vardhini and Rao, 1998; Rao et al., 2002; Sharma and Bhardwaj, 2007a). The

synergistic effects of BL and gibberellins (GAs) on seed germination in tobacco

revealed that there was induction of β-1,3-glucanase in the micropylar endosperm and

these two hormones acted in distinct pathways (Leubner-Metzger, 2001). They also

concluded that BRs directly enhanced the growth of the emerging embryo independent

of GA and β-1,3-glucanase.

Steber and Mc Court (2001) also reported that EBL and BL treatment promoted

the seed germination whereas ABA strongly inhibited germination of both BR-

biosynthetic and BR-insensitive mutant (det 2-1 and bri 1-1) than the wild type. In

Arabidopsis, applications of BL and EBL overcame the non-germination of GA

biosynthetic and sleepy 1 (sly1) GA signaling mutants (Steber and Mc Court, 2001), but

BR and GA stimulated tobacco seed germination by different mechanisms (Leubner-

Metzger, 2001; Finkelstein et al., 2008). Although both BRs and GA promoted

endosperm rupture of non-photo dormant tobacco seeds imbibed in the dark, only GA

induced the class I β-1,3 glucanase (bGLU I) activity that was critical for endosperm

rupture of photodormant tobacco seeds (Leubner-Metzger, 2001; Finkelstein et al.,

2008). Furthermore, microarray analysis revealed that these hormones induced the

expression of cell elongation associated genes, though they induced the expression of

distinct expansin family members (Goda et al., 2002). Thus, BRs may promote seed


26

germination by directly enhancing the embryo growth potential in a GA-independent

manner (Leubner-Metzger, 2001; Finkelstein et al., 2008).

Exogenous applications of 10-10 M and 10-8 M concentrations of HBL were most

effective in increasing the percent germination of wheat grains and Cicer arietinum

seeds (Hayat and Ahmad, 2003; Ali et al., 2005). Eight hour seed-presoaking with HBL

treatments showed significant improvement in percent germination by enhancing the

values for α-amylase, catalase, peroxidase, soluble sugars and proteins in wheat grains

(Hayat and Ahmad, 2003). The studies on endogenous levels of BRs and the expression

of biosynthesis/metabolism/perception genes involved in P. sativum revealed that seeds

that were rapidly growing had high level of active BRs i.e., BL and CS suggesting the

importance of BL and CS seed development (Nomura et al., 2007). Plants use

heterotrimeric G-protein signaling in the regulation of growth and development,

particularly in hormonal control of seed germination, but it is not yet clear which of

these responses utilize a seven-transmembrane (7TM) cell-surface receptors (Chen et

al., 2004). Generally, signal recognition by 7TM receptors is dependent on

heterotrimeric G-proteins in metazoan, fungal, and amoeboid cells whereas in some

responses in amoeboid cells and possibly in human cells this downstream signaling can

be independent. Chen et al. (2004) reported that putative G-protein-coupled receptor 1

(GCR1) could act independently of heterotrimeric G-protein in response to BRs and

GAs in A. thaliana during seed germination. Furthermore, Divi and Krishna (2009)

observed that over-expression of AtDWF4, BR biosynthetic gene, was able to overcome

the ABA induced inhibition of germination in A. thaliana seeds and also the plants so

produced were tolerant to cold stress.

Besides, GA and BRs, production of ethylene is also enhanced during seed

germination (Gianinetti et al., 2007; Chang et al., 2010; Wang et al., 2011). ACC (1-

aminocyclopropane-1-1carboxylic-acid) synthase (ACS) enzyme catalyzes the

conversion of S-adenosyl-methionine to ACC, while ACC oxidase (ACO) mediates the

subsequent oxidation of ACC to ethylene. Thus, Wang et al. (2011) investigated the

roles of BR and ethylene in seed germination under conditions of salt stress, effects of


27

EBL and ACC on seed germination of Cucumis sativus seeds. An increase in ethylene

evolution during seed germination was reported which was suppressed by salinity stress

in cucumber. However, this reduction in ethylene evolution from imbibed seeds by salt

stress was attenuated by EBL revealing that salt stress-induced decrease in ACC

oxidase (ACO) activity was reversed by EBL. Also, salt stress reduced the expression

of gene encoding ACO (CsACO2), and EBL reversed the salt stress-induced down-

regulation of CsACO2. The alleviative effect of EBL on seed germination under salinity

was diminished by antagonist of ethylene synthesis while aminoethoxyvinylglycine

indicated that both ethylene and BRs might be associated with suppression of seed

germination under salt stress. However, the stress-ameliorative effect of BR on NaCl-

induced inhibition of seed germination may occur through its interaction with ethylene

synthesis (Wang et al., 2011).

2.5.2.2 Brassinosteroids in Cell Division, Elongation and Differentiation

A permanent increase in size, volume, form or weight of an organ or a plant is

typically referred as growth where development is a series of changes that occur in an

organism during its life-cycle. Both growth and development of plant are a multifaceted

and well co-ordinated processes mediated through cell division, elongation and

differentiation. Prior reports confirmed that BRs affect these multifarious processes at

cellular as well as whole plant level (Clouse and Sasse, 1998; Khripach, 2000; Haubrick

and Assmann, 2006). They are reported to enhance the elongation of hypocotyls,

epicotyls and peduncle of dicots, as well as coleoptiles and mesocotyls of monocots

(Clouse, 1996; Mandava, 1988). The proton extrusion and hyperpolarization of cell

membranes mediated the BRs-induced cell-elongation and similar effects were also

observed in asymmetric expansion of the joint pulvini of rice lamina (Cao and Chen,

1995) and in C. vulgaris (Bajguz and Czerpak, 1996). In suspension cells of A. thaliana

it was observed that the applications of HBL and 28-homoCS promoted cell-expansion

through plasma membrane hyperpolarization which was regulated by both anion

channels and proton pumps (Zhang et al., 2005). Further, BRs also up-regulated the

expression of the BRU1, TCH4, LeBR1, OsXTR1 and OsXTR3 genes, encoding


28

xyloglucan endotrans-glycosylases or hydrolases (XTHs or XETs) in soybean,

Arabidopsis, tomato and rice, respectively (Zurek et al., 1994; Xu et al., 1995; Koka et

al., 2000; Uozo et al., 2000). These enzymes are involved in cell wall biosynthesis and

modification. These findings were further supported by microarray analysis confirming

that BL treatment up-regulated several additional genes related to cell expansion and

cell wall organization (Goda et al., 2002). BRs mediated cell-elongation or expansion

might be attributed to activity of aquaporins, the water channels associated with plant

cell osmo-regulation (Morillon et al., 2001) or reorientation of cortical microtubules in

epicotyls and expression of -tubulin genes (Mayumi and Shiboaka, 1995; Munoz et

al. 1998).

In addition to the cell elongation or expansion, BRs have also been reported to

enhance the cell division and also play a crucial role in directing cell fate. Transcription

of CycD3, a D-type cyclin in det2 suspension cultures in Arabidopsis was up-regulated

by EBL treatments (Hu et al., 2000). The promotion of cell division by EBL and BL

had also been reported in tobacco BY-2 cell lines, cultured parenchyma cells of

Helianthus tuberosus, Chinese cabbage and Petunia protoplasts in the presence of auxin

and cytokinin (Clouse and Zurek, 1991; Nakajima et al. 1996; Oh and Clouse, 1998;

Miyazawa et al., 2003). BRs regulated expression of BRU1 gene encoding a xyloglucan

endotransglycosylase (XET) in soybean, also strengthened the role of BRs in xylem

differentiation (Zurek et al., 1994). XETs were reported to be involved in cell wall

modification, expansion, vascular differentiation and fruit ripening (Fry et al., 1992).

Recently, BRs are also accounted for maintaining position dependent cell-fate

specification in Arabidopsis roots. A transcriptional complex composed of the MYB

transcription factor WEREWOLF (WER), a WD-40 repeat protein called

TRANSPARENT TESTA GLABRA (TTG), 2 basic helix–loop–helix transcription factors

called GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) promotes hairless cell

(N cell) differentiation. This complex induces expression of GLABRA2 (GL2), encoding

a homeodomain transcription factor, and CAPRICE (CPC), encoding a single-repeat


29

MYB protein. CPC moves laterally to the adjacent epidermal cells, where it inhibits

WER and GL2 expression, thus promoting hair cell (H cell) differentiation (Kuppusamy

et al., 2009). Studies in Arabidopsis roots demonstrated that BRs are requisite for

normal expression levels and patterns of WER and GL2, key regulators of epidermal

patterning. Loss of BR signaling results in loss of hair cells in H positions, might be

through reduced expression of CPC.

The role of BRs in vascular development in Arabidopsis has been shown in BR-

deficient mutants (cpd; dwf7) and perception mutants (Szekeres et al., 1996; Choe et

al., 1999; Cano-Delgado et al., 2004; Dettmer et al., 2009). Two BRASSINOSTEROID

INSENSITIVE 1 (BRI1) like genes namely BRL1 and BRL3 were expressed in the

vasculature of all organs, with BRL3 specifically expressed in the phloem suggested

that these function redundantly control differentiation of the vasculature (Cano-Delgado

et al., 2004; Dettmer et al., 2009). Recent investigation on A. thaliana described that

both loss- and gain-of-function BR-related mutants reduced meristem size, thus

indicating the need of balanced BR signalling for the optimal root growth (González-

García et al., 2011). In BR-insensitive bri1-116 mutant, the expression pattern of the

cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE had a decreased mitotic

activity resulting in reduced meristem size. However, overexpression of CYCD3;1

overcome the reduced mitotic activity. The activity of the quiescent centre (QC) was

low in the short roots of bri1-116 but plants treated with BL, or mutants with enhanced

BR signalling, such as bes1-D, resulted in early differentiation of meristematic cells.

These results confirmed that BRs have a regulatory role in the control of cell-cycle

progression and differentiation in the Arabidopsis root meristem.

2.5.2.3 Brassinosteroids in Vegetative growth

Studies on BRs biosynthesis mutants successfully justified the importance of

BRs in regulating various plant metabolic activities. Hayat et al. (2000) reported

enhancement in yield, carbonic anhydrase activity and net photosynthetic rate of

Brassica juncea after treatment with HBL. Asami et al. (2000) suggested that


30

brassinazole (Brz, a triazole-type BR biosynthesis inhibitor) can be used to shed light on

the function of BRs in plant growth and development. Wheat grown from HBL pre-

treated seeds had enhanced leaf number per plant, fresh and dry weight, and activities of

nitrate reductase and carbonic anhydrase (Hayat et al., 2001a). Furthermore, the

comparative analysis of effects of different concentrations of indole-3-acetic acid

(IAA), GAs, kinetin, ABA and BRs revealed that the BR were most effective even in

µM concentration to enhance the chlorophyll levels (Hayat et al., 2001b). However,

Chon et al. (2000) reported that BL at lower concentrations was effective in enhancing

both mesocotyl elongation and leaf sheath lengths of rice whereas at higher

concentration it had inhibitory effects. Also, in seedlings of soybean, the inhibitory

effects of EBL (0.1- 10 µM) on root/shoot length, dry weight, and nodule and lateral

root numbers were observed (Hunter, 2001).

Two BR analogues, BB-6 and BB-16 promoted the precocity and accelerated

growth during the first stage of vegetative bud development in cladodes of Opuntia

ficus-indica (Cortes et al., 2003). Foliar Spray of EBL enhanced the activity of ribulose-

1,5-bisphosphate carboxylase/oxygenase (Rubisco) and contents of sucrose, soluble

sugars, and starch in Cucumis sativus plants (Yu et al., 2004). In Arabidopsis, BRs

interacted synergistically with auxins to promote lateral root development by

stimulating acropetal auxin transport in the roots (Bao et al., 2004). The role of BRs in

micropropagation techniques for clonal propagation of woody angiosperms was studied

by Pereira-Netto et al. (2006). Treatment of 28-homoCS to in vitro-grown shoots of a

hybrid between Eucalyptus grandis and E. urophylla resulted in enhanced elongation

and formation of new main shoots at low doses but there was reduced elongation and

formation of primary lateral shoots. Similarly, Malabadi and Nataraja (2007)

highlighted the role of EBL in micropropagation of orchids and assumed that in vitro

regeneration and initiation of protocorm like bodies (PLBs) in Cymbidium elegans were

achieved using shoot tip sections and EBL supplemented basal medium.

Xia et al. (2009) found that EBL treatments improved growth of cucumber (C.

sativus) plants via increasing CO2 assimilation and quantum yield of Photosystem-II


31

(PS-II). However, treatment of brassinazole (Brz) reduced plant growth, CO2

assimilation and quantum yield of PS-II. Northern and Western blotting demonstrated

that EBL upregulated, the expressions of Rubisco large subunit gene (rbcL) and

Rubisco small subunit gene (rbcS) and other photosynthetic genes, while Brz

downregulated their expressions. Swamy and Rao (2010) emphasized that both HBL

and EBL enhanced the root formation and root growth of treated cuttings over control in

coleus (Plectranthus forskohlii). Recent reports by Sharma et al. (2010, 2011a, 2011b)

on increase in shoot and root length in R. sativus further confirmed the growth

enhancing effects of HBL and EBL.

2.5.2.4 Brassinosteroids in Reproductive Biology

First report on BRs isolation by Grove et al. (1979) established the fact that

pollens are the richest source of endogenous BRs. Further, reports by Abe et al. (1991)

and Takasuto (1994) also confirmed pollens as the main source of BRs (Table 2.2).

Hewitt et al. (1985) reported that 1.0 nM BR induced maximum elongation of the

pollen tubes. The haploid seeds of Arabidopsis and B. juncea were produced by Kitani

(1994) with the applications of BRs. The role of BRs, in reproduction was further

strengthened by the observations made on BR-deficient dwf4 mutant and cpd mutants

where filament failed to elongate such that pollens although viable fail to reach stigma

(Szekeres et al., 1996). The relative distribution of BRs has also been explored in pollen

grains and it was observed that conjugated testasterone was present at the microspore

stage. The level of conjugated testasterone got decreased as the pollens developed and

levels of free BRs were increased (Asakawa et al., 1996). It was observed by Singh and

Shono (2003) that in-vitro pollen germination was more tolerant to high temperature in

tomato pollen when treated with EBL. However, higher concentrations (1 and 100 µM)

of EBL treatment were recorded to inhibit flower formation in Pharbitis nil (Kesy et al.,

2004).

