10

Regulation of Oxidative Stress by

Brassinosteroids in Plants

RENU BHARDWAJ*, INDU SHARMA, NITIKA ARORA, PRIYA UPPAL,

MUKESH KUMAR KANWAR, GURJINDER SINGH HUNDAL AND NEHA SHARMA

ABSTRACT
In plants, adverse environments such as biotic and abiotic stresses result
in reduced growth, biomass, seed and fruit production. A common
consequence of most of environmental stresses is the increased production
of reactive oxygen species (ROS). Although under normal growth
conditions, their production in cells is low, many stresses that disrupt
the cellular homeostasis of cells enhance the production of ROS. Oxidative
stress occurs when there is a serious imbalance in any cell compartment
between the production of ROS and antioxidative defence, thereby leading
to cell damage. The resistance of plants to oxidative stress depends on
the overall balance between the production of ROS and antioxidant
capability of cell. Several plant hormones have been found to ameliorate
the oxidative damage by regulating antioxidative defence system of
plants. Brassinosteroids (BRs) are a novel group of polyhydroxysteroids
which regulate a broad spectrum of physiological responses in plants.
In addition to their growth regulatory activities, they have been reported
to play a significant role in stress-protection in plants. As compared to
other phytohormones, BRs has received relatively little awareness, but
recent developments indicate there crucial role in stress amelioration.
The present review deals with such oxidative stress-protective properties
of these hormones.

* Department of Botanical and Environmental Sciences, Guru Nanak Dev University,

Amritsar-143005, Punjab, India.
*
Corresponding author : E-mail : renu_bhardwaj@rediffmail.com,
renubhardwaj82@gmail.com
216 Oxidative Stress: Roll of Antioxidents in Plants

Keywords : Abiotic stress, Biotic stress, Heavy metals, Reactive oxygen

Species

INTRODUCTION
Plants are frequently exposed to various environmental factors, which
constitute their macro and microenvironment. Any deviation in these factors
from the optimum levels is harmful and ultimately leads to stress in plants
(Kumar et al., 2008; Ahmad and Sharma, 2008). Stress may be caused due
to various abiotic and biotic factors. Abiotic factors include high
temperature, cold, drought, water logging, inadequate mineral nutrients
in soil, heavy metals, pesticides, air pollutants and salinity whereas the
biotic factors include viruses, insects, nematodes, bacteria, fungi etc.
(Hopkins and Huner, 2004). At a given time, plant may face even a
combination of more than one of above mentioned stresses. These stresses
in plants result in growth retardation, reduced fresh weight and seed or
fruit production (Shanker et al., 2005; Salvatore et al., 2008). 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
aggravate the production of ROS as compared with ROS generated during
normal metabolic processes (Bhattacharjee, 2005; Mittler, 2006). The ROS
capable of causing oxidative damage include superoxide (O·2–), perhydroxy
radical (HO·2–), 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). All these, results in
the evolution of new metabolic pathways, the accumulation of low molecular
weight metabolites, the synthesis of special proteins and changes in
phytohormone levels (Banu et al., 2009; Bari and Jones, 2009). Burgeoning
evidences indicate that a class of plant steroids 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). These
plant steroidal compounds occur in free and conjugated forms, where they
are bound to sugars and fatty acids. Till date, 70 BRs (65 unconjugated
and 5 conjugated) have been reported in plants and fully characterized by
spectrometric methods. Presumably, there are a number BRs and their
conjugates in plants yet to be described (Bajguz and Tretyn, 2003).
Experimental studies on higher plants suggest that BRs play a critical
role in a range of developmental processes such as growth, seed germination,
rhizogenesis, senescence, flowering, abscission, maturation, germination,
Regulation of Oxidative Stress by Brassinosteroids in Plants 217

leaf bending and epinasty, root inhibition, induction of ethylene

biosynthesis, proton-pump activation, xylem differentiation, regulation of
gene expression and vegetative and reproductive developments (Sasse,
2003; Cao et al., 2005; ÇaÉg et al., 2007). The present chapter reviews the
contribution of BRs in the regulation of plant defence responses and their
implications in various biotic as well as abiotic stresses.