The development of anther and pollen is important for male reproduction and at

an early anther developmental stage SPL/NZZ is required for sporocyte formation and

cell proliferation (Ye et al., 2010). EMS1/EXS and DYT1 is essential for tapetum


32

formation and early tapetal development respectively. However, TDF1 that is highly

expressed in tapetum, meiocytes, and microspores regulates the callose dissolution

around microspores and exine formation of pollen wall. AMS, encoding a putative MYC

transcription factor, is specifically expressed in tapetum and microspores and required

for microspore mitosis. AtMYB103 is a member of the R2R3 MYB gene family, only

expressed in tapetum and required for tapetal development and microsporogenesis. MS1

acts downstream of AtMYB103 and plays a critical role in regulating exine formation

and pollen coat development whereas MS2 is involved in sporopollenin synthesis and

normal exine patterning.

Further analyses have revealed genetic interactions among these genes. DYT1

acts downstream of SPL/NZZ and EMS1/EXS, and is required for normal expression of

AMS, MS1, and other tapetum-preferential genes. TDF1 acts downstream of DYT1 and

upstream of AMS and AtMYB103 in the transcriptional regulatory networks of tapetal

development (Ye et al., 2010). ChIP analysis demonstrated that BRs control male

fertility in Arabidopsis through BES1, an important transcription factor for BRs-

signaling, via regulating the expression of key genes. BES1 could directly bind to the

promoter regions of genes encoding transcription factors essential for anther and pollen

development like SPL/NZZ, TDF1, AMS, MS1, and MS2 (Ye et al., 2010).

Flowering is a critical phase transition in the development of angiosperms. The

correct timing of this transition is essential factor for determining reproductive success.

In grapes, Rao et al. (2002) reported that numbers of flowers were increased when BR

was sprayed in autumn, whereas in late winter it reduced the number of flowers. Role of

BRs in growth and flowering by molecular intersection in Arabidopsis was proposed by

Clouse (2008) and Yu et al. (2008). They observed a connection between BR signal

transduction and pathways controlling floral initiation. A critical transcription factor

required for BR dependent gene expression directly interacts with two transcription

regulators having divergent roles in modulating time of flowering in Arabidopsis.

Genetic analyses of interactions among GA, ABA and BRs for the control of flowering


33

time in A. thaliana revealed the promotive role of BRs (Domagalska et al., 2010). Loss-

of-function studies demonstrated a complex relationship between GAs and ABA, and

between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs.

However, gain-of-function studies established the fact that GAs were clearly limiting in

their sufficiency of action, whereas increases in BRs and ABA led to a more modest

phenotypic effect on floral timings (Domagalska et al., 2010).

2.5.2.5 Brassinosteroids in Fruit Ripening

Extensive research has established that besides ethylene, BRs are also involved

in fruit ripening (Deluc et al., 2007). Fruit ripening in tomato induced by BRs was

associated with increase in ethylene production (Vardhini and Rao, 2002). BRs

accumulate during fruit development and seem to play a key role in determining the

onset of ripening in fleshy fruits (Pilati et al., 2007). BL applications (about 5 ppm)

were proposed for decreasing physiological drop from fruit trees such as citrus, peach,

apple, pear and persimmon (Susumu et al., 1991). Applications of BRs resulted in

accelerations in ripening in rice, tomato, grape, cucumber yellow passion fruit, grapes

and cucumber (Fujii and Saka, 2001; Vardhini and Rao, 2002; Montoya et al., 2005;

Gomes et al., 2006; Symons et al., 2006; Deluc et al., 2007; Fu et al., 2008).

Application of BRs to grape berries significantly promoted ripening, while brassinazole,

an inhibitor of BR biosynthesis, significantly delayed fruit ripening (Symons et al.,

2006). Putative grape homologs of genes encoding BR biosynthesis enzymes

(BRASSINOSTEROID-6-OXIDASE and DWARF1) and the BR receptor (BRI1) were

isolated, and the function of the grape BRASSINOSTEROID-6-OXIDASE gene was

confirmed by transgenic complementation of the tomato (L. esculentum) extreme dwarf

(dx/dx) mutant. Expression analysis of these genes during berry development revealed

transcript accumulation patterns that were consistent with a dramatic increase in

endogenous BR levels observed at the onset of fruit ripening establishing that changes

in endogenous BR levels influence this key developmental process (Symons et al.,

2006).


34

Effect of EBL and Brz on early fruit development, cell division, and expression

of cyclin and cyclin-dependent kinase (CDKs) genes in two cucumber cultivars that

differ in parthenocarpic capacity had been studied by Fu et al. (2008). The application

of EBL induced parthenocarpic growth accompanied by active cell division whereas

Brz treatment inhibited fruit set. This inhibitory effect had been rescued by the

application of EBL. RT-PCR analysis showed that both pollination and EBL induced

expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB)

after anthesis. BR6ox1 and SMT transcripts, two genes involved in BR synthesis,

exhibited feedback regulation. This study strongly suggested that BRs play an important

role during early fruit development in cucumber.

2.5.2.6 Brassinosteroids in Senescence

Senescence ‘phenomenon of aging’ occurs in all living organisms. At the

cellular level, a cell’s life history consists of two processes: mitotic division and post-

mitotic life pattern. A mother cell or germ-like cell can undergo a finite number of

divisions to produce daughter cells. When the cell ceases dividing, this cell is said to

undergo mitotic senescence (Gan, 2010). In plants, both mitotic and post-mitotic

senescence occur which is regulated through interactions between various

phytohormones. Leaf senescence is a type of post-mitotic senescence

visualized/estimated through loss of chlorophyll, and degradation of proteins, nucleic

acids and nutrient remobilization (Zhou and Gan, 2010). In contrast to cytokinins, BRs

promoted senescence in Xanthium, Rumex explants and in detached cotyledons of

cucumber seedlings and leaves of mung bean seedlings (Zhao et al., 1990; Ding and

Zhao, 1995; He et al., 1996; Rao et al., 2002).

Recently, EBL and HBL are anticipated to inhibit the oxidative degradation,

decreased lipid peroxidation levels and acted as membrane protectant thereby delaying

senescence (Anuradha and Rao, 2007). In Arabidopsis a model has been developed by

He et al. (2001) to understand the network of leaf senescence regulation. Out of 147

senescence-associated enhancer trap lines, EBL could activate only two and till now

associated genes have not been cloned. Higher concentrations of EBL activated


35

senescence of wheat leaves (Saglam-Cag, 2007). Effects of BRs on postharvest disease

(blue mould rot) caused by Penicillium expansum and senescence of jujube fruit in

storage were observed by Zhu et al. (2010). BL significantly inhibited the development

of blue mould rot and enhanced the activities of defense-related enzymes, such as

phenylalanine ammonia-lyase, PPO, SOD and CAT. Also, BL significantly delayed

fruit senescence by reducing ethylene production and maintained fruit quality.

2.5.3 Brassinosteroids in Agronomic Practices

Various field experiments were conducted to ensure the practical implications of

BRs in the agriculture. In USA (1970), Japan and USSR (in early 1980’s), various field

trials revealed valuable effects of BRs and confirmed their worth as eco-friendly

agricultural chemicals (Fujita, 1985; Gregory, 1981; Takeuchi, 1992; Khripach et al.,

1993, 2000). Gradually, EBL is employed as most accepted active ingredient in

officially registered field preparations and have large scale utility in agricultural

practices. It is also the principle active ingredient of the plant growth promoting

preparation “Epin” (0.025 % solution of EBL) which was recommended in Russia,

Belarus and Japan for treating of cash crop plants such as tomato, potato, cumber,

pepper and barley to enhance their yield and quality (Moiseev, 1998). In China, another

EBL-related chemical “Tianfengsu” was developed that constitutes the mixture of EBL

and its unnatural 22S, 23S isomer. Tianfengsu is widely used in China to increase yield

of economically important crops such as rice, maize, wheat, tobacco, vegetables and

fruits (Ikekawa and Zhao, 1991). Moreover, the employment of BRs in agriculture is

supported by Khripach et al. (2000) who reported that BRs are natural, non-toxic and

eco-friendly products when applied in extremely low doses are capable of improving

the crop yield even in non-fertilized fields.

Incorporation of BRs in various field trials and agronomical practices has

disclosed the fact that BRs can enhance yield by accelerating both quality and quantity

of various food crops. Large scale field trials, over a period of 10 years have shown that

percentage of grain setting, number of caryopsis per ear and weight of 1000 grains of


36

wheat increased by the application of EBL (Zhao and Chen, 2003) EBL treatment

enhanced the yield of wheat, tobacco, corn, rape orange, grape and sugar beet (Ikekawa

and Zhao, 1991; Hu et al., 1990; Zhao and Chen, 2003) and its foliar spray prior to the

tassel emergence, significantly decreased the kernel abortion of its ear tips and

increased the corn yield (Zhang, 1989). Also, EBL had significant growth promoting

effects on several vegetables like celery, cabbage, onion, lettuce and watermelon (Wang

et al., 1998; Huang and Li, 1998). Quality of potato was improved by HBL and EBL

treatments by significant acceleration in the contents of starch and vitamin C (Khripach

et al., 1996). Further, HBL-mediated enhanced yield responses of some economically

important crop plants like wheat, rice, groundnut, mustard, potato and cotton were also

studied (Ramraj et al., 1997).

Transgenic rice having about 30% higher grain yield with erect leaf phenotype

without any changes in grain by partial suppression of endogenous OsBRI1

(BRASSINOSTEROID INSENSITIVE1 ortholog) expression was produced by

Morinaka et al. (2006). Stress ameliorative effects of EBL on biomass and yield of

wheat grain and straw grown under drought and high temperature were observed

(Hnilička et al., 2007). Further, Štranc et al. (2008) reported the positive influence of

BRs and Lexin preparation (fulvic and humic acids mixture and auxins) on

physiological state and yield of soybean. Plants treated with these preparations were

found to be more resistant to short term drought, showed better physiological state and

energy balance of photosynthesis and higher seed yield.

2.6 ETHICAL ISSUES OR BIOSAFETY OF BRASSINOSTEROIDS

Though BRs have not been exploited practically and comprehensively in the

field trials, hitherto the reports available suggest their possible impending relevance in

human welfare from practical point of view. As BRs are natural nontoxic and eco-

friendly plant products so there would be no ethical issues attached with the

implications of BRs at large scale level. Hence, it makes BRs suitable candidate for

their application in agriculture and therapeutics. Before their commercial use in human

welfare, scientists have studied their bio-safety. Being normal constituent of all plants,


37

BRs are consumed by mammals, so no additional harmful effects can be expected from

their use in agriculture.

The confirmation of their safety had been obtained from toxicological studies

made in the Sanitary-Hygienic Institute of Belarus for EBL. It was observed that the

formulation, Epin (0.025 % solution of EBL), in mice and rats (orally and dermally) had

an LD of more than 5000 mg per litre. Repeated experiments confirmed the value of LD

50 for 24-epibrassinolide orally in mice and showed a value for Epin which was higher

than 15,000 mg per litre (white rats, orally or intra- nasally). Further 0.2 % EBL or a

solution of Epin did not irritate mucous membranes of rabbit eyes. Ames test for

mutagenic activity carried out at Scientific Research Center of Toxicologic and

Hygienic Regulation of Bio-preparations of Russia, was negative. Studies on fish

toxicity also showed no negative effects, but showed toxico-protective properties

(Khripach et al., 2000). In micro-nuclear or chromosome abberation tests (mice

CBAB1/6) neither EBL nor Epin caused spontaneous mutations. Complex biological

activities have confirmed the genetic safety of EBL and the absence of mutagenic

activity over seven generations. In prolonged experiments, EBL showed no toxicity but

a pronounced adaptogenic effect (increasing adaptive ability of the population).

Recently, the developmental toxicity of HBL in wistar rats was studied at

International Institute of Biotechnology and Toxicology (IIBAT), Tamil Nadu, India

(Murkunde and Murthy, 2010). HBL was administered by oral gavage at doses 0, 100,

and 1000 mg/kg body weight in water during gestation days (GD) 6 to 15 in groups of

20 mated females. Maternal and embryo-fetal toxicity was analyzed by studying the

effects such as clinical signs, mortality/morbidity, abortions, body weight, feed

consumption, and pregnancy data, gravid uterine weights, implantation losses, litter

size, external, visceral, and skeletal malformations. No treatment-related effect was

observed on any of the maternal/fetal end points in any dose group. Thus, Murkunde

and Murthy (2010) concluded that HBL is nonteratogenic at doses as high as up to 1000

mg/kg body weight in wistar rats.


38

2.7 COMMERCIALIZATION OF BRASSINOSTEROIDS

To increase the accessibility as well as affordability of BRs to human welfare,

various formulation of BRs are now being commercialized. Synthetic BRs are being

synthesized by various pesticidal companies of Japan and China (Hayat and Ahmad,

2003). In India, Godrej Agrovet Ltd., Mumbai has commercialized HBL in market for

yield enhancement of grapes, groundnut, tea and peanut (Hazara et al., 2005). Various

genes involved in BRs signal transduction pathway in plants have been invented. The

invention includes the nucleic acid molecules encoding the BZR1 polypeptide and

variants thereof, including the protein product of the dominant mutant bzr1-D, all of

which are involved in the regulation of cell expansion in plants through effects on

brassinosteroid response pathways. Also, the methods of modulating BR-related

responses, methods of identifying compounds involved in signaling pathways and

methods of altering plant phenotypes by altering the genes encoding the BZR1

polypeptides has been invented (Chory and Wang, 2005).

Further, a novel gene in plants which encodes a protein having the function of

controlling an in-vivo signal transduction system in a physiological reaction system

against BRs has been patented by Hirochika et al. (2006). By modulating and

expressing this gene various BRs mediated responses in plants (viz., growth promotion,

yield increase, quality improvement, maturation enhancement, and tolerance against

biotic and abiotic stresses) can be tailored. It may be possible to produce crops with

increased fruit size by linking fruit-specific promoters to genes with modified BR

responses. Recently, Kitron and Perga-Mentz (2010) had invented/patented a new

formulation of BRs or their derivatives for use in treating androgen-associated

conditions, particularly conditions afflicting nearly all men at certain age, such as

benign prostatic hyperplasia and androgenic alopecia.


39

2.8 BRASSINOSTEROIDS IN INSECT DEVELOPMENT

Zullo and Adam (2002) reviwed the effects of BRs on insect development,

particularly on molting, were reviewed by. EBL or 24-epicastasterone (24-epiCS) had

not affected the evagination of imaginal wing discs, and even after oral feeding it didn’t

show effect on intact last instar larvae of the cotton leaf worm, Spodoptera littoralis

(Smagghe et al., 2002). Similarly, treatment of root knot nematodes (Meloidogyne

incognita) with BL revealed much higher percentage of hatching in treated egg masses

as compared to control (Ohri et al., 2002). Further Ohri et al. (2004, 2005) found that

juvenile emergence of M. incognita was enhanced by treatment of BRs and also

stimulated their antioxidative defence system (Ohri et al., 2007). Ohri et al. (2008)

reported that EBL enhanced the percentage of hatching in treated egg masses of M.

incognita as compared to control. EBL treated juveniles of M. incognita induced more

and larger size of galls in tomato roots.

2.9 BRASSINOSTEROIDS IN MEDICINE

Several bioassays, including the bean first or second-internode bioassay

(Mitchell and Livingston, 1968; Thompson et al., 1981; 1982), radish hypocotyl assay

(Takasuto et al., 1983), wheat leaf unrolling assay (Wada et al., 1985), the tomato

hypocotyl test (Takasuto et al., 1983), mung bean epicotyl assay (Gregory and

Mandava, 1982) and the rice lamina inclination test (Wada et al., 1981; Arima et al.,

1984; Kim et al., 1990), have been used to determine the bioactivity of either isolated

BRs from plant sources or chemically synthesized analogs (Table 2.3). Fujioka et al.

(1998) evaluated the biological activities of BL, its biosynthetic intermediates and some

biosynthetically related BRs employing modified dwarf rice lamina inclination


40

bioassay. Modified bioassay was found to be very effective even for very earlier formed

bio-synthetic intermediates such as campestanol.

Recent studies on biological activities of BRs in various animal test systems

depicted their antibacterial, anticancerous/antiproliferative, antifungal, antigenotoxic,

antiviral and ecdysteroidal properties. Consequently, BRs have the prospect as a

potential future medicine for treating cancer, fungal, bacterial and viral infections and

neurodegerative diseases etc. (Nakashita et al., 2003; Wachsman et al., 2004a, 2004b;

Swaczynova et al., 2006; Wu and Lou, 2007; Romanutti et al., 2007; Ohri et al., 2008;

Malìková et al., 2008; Carange et al., 2011). The enduring researches in the field of

BRs have verified their ability to stimulate plant resistance to viruses (Rodkin et al.,

1997; Bobrik et al., 1998; Nakashita et al., 2003; Michelini et al., 2004, 2008).

However, antifungal potential of BRs is documented in few plants that indicate their

prospects for antifungal formulations against several fungal diseases, but the detailed

studies are required for another fungal infection in plants (Vasyukova et al., 1994;

Pshenichnaya et al., 1997; Volynets et al., 1997a, 1997b; Khripach et al., 2000).

Antigenotoxic potential of EBL has been demonstrated through Allium cepa

chromosomal aberration bioassay (Howell et al. 2007; Sondhi et al., 2008).

Furthermore, imminent investigations on BRs for their prospectives in developing

anticancerous and antiproliferative drugs have been carried out in various test systems

(Franěk et al., 2003; Swaczynova et al., 2006; Wu and Lou, 2007; Malìková et al.,

2008). Recent analysis of role of EBL as potent antioxidant and neuroprotective has

been shown in a mammalian neuronal cell line thus, indicates its future implications in

development of new BR-derived generation of drugs against neurodegenerative

disorders, like Parkinson’s disease (Carange et al., 2011). Some of the recent studies

done in this direction are tabulated in Table 2.4.


41

2.10 BRASSINOSTREOIDS AND OXIDATIVE STRESS MANAGEMENT

In agriculture several biotic as

well as abiotic stresses have been

reported to occur simultaneously, rather

than a particular stress condition, which

are most lethal to crops but the co-

occurrence of different stresses is rarely

addressed by molecular biologists

(Mittler, 2006). The abiotic/biotic

stresses against which BRs counteract

are drought, heavy metals, water

logging, thermal, infection, pesticides,

salt and even several pathogens

(Dhaubhadel et al., 1999, 2002;

Krishna, 2003; Wachsman et al., 2004a,

2004b).

Exogenous application of BRs improved the antioxidant system by regulating

the activities of antioxidant enzymes (SOD, POD, CAT, GR, APOX etc.) and

antioxidants (alkaloids, carotenoids, flavonoids, ascorbate, tocopherol, glutathione,

glycinebetaine, prolines etc.) to provide protection to the plants under stress conditions

(Núñez et al., 2004; Özdemir et al., 2004; Hayat et al., 2007; Sharma and Bhardwaj,

2007; Arora et al., 2008; Sharma et al., 2010, 2011a, 2011b). Antioxidants such as

ascorbate, tocopherol and glutathione react directly or via enzyme catalyis reactions

with hydrogen peroxide (H2O2), hydroxyl radical (OH.) or O2- while carotenes directly

act as effective quenchers of reactive oxygen species (Shakirova, 2002; Núñez et al.,

2004; Özdemir et al., 2004). The protective effects of BRs and its structural analogs had

been studied on growth, lipid peroxidation, compatible solutes, osmolytes, enzymatic

and non-enzymatic antioxidant defence system of maize, rice, soyabean, tomato, potato,


42

radish, mustard, chickpea and Arabidopsis under various stresses and it was observed

that application of these hormones lowered the oxidative stress and promoted plant

growth (Mazorra et al., 2002; Özdemir et al., 2004; Almeida et al., 2005; Kagale et al.,

2007; Arora et al., 2008a; Bhardwaj et al., 2008). Implications of BRs in a plethora of

abiotic stresses are discussed precisely as below:

2.10.1 BRs and Drought Stress

Treatment of BRs significantly relieved drought stress in sugar beet and wheat

etc. (Schilling et al., 1991). The anti-stress effect of BRs had been attributed for

enhancing the membrane stability via maintaining ion exchange pumps. Also, stomatal

transpiration was decreased by EBL treatment resulting in improved growth and yield

of plants under stress (Singh et al., 1993; Sairam, 1994; Nilovskaya et al., 2001). EBL

treatments to different varieties of spring wheat under drought revealed that plants

sprayed with EBL at start of booting stage of flowering resulted in higher water content

in leaves (Prusakova et al., 2000). In flax, positive effects of EBL and immunotzitofite

on drought resistance were observed by Khodiankov (2002). Foliar sprays of BRs at

flowering stage increased the root nodulation, cytokinin trans-zeatin riboside (ZR)

content and nitrogenase activity and yield, by ameliorating the water stress in French

bean (Upreti and Murti, 2004). Besides, EBL treatments also enhanced drought

tolerance in A. thaliana and B. napus seedlings by changing expression of drought

responsive genes (Kagale et al., 2007). EBL and BL also helped to overcome the

negative influence of drought, high temperature and water stress in case of three winter

wheat varieties (Ebi, Estica, Samanta) and 1-year old Robinia pseudoacacia

respectively (Hnilička et al., 2007; Li et al., 2008).

2.10.2 BRs and Pesticides Stress

Recent studies demonstrated that BRs are effective in reducing damage caused

by pesticides (simazine, butachlor, or pretilachor) in rice (Sasse, 2003). The phytotoxic

effects of nine pesticides including three herbicides (paraquat, fluazifop-p-butyl and

haloxyfop), three fungicides (flusilazole, cuproxat and cyazofamid) and three

insecticides (imidacloprid, chlorpyrifos and abamectin) were investigated on cucumber


43

leaves (Xia et al., 2006). EBL pre-treatments alleviated the inhibitory effects of

pesticides/fungicides/herbicides on photosynthetic rate except for the pesticides

paraquat and flusilazole. EBL induced resistance in cucumber plants to pesticides might

be mediated by enhanced activities of CO2 assimilation enzymes thereby enhancing the

rate of photosynthesis.

2.10.3 BRs and Salinity Stress

Almost 20% of the world’s cultivated area and half of the irrigated lands are

affected by salinity that leads to adverse plant growth and development (Akram et al.,

2008). BRs have been reported to overcome the salt stress by regulating the activities of

key stress protective enzymes and accumulation of osmolytes. Application of BRs had

promoting effects on seed germination and seedling or plant growth in A. hypogea, E.

camaldulensis, O. sativa plants and seedlings, salt sensitive rice cultivar IR-28, B.

napus, M. sativa, T. aestivum and B. juncea plants under saline conditions (Sasse et al.,

1995; Vardhini and Rao, 1997; Anuradha and Rao, 2003; Nùñez et al. 2004; Özdemir et

al., 2004; Kagale et al., 2007; Zhang et al., 2007; Shahbaz et al., 2008; Ali et al.,

2008c). Our earlier studies also revelaed that HBL ameliorated the salinity stress in Z.

mays seedlings and plants by increasing the activities of antioxidant enzymes (CAT,

POD, SOD, GR and APOX) and by reducing the level of lipid peroxidation (Arora et

al., 2008a).

2.10.4 BRs and Temperature Stress

BRs have also been reported to show growth promoting effects on eggplant,

cucumber, rice and maize under chilling stress (Mandava, 1988; Kim and Sa, 1989; He

et al., 1991; Hirai et al., 1991; Wang and Zeng, 1993). In winter conditions, Kamuro

and Takatsuto (1991) observed that tomato plants sprayed with BRs had higher fruit

settings. BRs- treated wheat leaves at 43 C by Kulaeva et al. (1991) revealed that

protein synthesis was maintained similar to those at 23 C, whereas in untreated leaves it

decreased 2.5 fold at 43 C as compared to control temperature. Treatment of EBL

reduced the MDA content, slowed the decrease in SOD activity, and increased the

proline content of rice under chilling stress (Wang and Zeng, 1993). EBL applications


44

enhanced basic thermotolerance in brome grass, B. napus and tomato seedlings grown

under high temperature (Wilen et al., 1995; Dhaubhadel et al., 1999). An analysis of

heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that

the HSPs did not preferentially accumulate in EBL-treated seedlings at the control

temperature. However, after heat stress, HSP accumulation was higher in EBL-treated

than in untreated seedlings. This report indicated the possibility of EBL-induced

expression of HSPs. Higher accumulation of HSPs in EBL-treated seedlings raises the

possibility that HSPs contribute, at least in part, to thermotolerance in EBL-treated

seedlings.

Dhaubhadel et al. (2002) observed that EBL treatment limits the loss of some of

the components of the translational apparatus during prolonged heat stress, and

increases the level of expression of some of the components of the translational

machinery during recovery, which correlates with a more rapid resumption of cellular

protein synthesis following heat stress and a higher survival rate. Müssig et al. (2002)

identified the oxidative stress related genes encoding MDHAR and thioredoxin-h, the

cold and drought response gene COR 47 and COR 78 and heat stress related genes

HSP83, HSP-70, HSF3, Hsc 70-3, Hsc70-G7 by micro-array analysis of either BRs-

deficient or BRs-treated plants. It was further studied by Singh and Shono (2005) that

EBL induced expression of MT-sHSP (mitochondrial small heat shock proteins), which

possibly induced thermotolerance in tomato plants at high temperature (38 C) in tomato

plants. Kagale et al. (2007) further extended the studies of Dhaubhadel et al. (2002) in

A. thaliana seedlings and BRs-deficient mutant seedlings. EBL treatment enhanced the

basic thermotolerance of A. thaliana seedlings under heat stress, similar to its effect on

B. napus by regulating the expression of transcripts of three cold responsive structural

genes- rd29a, a BN115 homolog and COR47 in A. thaliana seedlings. Janeczko et al.

(2007, 2009) observed a relationship between the temperature of oilseed rape growth

and the influence of EBL on biochemical changes in cells concluding that EBL-action is

highly temperature-dependent.


45

2.10.5 BRs and Water Stress

Several studies have been investigated on the effects of BRs on water-stressed

plants. Schilling et al. (1991) reported that HBL improved tap-root weight, sucrose

content, and yield of sugar beets grown under drought stress. Similarly, Sairam (1994)

observed that HBL significantly augmented the relative water content, chlorophyll A

content, photosynthetic rate, leaf area and biomass production of wheat under moisture

stress. HBL treated jackpine plants under water stress evolved more ethylene

(Rajasekaran and Blake, 1999). BRs treatment improved resistance to desiccation and

severe water stress in cucumber plants and Robinia seedlings respectively (Pustovoitova

et al., 2001; Li et al., 2008). In Robinia seedlings, leaf water content, predawn water

potential, soluble sugar content, free proline content, and SOD, POD and CAT activities

were alleviated in water-stressed seedlings in the 0.2 mg/l BL treatment compared to the

control (Li et al., 2008). Endogenous levels of BR and ABA in wild type (WT) and BR-

deficient mutant (lkb) and BR-perception mutant (lka) pea plants exposed to water

stress were observed by Jager et al. (2008) revealed that ABA levels enhanced whereas

BR levels didn’t. Further, it was proposed that change in endogenous levels of BR was

not normally part of the plant's response to water stress.

2.10.6 BRs and Heavy Metals Stress

Treatment of barley, sugarbeet, tomato and radish with EBL significantly

reduced the absorption of heavy metals (Khripach et al., 1996). Besides, EBL

treatments resulted in the absorption of radionuclides (cesium, Cs and strontium, Sr

ions) in barley grown on the model Cs and Sr- rich soils (Khripach et al., 1997).