Brassinostreoids and Oxidative Stress Management

BRs have also been reported to counteract stress effects in plants. The
plethora of abiotic/biotic stresses against which BRs act are water logging,
thermal, drought, heavy metals, infection, pesticides, salt and even
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 (superoxide
dismutase, guaiacol peroxidase, ascorbate peroxidase, catalase and
glutathione reductase etc.) and antioxidants (alkaloids, carotenoid
flavonoids, ascorbate, tocopherol, glutathione etc.) to provide protection to
the plants under stress conditions (Nunez et al., 2004; Ozdemir et al., 2004;
Hayat et al., 2007; Sharma and Bhardwaj, 2007; Arora et al., 2008).
Antioxidants such as ascorbate, tocopherol and glutathione react directly
or via enzyme catalysis 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 protective effects of brassinosteroids and its structural
analogs had been studied on growth, lipid peroxidation and antioxidative
system of rice, soyabean, tomato, potato and Cicer arietinum under various
stresses and it was observed that application of these hormones lowered
the oxidative stress and promoted plant growth (Mazorra et al., 2002;
Ozdemir et al., 2004; Almeida et al., 2005).

Oxidative Stress Generated with Biotic Factors: Regulation by

Brassinosteroids
Exogenous application of BRs stimulated inner potentials of plants that
are helpful not only in better survival in stress conditions, but also in
receding biotic stress caused by pathogens such as viruses, fungi and
bacteria (Nakashita et al., 2003; Wachsman et al., 2004a; Swaczynova
et al., 2006; Romanutti et al., 2007; Ohri et al., 2008).

Fungal stress
The potential of BRs to enhance plant resistance against fungal pathogen
infection was documented in several studies (Khripach et al., 2000).
Vasyukova et al. (1994) carried out investigations on the interaction
218 Oxidative Stress: Roll of Antioxidents in Plants

between Phytophthora infestans and potato tubers. The treatment of potato

plants with BRs, essentially reduced the incidence of Phytophthora
infection. The increase in resistance in BRs treated potato tubers was
associated with enhancement of ABA and ethylene levels and the presence
of phenolic and terpenoid substances. BR-induced disease resistance was
also noted in barley and cucumber plants. Spraying barley plants at tillering
phase with EBL decreased an extent of leaf disease induced by
Helminthosporium teres Sacc. and increased grain yield even at a dose of
5 mg ha–1 (Pshenichnaya et al., 1997; Volynets et al., 1997a, 1997b). The
protective effects of EBL in cucumber against fungi were studied by
Churikova and Vladimirova (1997). The increased activities of peroxidase
and polyphenoloxidase enzymes, which are involved in the metabolism of
polyphenols, was suggested as a factor contributing to BR induced disease
resistance in cucumber plants. Exogenous application of 24-epibrassinolide
was found to ameliorate the development of disease symptoms in tomato
plants inoculated with Verticillium dahliae, whereas untreated plants
showed the development of disease symptoms (Krishna, 2003).

Viral and bacterial stress

One of the important aspects of the protective action of BRs in plants is
related to their ability to stimulate resistance to virus infections (Rodkin
et al., 1997). The tobacco plants when given treatment of BRs against
tobacco mosaic virus (TMV), the bacterial pathogens Pseudomonas syringae,
and the fungal pathogen Odium species, showed lowered infection and
better growth. Similarly in rice, the infection caused by Magnaporthe grisea
and Xanthomonas oryzae which caused rice blast and bacterial blight
respectively, was significantly reduced by BR treatments (Nakashita
et al., 2003).
Applications of aqueous solutions of BRs (low conc. from 0.5 to
10 mg/l of both 24-epicastasterone and 24-episecasterone) containing
extracts to Lychnis viscaria L. seeds resulted in an enhanced resistance of
tobacco, cucumber and tomato to viral and fungal pathogens of up to 36%
compared with water-treated control plants (Roth et al., 2000). It has been
also reported that BR biosynthetic Arabidopsis mutant, cpd, showed a
considerable low expression of Pathogenesis Related genes i.e., PR1, PR2,
and PR5 with significant induction of them if CPD were over expressed in
transgenic plants (Szekeres et al., 1996). It has been shown that 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). BIN2 is one
of the 10 GSK3-like kinases in Arabidopsis (Jonak and Hirt, 2002) and is
considered to be a negative regulator of the BRs signal transduction
pathway (Li and Nam, 2002). Brassinolide (BL) enhanced resistance
Regulation of Oxidative Stress by Brassinosteroids in Plants 219