However, in grains of plants treated with 0.01 ppm of EBL had declined levels of Cs

and Sr than control plants grown on the polluted soil. EBL treatments at 10-8 M

concentration blocked the heavy metals (copper, Cu; lead, Pb; cadmium, Cd and zinc,

Zn) accumulation in C. vulgaris cells (Bajguz, 2000). Further, BRs induced the

phytochelatins synthesis in C. vulgaris under Pb toxicity (Bajguz, 2002). BRs also

ameliorated aluminium (Al) toxicity in P. aureus seedlings, Cd toxicity in winter rape

plants (Bilikisu et al., 2003; Janeczko et al., 2005). EBL and HBL treatments decreased


46

Cd toxicity in radish seedlings by enhancing seed germination, seedling growth, free

proline levels, activities of CAT, SOD, APOX and reducing MDA content, activity of

POD and ascorbic acid oxidase (Anuradha and Rao, 2007). Similarly in B. juncea

plants, HBL increased the activities of CAT, POD, SOD, nitrate content and nitrate

reductase enzyme activity; and enhanced photosynthetic rate as well as contents of

chlorophyll and proline under Cd stress (Hayat et al., 2007). HBL protected chickpea

from Cd toxicity by enhancing the activities of SOD, CAT, POD, nitrate reductase and

carbonic anhydrase and also by increasing the plant growth, leghemoglobin content,

proline content, nodule number, nitrogen and carbohydrates content in the nodules and

leaf chlorophyll content (Hasan et al., 2008).

HBL also improved the growth by increasing the net photosynthetic rate,

chlorophyll content and the activities of nitrate reductase and carbonic anhydrase in

nickel stressed B. juncea seedlings (Alam et al., 2007). Further, HBL and EBL

enhanced the activities of antioxidant enzymes (CAT, SOD, POD) and proline content

to overcome the Al-induced oxidative stress in mung bean (Ali et al., 2008a). The

heavy metal stress ameliorative properties of EBL were confirmed in 15 days old

Brassica juncea plants by Ali et al. (2008b). EBL improved the by enhancing the

proline level and activities of CAT, POD, SOD and GR and reducing the levels of

MDA in Ni stressed Indian mustard.

Our previous studies on stress-ameliorative effects of BRs in B. juncea seedlings

and plants had revealed that EBL and HBL treatments (pre-sowing) improved the shoot

emergence and plant biomass production under heavy metal stress (Cu, Zn, Mn, Co and

Ni). Furthermore, EBL and HBL reduced the heavy metal uptake and accumulation in

B. juncea seedlings and plants. The mechanism involved for reducing toxicity might be

the chelation of the metal ions by the ligands. Such ligands include organic acids, amino

acids, peptides or polypeptides (Sharma and Bhardwaj, 2007; Sharma et al., 2007,

2008; Bhardwaj et al., 2007, 2008). Further our studies on heavy metal stress indicated

that HBL ameliorated the Ni, Zn, Cu toxicity in maize seedlings by increasing the


47

activities of antioxidant enzymes such as SOD, POD, CAT, APOX and GR (Bhardwaj

et al., 2007, Arora et al., 2008a).

2.11 HEAVY METALS STRESS AND ANTIOXIDANT DEFENCE SYSTEM

In R. sativus an inhibition in the germination was observed by lead (Pb)

concentration higher than 1000ppm (Lane and Martin, 1980). The inhibitory effects of

lead contamination were readily seen in terms of a significant reduction in root hair

development on the emerging radicle. Subsequent development of secondary roots was

also severely restricted. Such effects led to a reduction in the seedlings ability to anchor

itself in the soil and cause problems of salt and water uptake in the developing

seedlings. Seed germination and seedling growth in relation to copper and cadmium

treatment individually and in combination have been studied by Agarwal and Gupta

(1994) in R. sativus. The germination values showed gradual reduction from 85 to 25

percent by copper treatment of 0.05-100mg/l and cadmium treatment of 0.01 to

100mg/l. Seedling growth in terms of seedling length, dry weight, total chlorophyll and

carotenoid in relation to these two heavy metal treatments also showed inhibitory

effects. Decrease in seedling length from 20 to 6cm and from 19 to 5cm, seedling dry

weight from 12 to 3mg per seedling, total chlorophyll from 0.5817 to 0.0182 mg/g and

from 0.4657 to 0.1276mg/g fresh weight and carotenoid content from 0.1676 to

0.0115mg/g and 0.1684 to 0.0571mg/g fresh weight was observed at 0.01 to 100mg/l

treatment of copper and cadmium respectively.

Since agronomic practices of various crops like R. sativus face the challenges of

startlingly high levels of toxic heavy metals like Hg, Cd, Ni, Pb, Cr etc., in groundwater

and agricultural soils including Punjab, India (Zahir et al., 2005), therefore it is most

warranted to explore the impact of heavy metals induced stress in a commonly edible

radish plant. R. sativus is the widely used agro-economic crop with culinary and

medicinal importance, also known for protective role against environmental mutagens

and their eventual use as therapeutics (Ghayura and Gilani, 2007; Alquasoumi et al.,

2008). Also, Raphanus has a strong defense system and has active constitutes viz.,


48

isothiocyanates, anthocyanins, catechols, flavanols, raphanusanol and BRs, alongwith

other minerals (Romero-Puertas et al., 2006; Csiszar et al., 2008). Recently, R. sativus

has emerged as plant with a potential for hyperaccumulation and phytoremediation

(Vamerali et al., 2010).

2.11.1 Cadmium and Antioxidant Defence System in Plants

Cadmium (Cd) is a relatively rare non-essential element and one of the most

toxic environmental and industrial pollutants for animal and plants (Ünyayar et al.,

2006; Daud et al., 2009). It is a byproduct of the mining and smelting, and is used in

electroplating, nickel-cadmium batteries, PVC plastics, paint pigments, cigarettes and

commercial fertilizers (Benavides et al., 2005). It can reach high levels in agricultural

soils and is easily assimilated by plants. Moreover, due to neurotoxic, mutagenic and

carcinogenic effects, high water solubility and thereby easy entry into human body via

food chain render Cd a dangerous environmental pollutant even at low concentrations

(Sanita di Toppi and Gabbrielli, 1999). The human health outcome of environmental

contamination by heavy metals, particularly Cd and Hg has been amply demonstrated

by the well-known Itai-Itai and Minamata episodes in Japan. In humans, cadmium

causes proteinuria, glucosuria, osteomalacia; arsenic affects the pulmonary organs,

nervous system and skin; lead and mercury affect nervous system and renal system

causing central and peripheral neuropathies, proteinuria, encephalopathy etc. (Misra and

Mani, 1992).It is well documented that Cd phytotoxicity leads to reduction in the yield,

seed germination, growth and development and inhibition of other plant metabolic

activities such as respiration, photosynthesis, water relations and gas exchange

(Drazkiewicz et al., 2003; Ünyayar et al., 2006; Monteiro et al., 2009). Cd exposure

decreased the rate of photosynthesis in O. sativa seedlings (Moya et al., 1993).

An increase in the activities of APOX, CAT, GR and DHAR in sunflower leaves

under Cd stress was observed by Gallego et al. (1999). However, there was no effect

observed on SOD activity. Further, in BY-2 cell cultures of Nicotiana tabaccum,

significant enhancement in SOD and APOX activities was observed under Cd (Piqueras


49

et al., 1999). It was reported by Zhu et al. (1999a) that B. juncea plants with an

overexpression of qsh2 gene encoding glutathione synthetase accumulated more

cadmium than non-transformed plants. Additionally, the transformed plants contained

higher levels of GSH, phytochelatins, thiol sulphur and calcium than wild plants.

Further, transgenic plants with simultaneous -ECS and glutathione synthetase over

expression, which led to an increased GSH production and accumulation, exhibited a

specifically enhanced efficiency of heavy metal phytoextraction (Zhu et al., 1999b).

Effects of Cd (0.1 and 0.2 mM CdCl2) and Ni (0.075 and 0.75 mM NiCl2) were

measured on N. tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over

a 72-h period. At different times during the period of metal exposure, activities of

enzymatic antioxidant defense systems tested; induced by the two concentrations of

Cd and Ni and these results indicated increase in activity of guaiacol peroxidase in

the latter half of the experimental period. Therefore, it is likely guaiacol-type

peroxidase appeared to play a more important role in the antioxidant response once

the stress became severe (Gratao et al., 2008). Detailed impact of Cd on enzymatic

and non-enzymatic antioxidant defence sytsem in diverse plant species have been

compiled in Table 2.5.

2.11.2 Chromium and Antioxidant Defence System in Plants

Among heavy metals, Cr is widely distributed in nature because of their

multifarious use in leather, refractory steel, drilling muds and electroplating industries.

It was first discovered in 1798 by French chemist Vanquelin. Cr is a transition element,

7th most abundant in the earth crust (Katz and Salem, 1994). Also, Cr is toxic to plants

and does not play any role in plant metabolism (Dixit et al., 2002). The adverse effects

of Cr on the yield, seed germination, inhibition of some metalloenzymes, growth and

development have been well documented (Shanker et al., 2005). It occurs in two stable

forms which are trivalent Cr (III) and hexavalent Cr (VI) species. The hexavalent

species of Cr or Cr (VI) usually occurs associated with oxygen as chromate (CrO42-) or

dichromate (Cr2O72-) oxyanions. Cr (VI) is reported to be more mobile and toxic


50

compared to Cr (III) species (Yu et al., 2007). It is also a strong oxidant with high

redox potential which accounts for the generation of free radicals. Thus, Cr toxicity is

mediated by the formation of ROS (Yu et al., 2007) and by catalysis of the Haber-

Weiss reaction (Halliwell and Gutteridge, 2007). ROS like H2O2 to form OH• and O2

-

directly by interaction of Cr (VI) or Cr (III) with glutathione, NADPH and H2O2 (Shi

and Dalal, 1989; Aiyar et al., 1991) resulting in oxidative damage and stimulation of

lipid peroxidation (Panda and Patra, 2000; Dixit et al., 2002; Panda et al., 2003;

Choudhury and Panda, 2004). Cr affects Fe uptake in dicots either by inhibiting

reduction of Fe (III) to Fe (II) or by competing with Fe (II) at the absorption site

(Shanker et al., 2005).

Inhibitory effects of Cr on cell division by inducing chromosomal aberrations in

A. cepa were reported by Liu et al. (1993). Cr phytotoxicity caused reduction in seed

germination and growth in wheat (Jamal et al., 2006), Vigna radiata L. (Panda and

Khan, 2002) and Echinochloa colona (Rout et al., 2000). A significant decrease in

biomass productivity and total chlorophyll contents with increase in Cr was observed

in Wolffia globosa (Boonyapookana et al., 2002). Chlorophyll and carotenoid content

were reduced by Cr via degrading the enzyme (∂-aminolevulinic acid dehydratase)

involved in chlorophyll biosynthesis (Vajpayee et al., 2000) and via replacing Mg2+

ions from the active sites of this important enzyme (Baszynski et al., 1981; Rai et al.,

1992). Cr induced toxicity also reduced CO2 fixation, and activities of CAT and POD

(Sharma and Sharma, 1996; Ghosh and Singh, 2005; Scoccianti et al., 2006).

Declined rates of photosynthesis, inactivation of calvin cycle enzymes, reduced

CO2 fixation and chloroplast disorganization were observed in wheat, peas, rice, maize,

beans and sunflower under Cr stress (Bishnoi et al., 1993a, 1993b; Zeid, 2001; Davies

et al., 2002; Shanker, 2003). The effects of different heavy metals including Cr on

antioxidant defence system in plants have been tabulated in Table 2.5.


51

2.11.3 Mercury and Antioxidant Defence System in Plants

Bioaccumulation of non-essential and non-biodegradable heavy metals

particularly mercury (Hg) has become a contentious issue of global concern for the

environmentalists. Regular use of cosmetics, fungicides, pesticides and soil fertilizers;

mismanaged dumping of wastes from paper and leather tanning industries, wastewater

treatment plants; disposal of hazardous wastes viz. batteries and thermometers to

municipal landfills, are the critical sources of mercury in the environment (Arabi,

2005). Although in atmosphere, Hg can exist in diverse forms, e.g. HgS, Hg(II), Hg(0),

and methyl-Hg but its predominant form is Hg (II) or Hg2+, which is readily soluble in

water and accumulates in higher plants (Wang and Greger, 2004; Han et al., 2006;

Elbaz et al., 2010). Organo-mercurial compounds like methyl-Hg may be several

times more toxic than inorganic Hg and is a key noxious agent in most

environmental Hg-release disasters (Heaton et al., 1998; Patra and Sharma, 2000).

Mercury binds to sulfhydryl groups of proteins and disulfide groups in amino acids

thereby resulting in inactivation of sulfur and blocking related enzymes or

cofactors/hormones (Zahir et al., 2005).

Mercury has no nutritional role and exposure of biological systems to

relatively low Hg concentrations results in serious toxicity. Of critical concern is the

risk of developing hypoesthesia, ataxia, impairment of hearing and visual changes,

and severe neurodegenerative diseases viz., Minamata, Alzheimer’s, Parkinson’s,

Rheumatoid arthritis, Autism etc. as impact of human exposure to Hg (Zahir et al.,

2005). Since the confirmation that Minamata disease was the direct result of methyl

mercury accumulation in fish, researchers have recognized mercury contamination of

food chains as a potential threat to humans. At the end of 1953, the first victim of

methyl mercury poisoning appeared in the ill-fated Minamata city of Japan, followed by

several more cases subsequently. On May 1, 1956, Dr. Hajime Hosokawa, the head of

the Chisso Factory Hospital, reported to the Minamata Public Health Department: “An

unclarified disease of the Central Nervous System has broken out”. This was the official


52

discovery date of what is now called Minamata disease (Hosokawa, 1957). The level of

mercury in natural fresh water is generally below 0.1 g/l, but in contaminated areas

such as Minamata bay in Japan, levels as high as 1 to 10 g/l were observed (Kiyoura,

1963). The mud of Minamata bay was found to contain from 30 to 40 g/kg of mercury

(Kitamura, 1968). It is estimated that worldwide more than 1,400 humans had died and

over 20,000 had been afflicted by Hg poisoning over the last 40 years (Lacerda, 1997).

In plants, the absorbed Hg is usually retained by the roots, albeit the precise

mechanism of Hg-induced phytotoxicity is uncertain (Boening, 2000; Carrasco-Gil

et al., 2011). It might be possible that Hg alters the permeability and structural

integrity of plant cell-membranes by binding to sulfhydryl (SH) radical (Zahir et al.,

2005). Consequently, Hg poisoning reduces mineral/nutrient/water uptake, inhibits the

development of roots and leaves, inhibits pollen germination, alters the contents of

various biochemical constituents and reduces the rates of photosynthesis and

transpiration (Patra and Sharma, 2000; Chen et al., 2009; Elbaz et al., 2010). Besides

this, the activities of several enzymes involved in various plant metabolic pathways are

inhibited by Hg toxicity (Lenti et al., 2002; Ansaria et al., 2009). Ultimately, Hg stress

results in the over-production of reactive oxygen species (ROS) and free radicals which

generate oxidative stress in plants (Elbaz et al., 2010).

Decrease in active mitotic index and increase in mitotic anomalies like clumping

of chromatin and chromosomes, chromosome erosion and chromatin fragmentation

under Hg stress was observed by Aggarwal and Srivastava (2000). Further study by

Patra (2000) confirmed that mitotic index to be inversely proportional and chromosomal

aberrations to be directly proportional to the Hg concentration and the duration of

exposure while analyzing the genotoxic effects of inorganic mercurial salts in A. cepa.

Treatment of Hg affected cell division in C. vulgaris, abscission in Coleus blumei plant

to greater extent as compared to content of chlorophyll in these cells (Speitel and

Siegel, 1975; Rosko and Rachlin, 1977). Also, inhibitory effects of Hg on the pollen

tube germination and tube growth in Apricot (Armenica vulgaris Lam.) and Cherry


53

(Cerasus avium L.) were reported (Gur and Topdemir, 2008).However, Ali et al. (2000)

has reported the induction of oxidative stress and toxicity to submerged macrophyte

Potamogeton crispus by bioaccumulation of Hg. The reports on Hg induced modulation

of antioxidant defence system in plants are tabulated in Table 2.5.

2.11.4 Nickel and Antioxidant Defence System in Plants

Nickel (Ni), first isolated by the Swedish chemist Cronstedt in 1751, is 22nd

most abundant element in the earth's crust. Ni is essential micronutrient and functions as

an active centre of the enzyme urease required for the hydrolysis of urea and nitrogen

metabolism in higher plants (Brown et al., 1987; Gerendás et al., 1999). Though Ni is

essential but its concentration in the majority of plant species is very low (0.05 – 10

mg/kg dry weight) (Nieminen et al., 2007). A higher levels, Ni produces toxic

symptoms like stunting growth, leaf chlorosis, mitotic inhibition, vein necrosis etc. in

plants (Seregin and Kozhevnikova, 2006; Llamas and Sanz, 2008). In general, naturally

occurring concentrations of Ni in soil and surface waters are lower than 100 and 0.005

ppm, respectively (McIlveen and Negustani, 1994; McGrath, 1995; Chen et al., 2009).

Ni is also released into the environment from anthropogenic activities, such as metal

mining, smelting, fossil fuel burning, vehicle emissions, disposal of household,

municipal and industrial wastes, fertilizer application and organic manures (Chen et al.,

2009).

In recent years, Ni pollution has been reported from across the world, including

Asia, Europe, and North America (Chen et al., 2009). From contaminated soils, Ni is

easily absorbed by the plants and its excessive uptake leads to altered plant growth, fruit

quality and quantity (Chen et al., 2009). Also, Ni metal leads to the alteration of

biochemical parameters including accumulation of reactive oxygen species (ROS) and

enhancement of lipid peroxidation in plant tissues (Gajewska and Skłodowska, 2008).

Since Ni is not a redox-active metal, it cannot directly generate ROS, however, it

interferes indirectly with a number of antioxidant enzymes (Pandolfini et al., 1992;

Baccouch et al., 2001; Pandey and Sharma, 2002; Hao et al., 2006; Chen et al., 2009).


54

Effect of Ni toxicity on antioxidant defence system in various plant species have been

assembled in Table 2.5.

2.12 EXPRESSION OF ANTIOXIDANT ENZYMES DURING OXIDATIVE

BURST

Recent investigations of knockout and antisense lines for CAT2, APOX1 and

various NADPH oxidases have revealed a strong link between ROS production and

responses during physiological, developmental and morphological processes, and biotic

or abiotic stress responses (Mittler et al., 2004). Thus, ROS-induced redox signals

provide important information on plant metabolism during development and in

dependence on environmental parameters and trigger compensatory responses and

antioxidant defence (Kandlbinder et al., 2004). Redox information is derived from

primary redox intermediates of metabolism such as plastoquinone and cytochrome b6f-

complex in photosynthesis (type I redox signals), from integrating redox buffers such as

ferredoxin, thioredoxins, glutathione and ascorbic acid (type II redox signals) and from

ROS liberated at elevated rates only under stress (type III redox signal) (Pfannschmidt

et al., 2001, Dietz, 2003; Kandlbinder et al., 2004). Simultaneous expression of Cu/Zn

SOD and APOX genes in tobacco chloroplasts enhanced tolerance to methyl viologen

stress compared to expression of either of these genes alone (Kwon et al., 2002).

Three isoforms of SOD i.e., Cu/Zn SOD, Fe SOD and Mn SOD on the basis of

metal co-factors have been distinguished in plants (Joseph and Jini, 2011). Cu/Zn SOD

is most abundant and has little resemblance with Fe SOD and Mn SOD. Both Fe SOD

and Mn SOD are closely related by their structural and evolutionary point of view.

Cu/Zn-SOD is found primarily in eukaryotes, localized mainly in the cytosol and

nucleus, as a dimer of identical 16,000 MW subunits. Fe-SOD, a dimer of identical

20,000 MW subunits, is present mainly in prokaryotes. Mn-SOD is found in both

prokaryotes and eukaryotes; most abundantly in the mitochondria, and has been isolated

as both dimers and tetramers of 21,000 MW identical subunits. In plants, CAT have

three isozymes which can be either Cat1, Cat2, Cat3 in Arabidopsis, Nicotiana,


55

Raphanus etc. or Cat-A, Cat-B, Cat-C in maize. The expression levels of Cu/ZnSod,

FeSod, MnSod, Mdhar, tApox (thylakoid-bound-APOX), Dhar, Gr and Cat were

observed in wheat under cold stress. The expression levels were up-regulated (MnSod,

Mdhar, tApox, Dhar and Gr), down-regulated (Cat), or relatively constant (FeSod and

Cu/ZnSod). After 4 weeks’ cold acclimation the expression level of tApox, Cat and

MnSod were higher than others (Baek and Skinner, 2003). Further in 2005, Shi et al.

reported that Mn-Sod activity was enhanced with the increasing Mn concentrations in

plants, whereas Fernando and Miguel (2000) observed that SOD activity was not

affected by Mn level in rice.

Gene Expression and activities of SOD in cucumber seedlings were found to be

related with concentrations of Mn2+, Cu2+, or Zn2+ under low temperature stress by Gao

et al. (2009). Both gene expressions and activities of Cu/ZnSod and MnSod in cucumber

seedling leaves were induced by increasing Mn2+, Cu2+, or Zn2+ under low temperature

stress, especially 48 h afterwards. The activities of Cu/Zn-SOD and MnSod at 0 and 48

h after treatment were in accordance with their gene expression levels, which implied

that the transcriptional regulation plays key roles in regulating their activities at the

early stage of low temperature stress. Gene expressions of Cu/ZnSod and MnSod

declined at 96 h, but Cu/ZnSod and MnSod activities still remain high, which suggested

that Cu/ZnSod and MnSod activities might be regulated by other factors after

transcription at the later stage of low temperature stress (Gao et al., 2009). Furthermore,

in A. thaliana, organellar peroxiredoxins and tAPOX were activated early in seedling

development in light, while sAPOX (stromal APOX), Cu/Zn-SOD, MDHAR cytosolic

peroxiredoxins were fully activated between 2.5 and 3 days after radical emergence

(Pena-Ahumada et al., 2006). In Thellungiella halophila, expression of microRNA from

MIR398 family, which regulated expression of superoxide dismutase (Cu/ZnSod) in

Arabidopsis thaliana at post-transcriptional level, was observed by Pashkovskii et al.

(2010). The analysis of Cu/ZnSod1 gene expression in T. halophila by RT-PCR showed

that, in the presence of 100 mM NaCl, the level of Cu/ZnSod1 mRNA in roots and

leaves increased. Change in level of MIR398 expression in roots and leaves in under


56

various salinity levels, illumination intensity, and UV-B radiation on MIR398

expression was observed. During salinity stress and UV-B irradiation, a negative

correlation was observed between expression of MIR398 and its target, mRNA of

Cu/Zn-SOD. The changes in expression of important antioxidant enzymes in response

to different stresses are highlighted in Table 2.6.

2.13 MECHANISM OF BRs INDUCED OXIDATIVE STRESS TOLERANCE

BRs have been speculated to act via receptor/ligand complex that binds to

nuclear or cytoplasmic sites to regulate the expression of specific stress related genes.

Dhaubhadel and Krishna (2008) recently identified the six differentially expressed

genes in epibrassinolide treated heat stressed B. napus seedlings. Six differentially

expressed cDNAs were isolated and characterized, and were found to encode a

mitochondrial transcription termination factor (mTERF)-related protein, glycine-rich

protein 22 (GRP22), myrosinase, 3-ketoacyl-CoA thiolase and a copia like polyprotein.

Transcripts of mTERF-related protein, GRP22 and myrosinase were present at higher

levels in EBL treated seedlings under non stress conditions. Whereas heat stressed,

epibrassinolide treated seedlings showed higher level of transcript of 3-ketoacyl-CoA

thiolase. So, BRs lead to the change in expression of genes involved in various

physiological responses under stress conditions. BRs are perceived by extracellular

domain of plasma membrane localized BRI1 (Brassinosteroid Insensitive leucine rich

repeat receptor kinase). A second protein or co-receptor, BAK1 (BRI1-Associated

Receptor Kinase) heterodimerize with BRI1 which in turn induce the

autophosphorylation of BAK1 and BRI1 and activate the intracellular signal

transduction cascade (Li, 2005). The activation of BRI1 and BAK1 receptor kinases

leads to the dephosphorylation and nuclear localization of BR-response proteins, BZR1

and BES1, possibly by the inhibition of BIN2, which otherwise phosphorylates and

destabilizes BZR1 and BES1. The stabilized BZR1 and BES1 translocate to the nucleus

and activate the target gene expression. The phosphorylated receptor ligand complex

may also activate the NHX (vacuolar membrane bounded Na+/H+ antiporter) and V-


57

ATPase pump which in turn move the excess of heavy metals and Na+ to vacuole.

Stress responsive genes activated by BRs may code for the PCs, organic acids,

osmolytes and stress protective proteins (LEA proteins and HSPs) (Gendron and Wang,

2007; McSteen and Zhao, 2008).

Though diverse genomic and proteomic approaches have unsolved various

components in the process of phosphorylation during BRs signaling pathway yet many

puzzles remained uncertain (Tang et al., 2010). Thus, stress-mitigation via applications

of BRs is mediated through a tremendously complex sequence of biochemical shifts,

such as activation or suppression of key enzymatic reactions, induction of protein

synthesis and the production of various chemical defence compounds. Since BRs are

natural, nontoxic and eco-friendly and when applied in extremely low doses are capable

of improving the crop yield even under stressed conditions (Khripach et al., 2000),

therefore, BRs may be implicated in plant stress-protection. Further, studies on effects

of BRs and its interactions with other plant growth regulators might provide more

efficient phytohormone-based practices to reduce the risk of plants being exposed to

habitats contaminated with heavy metals.

2.14 INTERACTIONS OF BRs WITH OTHER PHYTOHORMONES

BRs have been reported to affect the expression of several genes involved in

plant defence as well as biosynthesis of other hormones (Bari and Jones, 2009). For

example, BRs induced ACC synthase and OPR3, the genes involved in biosynthesis of

ethylene (ET) and jasmonates (JA) respectively in Arabidopsis (Yi et al., 1999; Muessig

et al., 2006). But, the need of JA or ET for BRs regulated stress resistance is not known.

Molecular and genetic approaches have resulted in the identification of many of the

proteins involved in hormone signalling and the analysis of these proteins has

contributed significantly to our current models of hormone action (Santner and Estelle,

2009). Recent advances in the identification of various receptors for auxin, cytokinins

(CKs), JAs, BRs, ABA, ET and gibberellins (GAs) have enhanced our understanding of

hormone perception and signalling (Dharmasiri et al., 2005; Kepinski and Leyser 2005;


58

Ueguchi-Tanaka et al., 2005; Chini et al., 2007; Thines et al., 2007; Pandey et al.,

2009; Santner and Estelle, 2009). It is elucidated that CKs, ET and BRs use well-

characterized signalling mechanisms, whereas, the identification and characterization of

the auxins, GAs and JA receptors, have highlighted a novel mechanism for hormone

perception in which the ubiquitin–proteasome pathway play a central role (Chow and

McCourt, 2006; Santner and Estelle, 2009).

Acharya and Assmann (2009) reported that ABA, JAs, BRs and SA are positive

regulators of stomatal closure, while auxins and CKs are generally positive regulators of

stomatal opening. In contrast, ET plays a dual regulatory role on stomatal apertures in a

condition- and species-specific manner. Interaction of auxin, CKs, or ET with ABA

inhibits ABA-mediated stomatal closure, whereas, the interaction of ABA and SA

positively regulates stomatal closure, and hinders the invasion of bacterial pathogens

(Acharya and Assmann, 2009). While genetic analyses of components involved in

stomatal regulation by ABA and JAs have identified commonalities in the signaling

pathways of these two hormones. In addition to ABA, the hormones JA, auxin, CKs,

ET, BRs and GAs all alter expression of drought-related genes (Huang et al., 2008;

Nemhauser et al., 2006), suggesting cross-talk by different signaling pathways during

drought stress, but the effects of such cross-talk on the guard cell transcriptome are yet

to be explored.

Genome-sequencing projects such as those of Physcomitrella patens (moss),

Selaginella (fern), A. thaliana and O. sativa (rice) etc. have improved our knowledge of

hormone signaling pathways (Santner and Estelle, 2009). Through biochemical, genetic,

and genomic approaches in several organisms, such as Arabidopsis, Populus, and

Zinnia various signals controlling vascular development have started to emerge

(Dettmer, 2009). BRs are reported to promote xylem formation by enhancing HD-

ZIPIII genes expression in Zinnia elegans (Ohashi-Ito et al., 2005). Furthermore,

overexpression of one Zinnia HD-ZIPIII gene resulted in upregulation of BR- receptor:

BRI1-like 3 (BRL3) and a BAK-like leucine-rich repeat receptor-like kinase (LRR-

RLK), indicating that HD-ZIPIII genes promote BR-signaling (Dettmer, 2009).


59

Interestingly, along with BRs the miRNA165/166 and auxin also have been implicated

in positively regulation of the expression of HD-ZIPIII and KANADI genes, which

determine the abaxial and the adaxial position of phloem or xylem, respectively

(Dettmer et al., 2009). The availability of P. patens (an ancient plant ancestor) genome

sequence has helped in revealing the facts that there are common proteins in auxin,

ABA and CKs signaling in the moss, but there were no such proteins in the genome of

green algae, further suggesting that these signaling pathways emerged when plants were

inhabiting the land (Vandenbussche et al., 2007; Rensing et al., 2008; Santner and

Estelle, 2009).

Tryptophan

AUX / IAAs

ARFs

Sterols

BRI1

BIN2

BES1 BZR1

Common Targeted

Genes

BRs

Auxin ABA

GA

JA

Zeaxanthin

ETSAMACC-S

ACCACC-O

Geranylgeraniol

diphosphate

Elongation

Seed germination

Linoleic

acid

Defence

against Insect

attack

BRs

Plant Growth and Development

CK

Isopentenyladenosine-

5-triphosphate

ATP

SA

L-Phenylalanine

Fig. 2.4. Interactions of Phytohormones for normal plant growth and development


60

In contrast, a comparison of the moss genome with more recently diverged plant

genomes suggests that signaling mechanisms for GAs, ET and the BRs probably did not

evolve until after the evolutionary split of moss and vascular plants (Santner and

Estelle, 2009). Further, genome sequencing of more organisms/plants will further give

advance the knowledge of additional signaling molecules in phytohormone interactions.

So far several key components of the plant hormone signaling have been identified

during plant defense providing the strong evidences for the involvement of abscisic

acid, auxin, gibberellic acids, cytokinins, salicylic acid and BRs in plant defence

signaling pathways (Fig. 2.4). These phytohormone signaling pathways are not isolated

but rather well organized into a complex regulatory network involving various defence

signaling pathways and developmental processes. Though, the major hormonal

signaling pathways have been established, but the comprehension of the interactions

between signaling components of various plant growth regulators is still relatively

sparse.

FIELD

PREPARATION

Soil

Mixing(clay: sand:

manure ::

2:1:1)

Beds preparation

CRESTS & TROUGHSSOIL TREATMENTS

LABELS

SEED

SOWING

Fig. 3.7. Plan and preparation of the fields

10 days old Radish

plants

hh

Fig. 3.8. A prepared field and 10-days old radish plants raised in field

Fig. 3.9. Thirty-days old radish plants raised in field

Fig. 3.10. Sixty-days old radish plants raised in field

Fig. 3.11. Ninety-days old radish plants raised in field

Fig. 3.12. Growth in 60-days old radish plants pre-treated with HBL and EBL in

comparison to untreated plant (DDW)

Raphanus sativus L.

(Pusa Chetaki) seeds

Rinsed

with

Distilled

water

Surface

sterilization

Seed

soaking

treatments

for 8

hours

EBL or HBL

Seed

sowing

Heavy

metals

solution

in petri

-plates

25oC,

16 h

light/8h

dark period

Inside seed germinator

Radish seedlings

Radish

seedlings

harvested

on 8th day

Analysis

PARAMETERS STUDIED

MORPHOLOGICAL MOLECULARBIOCHEMICAL

Fig. 3.4. Raising of R. sativus cv. Pusa chetki in laboratory conditions

Certified Seeds of Raphanus sativus L. (Pusa Chetki)

Surface sterilization with Sodium hypochlorite

8-hour Seed pre-soaking treatments with different concentrations

(0, 10-11, 10-9 and 10-7 M) of either HBL or EBL

Seed sowing on metal (Cd, Cr, Hg, Ni) treated soil or filter-papers

treatm

d 10 7

r, Hg, NHg

Whatman#1 filter-paper lined Petri-dishes Soil beds / troughs in fieldg

Controlled conditions in laboratory Natural conditions

Observations after 7-days Observations after 30, 60 and 90 days

I. Morphological Parameters

IV. Morphological Parameters

II. Biochemical Parameters(In shoots only)

V. Biochemical Parameters

(Both roots and shoots)

III. Molecular Biology Studies

a. Root lengthb. Shoot lengthc. Fresh Biomassd. Dry Biomass

a. Number of leavesb. Root lengthc.Shoot length

a. Total Soluble Protein Contentb. Free Prolines Contentc. Malondialdehye (MDA) Contentd. Chlorophyll Content (Total, A and B)e. Reducing sugars Contentf. Osmomolalityg.Superoxide anion production rateg. Activities of Antioxidant Enzymes:

1. Ascorbate peroxidase (APOX)2. Catalase (CAT)3. Dehydroascorbate reductase (DHAR)4. Glutathione reductase (GR)5. Guaiacol peroxidase (POD)6. Monodehydroascorbate reductase (MDHAR)7. Superoxide dismutase (SOD)

a. Total Soluble Protein Contentb. Free Prolines Contentc. Malondialdehye (MDA) Contentd. Activities of Antioxidant Enzymes:

1. Ascorbate peroxidase (APOX)2. Catalase (CAT)3. Dehydroascorbate reductase (DHAR)4. Glutathione reductase (GR)5. Guaiacol peroxidase (POD)6.Monodehydroascorbate reductase (MDHAR)7. Superoxide dismutase (SOD)

Semi-quantitative RT-PCR analysis of Fe-SOD, Mn-SOD, Cu/Zn-SOD, CAT1, CAT2 and CAT3 taking26S-rRNA as constitutive gene

Flow-Chart 3.1. Plan and Parameters of Experiment

Table 2.1. Effect of Animal Steroid Hormone on various biochemical, morphological and physiological parameters of plants

S.No. Animal Steriod Hormone Study Material Observations Reference

1. Esterone,

17 β-estradiol

Dwarf Pea

Ø Estrogens increased the contents of gibberllins

Kopcewicz (1969a)

2. Esterone,

17 β-estradiol

Cichorium intybus L.

(Chicory) plants

Ø Influenced flowering in Chicory plants Kopcewicz (1969b)

3. Estrone, estradiol and

testosterone

Pinus pinea seeds Ø Higher growth rate

Ø Higher germination degree

Ø Raised levels of nucleic acids with most increase in

fractions of RNA

Martínez-Honduvilla et

al. (1976)

4. Esterone,

17 β-estradiol,

Androstenedione,

Testosterone,

17-α-hydroxy progesterone

Pregnenolone acetate

Germinating Wheat

seedlings and plants

Ø Activities of antioxidant enzymes such as catalase

(CAT) and peroxidase (POX) increased

Ø Changed protein content

Ø Increased the plant growth and rate of germination

Dogra and Kaur (1994);

Dogra and Thukral (1994)

5. Esterone,

17 β-estradiol,

Androstenedione,

Testosterone,


Wheat plants Ø Pre-sowing treatment of steroid hormones enhanced

total soluble proteins and activities of antioxidant

enzymes (CAT and POX)

Ø Increased contents of DNA and RNA

Dogra and Thukral (1994;

1996)

6. Esterone,

17 β-estradiol,

Androstenedione,

Testosterone,


Pregnenolone acetate

Maize Plants Ø Pre-sowing treatments significantly accelerated the

contents of inorganic components such as N, P, Fe, Na,

and K

Dogra and Thukral (1998)

Continued…..

S.No. Animal Steriod Hormone Study Material Observations Reference

7. Progesterone Arabidopsis thaliana

seedlings

Ø Vegetative growth was promoted at low concentrations

but suppressed at higher concentrations under both light

and dark growth conditions

Ø Growth of the gibberellin-deficient mutant lh of pea

(Pisum sativum) was also promoted

Ø Progesterone binds to MEMBRANE STEROID

BINDING PROTEIN 1 (MSBP1) of Arabidopsis. All

the cloned homologous genes of Arabidopsis, MSBP2

and STEROID BINDING PROTEIN (SBP), as well as

of rice (Oryza sativa) OsMSBP2 and OsSBP, except

OsMSBP1, were expressed abundantly in plant tissues.

Lino et al. (2007)

8. Progesterone, β-estradiol,

Androsterone

Germinating seeds,

seedlings and leaves

of Chickpea

Ø Increase in inorganic constituent (K, S, Na, Ca, Mg, Zn,

Fe, P, Cu, and Ni) of after steroid treatment have been

reported by using wavelength dispersive X-ray

fluorescence spectroscopy

Ø Decrease in the contents of Mn and Cl

Erdal et al. (2010a)

9. Progesterone, β-estradiol Germinating Maize

seedlings

Ø Stimulated germination velocity and growth parameters

(percentage of germination, root and shoot or coleoptyle

length)

Ø Activities of α-amylase, superoxide dismutase (SOD),

POX, CAT and polyphenoloxidase (PPO)

Erdal et al. (2010b)


Androsterone

Chickpea plants Ø Increased plant growth and the contents of soluble

protein and sugars, and activities of SOD, POX and CAT

Ø Decreased H2O2 content and lipid peroxidation levels

Erdal and Dumlupinar

(2011)


Androsterone

Barley leaves Ø Augmented the concentrations of Ca, Mg, P, S, Cu, Mn,

Al, Zn, Fe, K and Cl

Ø Decline in Na determined through wavelength dispersive

X-ray fluorescence spectroscopy technique

Dumlupinar et al. (2011)

Table 2.3. Bioassays to determine biological activities of BRs

S.No. Concentration

of BRs

Bioassay Plant System Response Generated Reference

1. 10 -10 M and

10 -12 M

Bean First

Internode

First internode section

of seedlings

Curvature of first bean internode

sections after application of BRs near to their base

Thompson et al. (1982); Strnad

and Kamínek (1985)

2. 10 -11 M and

10 -14 M

Bean Second

Internode

Second internode section of seedlings

Curvature of second bean internode sections

Thompson et al. (1981); Kohout et al. (1991)

3. 10 -12 M Mung Bean

Epicotyl

Hypocotyls, Epicotyls,

Leaves and Terminal

Buds

Increase in the length of cuttings of

etiolated mung bean due to BRs

response

Gregory and Mandava (1982)

4. 10 -12 M Radish Hypocotyl Seedlings Length of hypocotyls is increased Takatsuto et al. (1983)

5. 10 -14 M,

10 -13 M and

10 -10 M

Rice Lamina

Inclination Test

Lamina, lamina joint

and leaf sheath from

etiolated rice seedlings

Lamina inclination of rice seedlings

Cells aligned in the adaxial side of the lamina joint between leaf sheath and

lamina are swollen causing bending by

BRs

Fujioka et al. (1998); Kim et

al. (1990); Wada et al. (1981,

1984); Takeno and Pharis (1982)

6. 10 -11 M Tomato Hypocotyl Seedlings Length of hypocotyls is increased Takatsuto et al. (1983)

7. 10 -12 M Wheat Leaf

Unrolling

Leaf Segments BR treatment unrolls the first leaf

segments of dark-grown wheat

Wada et al. (1985)

Table 2.4. Bioactivities of Brassinosteroids in different test systems

S.No. Pathogen/ Disease Host Plant / Test

System /Cell Lines

Observations/Inferences Reference

Antibacterial Activities of Brassinosteroids

1. Pseudomonas syringae

(Bacterial pathogen)

Tobacco Ø BR-treatment significantly reduced bacterial infection

Ø BR-treatment enhanced growth of tobacco

Rodkin et al. (1997)

2. Xanthomonas oryzae Rice Ø Treatments of BRs significantly lowered bacterial

blight in rice plants

Nakashita et al. (2003)

Antifungal Activities of Brassinosteroids

1. Phytophthora infestans

Potato tubers Ø Incidence of late blight was reduced with the

application of BRs

Ø Resistance to Phytophthora infection was increased by

BRs treatment

Vasyukova et al. (1993,

1994)

2. Helminthosporium teres

Sacc.

Barley plants Ø EBL foliar spray to barley plants at tillering phase

decreased an extent of leaf disease induced by H. teres

Ø EBL treatments increased grain yield even at a dose of

5 mg ha-1

Pshenichnaya et al. (1997);

Volynets et al. (1997a,

1997b)

3. Odium species

Tobacco Ø BR - treatment significantly lowered fungal infection

Ø BR-treatment enhanced yield of tobacco plants


4. Peronosporous epiphytotia Cucumber Ø EBL increased activities of peroxidase and PPO

enzymes that are involved in the metabolism of

polyphenols was suggested as a factor contributing to

BR induced disease resistance in cucumber plants

Churikova and Vladimirova

(1997)

5. Verticillium dahliae Tomato Ø Exogenous applications of EBL lowered the

development of disease symptoms as compared to

untreated plants

Krishna (2003)

6. Magnaporthe grisea Rice Ø Rice blast was significantly reduced by application of

BRs

Nakashita et al. (2003)

Antiviral Activities of Brassinosteroids

1. Tobacco Mosaic Virus

(TMV)

Tobacco Ø BRs treatment lowered TMV induced infection

Ø BR-treatment increased crop yield by 56%


2. Measles Virus, Herpes

simplex virus Type 1

(HSV-1), and Arena virus

Cell cultures Ø 27 derivatives/analogues of BRs revealed antiviral

properties

Ø Selectivity index (SI) values of BRs were higher than

reference drug ribavirin

Ø Inhibited HSV-1 and arena virus replication in cell

cultures

Wachsman et al. (2000,

2002)

3. Junin virus (JV)

(Arenaviridae);

Measles virus (MV)

(Paramixoviridae), HSV-1

and HSV-2

(Herpesviridae)

Cell cultures Ø Synthetic methods to obtain BRs analogues and their

in vitro antiviral activity against RNA and DNA

viruses were used

Ø Animal viruses susceptibile to BRs analogues

comprised two RNA monocistronic viral families,

Paramyxoviridae and Arenaviridae, and one DNA

virus family Herpesviridae

Wachsman et al. (2004a)

4. HSV-1 and HSV-2

(Herpesviridae)

Human conjunctive

cell lines (IOBA-

NHC) and Murine

herpetic stromal

keratitis (HSK)

experimental model

Ø Three new synthetic BRs analogues viz. (22S,23S)-

3 -bromo-5 ,22,23-trihydroxystigma-stan-6-one,

(22S, 23S)- 5 -fluoro-3 -22,23-

trihydroxystigmastan-6-one, (22S, 23S)-3 -5 ,22,23-

trihydroxystigmastan-6-one revealed antiherpetic

activity in vitro and in vivo

Ø Synthetic BRs prevented the multiplication of HSV-1

in NHC cells in a dose dependent manner when

added after infection without cytotoxicity

Michelini et al. (2004)

5. Human-immuno Cultured Cell lines Ø In vitro studies confirmed the ability of EBL to arrest Khripach et al. (2005)

deficiency virus (HIV) or reduce the growth of the HIV in cultured infected

cells

Ø BRs can be used in the cure or prevention of HIV

infection and related conditions (AIDS related

complex)

6. Vesicular stomatitis virus

(VSV)

Vero cells Ø Synthetic BR: (22S, 23S)-3 -bromo-5 ,22,23-

trihydroxystigmastan-6-one had antiviral effects

against replication of VSV that causes an

economically important disease in cattle, horses and

swine

Romanutti et al. (2007)

7. Beet curly top virus

(BCTV)

Arabidopsis Ø Beet curly top virus (BCTV) C4 functionally interacts

with BRs insensitive 2 (BIN2), a glycogen synthase

kinase 3-like (GSK3-like) protein kinase involved in

brassinosteroid signaling in Arabidopsis

Piroux et al. (2007)

8. HSV-1 Cell lines Ø HSV-1 causes an ocular chronic

immunoinflammatory syndrome named herpetic

stromal keratitis that might lead to vision impairment

and blindness in mice

Ø BRs had antiherpetic activity against HSV-1

Michelini et al. (2008)

Antiproliferative/Anticancerous Activities of Brassinosteroids

1. Cancer (Mouse

hybridoma)

Mouse Hybridoma

cultured on standard

serum free medium

or 30% diluted

medium

Ø EBL treatments (10–16 to 10–9 mol l–1) resulted in

increase in the value of mitochondrial membrane

potential, drop of intracellular antibody level,

increase in the fraction of the cells in the G0/G1 phase,

and decrease in the fraction of the cells in the S phase

Ø EBL inhibited the viability of cell lines, and affected

proliferation, differentiation, apoptosis and

expression of some cell cycle related proteins

Franěk et al. (2003)

2. Breast adeno carcinoma,

Acute lymphoblastic

leukemia and myeloma

cancer

Human Breast adeno

carcinoma cell lines

(MCF-7–estrogen-

sensitive, MDA-MB-

468–estrogen-

insensitive), human

acute lymphoblastic

leukemia cell line

(CEM) and human

myeloma cell line

(RPMI 8226)

Ø Cytotoxic activity of BRs was tested in vitro by

Calcein AM assay whereas TUNEL, DNA ladder

assay and immunoblotting techniques were used for

the analysis of changes of cell viability, proliferation,

differentiation and apoptosis

Ø Significant reduction in the expression of p21, p27,

p53, cyclins, proteins of Bcl-2 family and ER-alpha

was observed by BRs application

Swaczynova et al. (2006)

3. Prostate cancer Human prostate

cancer PC-3 cells

Ø BL induced apoptosis in human prostate cancer PC-3

cells

Wu and Lou (2007)

4. Breast and Prostate Cancer

Hormone-sensitive/

insensitive breast

(MCF-7/MDA-MB -

468) and prostate

cancer cell lines

(LNCaP/DU-145)

Ø 28-homoCS and EBL inhibited cell growth in a dose

dependent manner in the cancer cell lines

Ø Flow cytometry analysis showed that BR treatment

arrested, MDA-MB-468, LNCaP and MCF-7 cells in

G1 phase of the cell cycle and induced apoptosis in

MDA-MB-468, LNCaP, and slightly in the DU-145

cells

Ø BRs can inhibit the growth, at micromolar

concentrations, of several human cancer cell lines

without affecting the growth of normal cells

Malìková et al. (2008)

Antigenotoxic Activities of Brassinosteroids

1. Aberrations in

Chromosomes or

genotoxicity

Allium cepa (Onion)

root tips

Ø Allium cepa chromosomal aberration bioassay

revealed the antigenotoxicity of EBL treatments

Ø Highest dose of EBL (0.5 ppm) was reported to be

Howell et al. (2007)

effective in increasing mean root lengths and

lessening the number of mitoses as compared to

control

Ø Low doses of EBL (0.005 ppm) and intermediate

doses of EBL (0.05 ppm) nearly doubled the mean

root length and the number of mitosis over that of

controls

2. Genotoxicity induced by

maleic hydrazide (MH)

Allium cepa (Onion)

root tips

Ø EBL treatment significantly reduced the % of

chromosomal aberrations induced by MH (0.01%)

Ø EBL (10-7 M) proved to be the most effective

concentration with 91.8% inhibition

Sondhi et al. (2008)

Neuroprotective Activity of Brassinosteroids

1. Parkinson’s disease Neural PC12 cells Ø EBL protected neuronal PC12 cells from 1-methyl-4-

phenylpyridinium- (MPP+-) induced oxidative stress

and consequent apoptosis in dopaminergic neurons

Ø EBL reduced the levels of intracellular ROS and

modulated SOD, CAT and glutathione peroxidase

activities

Ø Antioxidative properties of EBL inhibited MPP+-

induced apoptosis by reducing DNA fragmentation as

well as the Bax/Bcl-2 protein ratio and cleaved

caspase-3

Ø Potent antioxidant and neuroprotective role of EBL in

a mammalian neuronal cell lines was observed

(Carange et al., 2011)

Table 2.5. Modulation of Antioxidant defence system of plants under Heavy Metals Stress

S.No. Heavy Metal

Concentration

Plant Species Exposure

Time

Modulation of Antioxidant Defence System References

CADMIUM (Cd) METAL TOXICITY

1. 500 µM Cd Helianthus annuus 12 h Ø Ascorbate peroxidase (APOX), Catalase (CAT),

Glutathione reductase (GR), Superoxide

dismutase (SOD), Glutathione peroxidase

(GPOX), Glutathione reducatse (GR)

Gallego et al. (1996)

2. 50, 100 and 200

µM Cd

Glycine max 48 h Ø APOX, CAT and SOD Balestrasse et al. (2001)

3. 4 and 40 µM Cd Pisum sativum 7 d Ø APOX, CAT, GPOX and SOD;

Ø Raised levels of malondialdehyde (MDA)

content

Dixit et al. (2001)

4. 50 M Cd Pinus sylvestris 6 h Ø APOX, CAT, GR and SOD Schützendübel et al.

(2001)

5. 100 and 200 µM

Cd

Oryza sativa 20 d Ø CAT, GPOX and SOD Shah et al. (2001)

6. 2000 and 5000

µM Cd

Saccharum officinarum 0-96 h Ø CAT, GR and SOD Fornazier et al. (2002)

7. 50 µM Cd Phragmites australis 21 d Ø APOX, CAT, GR and SOD Ianelli et al. (2002)

8. 5 and 50 µM Cd Populus canescens 48 h Ø APOX, CAT, GR, MDHAR and SOD Schützendübel and Polle

(2002)

9. 1 and 10 µM Cd Triticum durum 10 d Ø APOX, CAT, GPOX and SOD Milone et al. (2003)

10. 300 and 500 µM

Cd

Arabidopsis thaliana 21 d Ø APOX, CAT, GPOX, GR and SOD Cho and Seo (2004)

11. 10, 100 and

1000 µM Cd

O. sativa: Roots 24 h

Ø Increased Contents of MDA and H2O2

Ø Accelerated superoxide (O2.-) radical formation

Choudhury and Panda

(2004)

12. 0.01, 0.1, 0.5

and 1 mM Cd

Ceratophyllum,

Hydrilla, Wolffia;

Brassica juncea, Vigna

radiata, T. aestivum

16 weeks Ø Accumulation of prolines in only mesophytes;

Ø About 2-fold increase in MDA production in

mesophytes exposed to 1mM Cd

Dhir et al. (2004)

13. 5000 µM Cd O. sativa 0-24 h Ø APOX, CAT, GPOX, GR and SOD Hsu and Kao (2004)

14. 5 µM Cd P. sativum 10 d Ø APOX, CAT, GPOX and accumulation of MDA Metwally et al. (2004)

15. 1, 10, 100 and

200 µM Cd

Allium sativum: Roots 0-48 h Ø Accumulation of MDA under Cd stress but not

in dose-dependent manner

Ünyayar et al. (2006)

16. 1, 10, 100 and

200 µM Cd

Vicia faba: Roots 0-48 h Ø MDA contents in about 2.5 times after Cd

exposure

Ünyayar et al. (2006)

17. 25 µM Cd Solanum lycopersicon:

Leaves

10 d Ø APOX, CAT, GR, guaiacol peroxidase (POD),

SOD

Ø Enhanced levels of MDA

Chamseddine et al.

(2008)

18. 1 and 5 µM Cd O. sativa (japonica rice

cv. Xiushui 11 and its

BADH-transgenic line

Bxiushui 11)

10 d Ø SOD, POD

Ø Higher proline and MDA contents in both roots

and leaves of wild type than transgenic rice

Shao et al. (2008)

19. 68 µmol Cd kg-

1 soil

P. sativum plants 15, 30 and

45 d

Ø CAT, SOD, POD, contents of ascorbic acid,

flavonoids, thiols, MDA and proline

Accumulation of MDA and prolines in Cd stress

Agrawal and Mishra

(2009)

20. 50 - 200 µM Cd Solanum nigrum L. 3 d Ø Cd (50 and 200 μM) significantly increased the

contents of thiobarbituric acid-reactive

substances (TBARS), the production of H2O2

and O2− anion

Ø APOX, CAT, glutathione peroxidase (GPOX),

POD and SOD

Deng et al. (2010)

21. 10 and 100 µM

Cd

Lycopersicon

esculentum Mill (cv.

Tres Cantos)

10 d Ø Low Cd treatment (10 μM) resulted in 2-fold

changes in the relative amounts of 36

polypeptides, whereas 100 μM Cd demonstrated

Rodríguez-Celma et al.

(2010)

change in the relative amounts of 41

polypeptides

Ø 2-DE based proteomic approach allowed

assessing the main metabolic pathways affected

by Cd toxicity.

Ø 10 μM Cd treatments suggested activation of

glycolytic pathway, TCA cycle and respiration,

whereas the 100 μM Cd resulted in shutdown of

carbon metabolism and increased accumulation

of stress related and detoxification proteins.

22. 0, 10, 25 and 50

µM Cd

Tagetes patula 0-14 d (in

35 d old

plants)

Ø Reduction of chlorophyll content and decreased

cell viability by 25 and 50 µM Cd

Ø Increase level of lipid peroxidation

Ø APOX, CAT, GR, and SOD

Ø In-gel zymography analysis revealed that Cd

induced the enzymatic activities of APOX,

MnSOD, CuZnSOD and different isozymes of

GR in leaves.

Liu et al. (2011)

23. 1 mM Cd Phaseolus vulgaris L.

(cv. Bronco)

30 d Ø CAT, GR, POD and SOD

Ø MDA content raised under Cd stress

Rady (2011)

CHROMIUM (Cr) METAL TOXICITY

1. 25 and 150 µM

of Cr (III)

H. annuus calli 2 months Ø APOX, CAT, POD, GR and SOD;

Ø Decreased the levels of reduced glutathione

(GSH), oxidized glutathione (GSSG) and

ascorbate;

Ø Dehydroascorbate (DHA) levels increased

Gallego et al. (2002)

2. 0.1, 1.0, 10 and

100 mM Cr T. aestivum (cv. 8 d Ø CAT, POD and SOD;

Ø Carotenoid, ascorbate, proline, total peroxide

Panda et al. (2003)

Sonalika) seedlings and MDA contents increased whereas

glutathione content declined

3. 10, 20, 50 and

100 μM Cr Ocimum tenuiflorum 45 d Ø APOX, CAT, POD, SOD and nitrate reductase

Ø Cr induced lipid peroxidation coupled with K+

leakage

Ø Reduced amounts of photosynthetic pigments,

protein, cysteine, ascorbic acid and non-protein

thiol (NPSH)

Ø Increased proline content.

Rai et al. (2004)

4. 50 μM Cr3+ and

Cr6+ V. radiata (cv. CO4) 5 d Ø APOX, CAT, MDHAR and SOD

Ø Significant increase in lipid peroxidation and

H2O2 generation was seen 5 h after stress in

Cr6+as against 12 h in Cr3+-treated plants

Ø Higher levels of ascorbic acid and oxidized

glutathione (GSSG)

Ø Lowered levels of reduced glutathione (GSH)

Shanker et al. (2004)

5. 10,40,80 and

160 µM Cr

Pistia stratiotes: Roots 48,96 and

144 h

Ø APOX, POD and SOD;

Ø Contents of MDA and antioxidants (ascorbic

acid and cysteine) enhanced;

Ø Chlorophyll and protein levels decreased

Sinha et al. (2005)

6. 0.2, 2 and 20µM

Cr+6

B. juncea plants 15 d Ø APOX, CAT, GR, glutathione-S-transferase

(GST) and SOD

Ø Higher rate of H2O2 production

Ø Raised contents of MDA

Pandey et al. (2005)

7. Cr+6 O. sativa : Roots 24-48 h Ø SOD and POD

Ø Cr also increased the production of H2O2, O2.-

and MDA content in root cells

Panda (2007)

8. Cr+6 Hybrid willows (Salix

matsudana X Salix

192 h Ø CAT, POD and SOD

Ø Levels of soluble proteins lowered

Yu et al. (2007)

alba)

9. 10-6 ,10-5 , 10-4

M of Cr+6

Amaranthus viridis 20 d Ø POD and SOD

Ø Enhanced MDA content

Ø Net photosynthetic rate, transpiration rate,

stomatal conductance and intercellular CO2

concentration were reduced only by high Cr+6

treatments (10−5 M and 10−4 M)

Liu et al. (2008)

10. 20-100 ppm Phyllanthus amarus 30 d Ø Decreased contents of protein, sugar,

chlorophyll and carotenoids

Rai and Mehrotra (2008)

11. 1, 10 and 100

μM Cr+6

Zea mays seedlings 7, 14 or 21 d Ø CAT, POD and SOD

Ø High levels of MDA and increased electrical

conductivity of the cell membrane

Ø Minimization of photosystem II (PSII) activity

caused by Cr

Ø Cr phytotoxicity decreased Chl a/b ratio and

quenched the chl a fluorescence emission

spectra

Zou et al. (2009)

12. 50, 100 and 200

μM Cr

B. juncea (cv. Pusa Jai

Kisan)

1-7 d Ø APOX, CAT, GR and SOD

Ø Altered levels of MDA and GSH

Ø Induced Phytochelatins (PCs)

Diwan et al. (2010)

13. 50, 100 and 200

μM Cr

V. radiata (cv. Pusa

Ratna)

1-7 d Ø APOX, CAT, GR and SOD

Ø Cr stress modulated the contents of MDA, GSH

and PCs

Diwan et al. (2010)

14. 25, 50, 100, 200

and 250 μM

Cr+6

O. sativa

(cv. IR-64)

10 d Ø Increased levels of proline, MDA and NPSH

Ø Rice root metabolome analysis was carried out

to relate differential transcriptome data to

biological processes affected by Cr+6

Ø The integrated matrix of both transcriptome and

metabolome data provided visual picture of the

Dubey et al. (2010)

correlations between components.

Ø Predominance of different motifs in the subsets

of genes suggested the involvement of motif-

specific transcription modulating proteins in Cr

stress responses.

MERCURY (Hg) METAL TOXICITY

1. 10 M Hg L. esculentum 30 d Ø CAT, POD and SOD Cho and Park (2000)

2. 0 - 500 M Hg Cucumis sativus

seedlings

10 and 15 d Ø APOX and CAT

Ø Hg-treated seedlings showed elevated levels of

lipid peroxides with a concomitant increase in

protein oxidation levels and decreased

chlorophyll content at 250-500 M Hg

exposure.

Cargnelutti et al. (2010)

3. 0 - 50 mg l−1 Hg Sesbania drummondii 3 weeks Ø Both non-enzymatic antioxidants (GSH and

non-protein thiols, NPSH) and enzymatic

antioxidants (APOX, GR and SOD) were

modulated

Israr et al. (2006)

4. 0 - 50 µM Hg S. drummondii cell

culture

3 weeks Ø APOX, GR and SOD

Ø GSH and NPSH contents

Ø Both antioxidants and antioxidative enzymes

were first increased up to 40 μM Hg and then

declined at 50 μM Hg

Israr and Sahi (2006)

5. 0 - 40 µM Hg Medicago sativa 24 h Ø APOX, GR, POD and SOD Zhou et al. (2006)

6. 1, 2, 5, 10, and

20 mg Hg/L

B. juncea (cv.

Longstanding and Florida

Broadleaf)

20 and 30 d Ø CAT, POD and SOD

Ø Lower H2O2 in shoots with higher mercury

concentration in Indian mustard

Shiyab et al. (2008)

7. 5, 10, 25 and 50

µM Hg

B. juncea (cv. Pusa Jai

Kisan)

---- Ø APOX, CAT, GR and SOD

Ø Increased lipid peroxidation (MDA) and

Ansari et al. (2009)

accumulation of H2O2

8. 10 µM Hg M. sativa cv. Daiyi 24 h Ø APOX, POD and SOD

Ø Contents of prolines, ascorbate and glutathione

were enhanced under Hg stress

Zhou et al. (2009)

9. 1, 25 and 50 µM

Hg

Pfaffia glomerata

plantlets

9 d Ø APOX, CAT and SOD

Ø Toxicity indicators (δ-aminolevulinic acid

dehidratase activity), oxidative damage markers

(lipid peroxidation, and H2O2 concentration) and

non-enzymatic (NPSH, ascorbic acid, and

proline concentration) antioxidants were altered

under Hg stress.

Calgaroto et al. (2010)

10. 1, 2, 4, 6, and 8

µM Hg

Chlamydomonas

reinhardtii

96 h Ø APOX, CAT and SOD

Ø Treatment with Hg induced accumulation of

ROS, prolines and peroxidative products.

Ø RT-PCR-based assay revelaed the transcript

abundance of Mn-SOD, CAT and APOX

Elbaz et al. (2010)

11. 50, 100, 200 and

400 μM Hg

Jatropha curcas

seedlings

During

development

of

cotyledons

from

embryo

Ø Activities of CAT, POD, SOD and

Phenylalanine ammonia-lyase were modified

Ø Isoenzymes’ Analysis of SOD, POD and CAT

suggested that different patterns depend on Hg

concentrations and tissue types, and the staining

intensities of these isoenzymes are consistent

with the changes of these enzyme activities

assayed in solutions.

Gao et al. (2010)

12. 10 μM Hg B. juncea (cv. long-

standing)

24 h Ø APOX, CAT, POD and SOD

Ø Hg(II) triggered production of O2.-, H2O2 and

peroxides.

Ø Alterations in the contents of proline, NPSH and

GSH.

Meng et al. (2011)

NICKEL (Ni) METAL TOXICITY

1. 20 mmol m-3 to

40 mmol m-3

Ni2+

T. aestivum --------- Ø Activities of intracellular and extracellular POD

in root and shoot

Ø Enhanced level of lipid peroxidation and K+

leakage in roots

Pandolfini et al. (1992)

2. 200 µM Ni T. aestivum seedlings 4 d Ø CAT, GST, POD and SOD

Ø Enhanced level of proline in shoots

Gajewska et al. (2006)

3. 0.5 mM Ni Coffea arabica cell

suspension cultures

7 d Ø APOX, CAT, GR, POD and SOD

Ø Accumulation of MDA

Ø Native PAGE revealed one CAT isoenzyme,

four GR isoenzymes and nine SOD isoenzymes.

Ø SOD isoenzymes were differentially affected by

NiCl2 treatment and also one GR isoenzyme was

increased by NiCl2 treatment

Gomes-Junior et al.

(2006)

4. 50 and 100 µM

Ni

T. aestivum plants 4 d Ø Decreased chlorophyll content in shoots

Ø Increased levels of MDA and tocopherol in Ni

stressed wheat shoots

Gajewska and Sklodowska

(2007)

5. 10-20 µM Ni T. aestivum seedlings 6 d Ø APOX, CAT, GST, POD and SOD

Ø Proline levels raised

Ø Enhanced production of H2O2

Gajewska and Sklodowska

(2008)

6. 200 and 400 µM

Ni

O. sativa (cv. Malviya-

36) and (cv. Pant-12)

24 h, 5d and

20 d

Ø Increased levels of RNA, soluble proteins and

free amino acids in both endosperm and embryo

axes

Maheshwari and Dubey

(2008)

7. 200 and 400 µM

Ni

O. sativa

(cv. Pant-12)

Ø APOX, CAT, GR, POD, DHAR, MDHAR and

SOD; Activities of all isoforms of SOD (Cu-

ZnSOD, Mn SOD and Fe SOD), POD and

APOX increased in Ni stress

Ø Increased rates of O2.- production

Ø Elevated levels of H2O2, thiobarbituric acid

Maheshwari and Dubey

(2009)

reactive substances (High rate of lipid

peroxidation), NPSH and ascorbate

Ø levels of GSH and protein thiol were lowered

8. 0.5 mM Ni B. napus cv. PF 21 d Ø APOX, CAT and POD

Ø Levels of lipoxygenase activity and MDA, H2O2

and proline contents significantly increased

under Ni stress, while the activities of APOX,

CAT and POD decreased in leaves

Kazemi et al. (2010)

9. 50, 100, 200,

400 and 800 µM

Ni

Luffa cylindrica 7 d Ø CAT, POD and SOD

Ø Phenylalanine ammonia-lyase (PAL) activity in

the cotyledons, stems and roots was

significantly induced and was positively

correlated to increasing Ni concentrations

except for in the roots under 800 μM Ni

Wang et al. (2010)

10. 1 mM Ni Hypnum plumaeforme,

Thuidium cymbifolium,

and Brachythecium

piligerum

-------- Ø CAT, POD and SOD

Ø Increased levels of ROS and MDA under Hg

treatments in three mosses

Sun et al. (2011)

Table 2.6. Expression of Antioxidant Enzymes under Oxidative Stress in Different Plant Species

S.No. Enzyme ROS-

Detoxification

Mechanism

Localization Gene(s) Expression of Gene(s) under oxidative burst References

1. SOD O2●‾ + O2

●‾ +

2H+

↓

H2O2 + O2

Chloroplast

(Chl)

Cytosol

(Cyt)

Mitochondria

(Mit)

Peroxisomes

(Per)

Cu/Zn- SOD,

(In Chl, Cyt, Mit,

Per)

Fe-SOD

(In Chl) and

Mn-SOD

(In Mit -matrix)

Ø Fe-SOD, Cu/Zn- SOD and Mn-SOD in

Arabidopsis under heat, drought, salt , high light

and cold stress

Ø 4-times change in Cu/Zn- SOD in tobacco under

chilling stress

Ø 40-fold higher levels of Cu/Zn- SOD in Petunia

under O3 stress

Ø Higher levels of Cu/Zn- SOD in potatoes

exposed to paraquat

Ø Reduction in expression of Fe-SOD, Cu/Zn- SOD

and Mn-SOD in pea under Cd metal stress

Sen Gupta et al.

(1993),

Tepperman and

Dunsmuir

(1990), Ruth et

al. (1997),

Sandalio et al.

(2001), Mittler et

al. (2004)

2. APOX 2 Asc

(Ascorbate) +

H2O2

↓

2 MDA + 2H2O

Chloroplast

Cytosol

Peroxisomes

APOX1, APOX2,

APOX3, APOX4,

APOX5, APOX6,

APOX7,

Stromal-APOX

(sAPOX), Cyt-

APOX (cAPOX),

Per- APOX

(pAPOX)

and Thylakoid-

APOX (tAPOX)

Ø APOX1, APOX2,APOX3, APOX4,

APOX5,APOX6, APOX7, Stromal-APOX and

Thylakoid- APOX in Arabidopsis under heat,

drought, salt , high light and cold stress

Ø Higher increases in steady-state transcript levels

of the cAPOX, sAPOX, tAPOX and pAPOX

genes were observed in cowpea (Vigna

unguiculata) leaves under water deficit. On the

other hand, the expression of the cAPX genes was

stimulated earlier in the tolerant cultivar when

submitted to progressive drought

Ø cAPOX was highly induced in leaves by

wounding, bacterial pathogen (Pectobacterium

Mittler et al.

(2004) ; Arcy-

Lamet et al.

(2006)

chrysanthemi) and treatment with methyl

viologen, H2O2, ABA or exposure to high

temperature in sweet potato (Ipomoea batatas)

3. CAT 2 H2O2

↓

2 H2O + O2

Peroxisomes/

Glyoxysomes,

Cytosol and

Mitochondria

CAT1, CAT2 and

CAT3 in

Arabidopsis,

Nicotiana, Maize;

CAT-A, CAT-B

and CAT-C in

Oryza sativa

Ø CAT1, CAT2 and CAT3 in Arabidopsis under

heat, drought, salt , high light and cold stress

Ø CAT1 and CAT2 induced by light in Arabidopsis

Ø Differential expression of CAT1, CAT2 and

CAT3 in Nicotiana plumbaginifolia

Ø Rapid increase in CAT2 levels by UV-B light, O3,

SO2 treatments in N. plumbaginifolia

Ø Over-expression of Hot pepper CAT1 in

Arabidopsis in response to paraquat

Ø Transgenic tobacco plants expressing maize CAT2

gene enhanced resistance to Psedomonas syringae

induced oxidative stress in tobacco

Willekens et al.

(1994a), (1994b);

McClung (1997),

Polidoros et al.

(2001), Mittler et

al. (2004) , Park

et al. (2004) ,

Lee and An

(2006); Kwon et

al. (2007)

4. MDHAR MDA +

NADPH + H+

↓

Asc + NADP‾

Chloroplast

Cytosol

Mitochondria

MDHAR1,

MDHAR2,

MDHAR3,

MDHAR4 and

MDHAR5

Ø MDHAR1, MDHAR2, MDHAR3, MDHAR4 and

MDHAR5 in Arabidopsis under heat, drought, salt

, high light and cold stress

Mittler et al.

(2004)

5. DHAR DHA + 2 GSH

↓

Asc + GSSG

Chloroplast

Cytosol

Mitochondria

DHAR1, DHAR2,

DHAR3, DHAR4

and DHAR5

Ø DHAR1, DHAR2, DHAR3 and DHAR5 in

Arabidopsis under heat, drought, salt , high light

and cold stress

Ø DHAR4 in Arabidopsis under salt and cold stress

Mittler et al.

(2004)

6. GR GSSG +

NADPH

↓

2GSH + NADP‾

Chloroplast

Cytosol

Mitochondria

GR1 and

GR2

Ø GR1 and GR2 in Arabidopsis under heat, drought,

salt , high light and cold stress

Mittler et al.

(2004)

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REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10220/8/08_chapter 2.pdf · ground water or soil solution available for plant uptake, thus strongly influence