independently of SA and the combinations of BL and SAR inducers exhibit

additive or promotive effects against various pathogens. This reveals that
the SA-mediated signalling pathway for induced resistance is completely
distinct from the JA ethylene- and BL-mediated signalling pathways.
However, crosstalks may exist between these phytohormones (Nakashita,
2003).
Application of EBL increased activities of polyphenoloxidase and
peroxidase enzymes resulting in increased resistance to fungal infection
in the leaves of cucumber plants (Korableva, 2002). BRs and their synthetic
derivatives were reported to be good inhibitors of herpes simplex virus
type 1(HSV-1) and arena virus replication in cell culture (Wachsman
et al., 2004a, 2004b). Further, Michelini et al. (2004) had reported the in
vitro and in vivo antiherpetic activity of three new synthetic BRs analogues
{(22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one, (22S,23S)-5α-
fluoro-3β-22, 23-trihydroxystigmastan-6-one, (22S,23S)-3β-5α, 22,23-
trihydroxystigmastan-6-one}. Khripach et al. (2005) reported that EBL may
be used in the prevention and cure of HIV infection and related conditions
(AIDS related complex), both symptomatic and asymptomatic, or when
exposure to HIV virus was suspected. Romanutti et al. (2007) reported the
antiviral effects of a synthetic BR [(22S, 23S)-3β-bromo-5a,22,23-
trihydroxystigmastan-6-one] against replication of vesicular stomatitis
virus (VSV) in vero cells. Some synthetic BRs were tested against
herpes simplex virus type 1 (HSV-1) by Michelini et al. (2008) which
induced an ocular chronic immunoinflammatory syndrome named herpetic
stromal keratitis that might lead to vision impairment and blindness
in mice.

Insect development
Effects of BRs on insect development, particularly on molting, were reviewed
by Zullo and Adam (2002). EBL or 24-epicastasterone did not affect the
evagination of imaginal wing discs, nor was there any effect on intact last
instar larvae of the cotton leaf worm, Spodoptera littoralis, after oral feeding
(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).
Ohri et al. (2004, 2005) further revealed enhanced juvenile emergence of
M. incognita by brassinosteroids treatments. The treatment of
brassinosteroids to root knot nematodes (M. incognita) also stimulated their
antioxidative defence system (Ohri et al., 2007). Further Ohri et al. (2008)
studied the influence of EBL on development of Meloidogyne incognita.
EBL enhanced the percentage of hatching in treated egg masses as
compared to control. EBL treated juveniles induced more gall numbers
and larger size of galls in roots of tomato plants.
220 Oxidative Stress: Roll of Antioxidents in Plants

Oxidative Stress Generated with Abiotic Factors: Regulation by

Brassinosteroids
In agriculture several 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).

Drought stress
Mild drought stress in sugar beet was relieved by treatment with a synthetic
BR (Schilling et al., 1991). A wheat variety sensitive to water stress on
treatment with HBL showed an increase in grain yield, relative water
content and soluble protein contents, while ion leakage was reduced. The
antistress effect of BRs had been attributed for enhancing the membrane
stability. EBL was reported to decrease stomatal transpiration and improve
the performance of plants under stress (Singh et al., 1993; Sairam, 1994).
BRs had been established to increase plant resistance to drought and yield
value for different crops growing under drought conditions (Nilovskaya
et al., 2001). Application of EBL to different varieties of spring wheat under
normal and stress conditions (drought soil) was studied by Prusakova
et al. (2000). The plants sprayed with EBL solutions in the beginning of
booting stage of flowering resulted in higher water content in leaves.
Khodiankov (2002) has also studied the positive effect of EBL and
immunotzitofite on drought resistance of flax. Foliar sprays of BRs at
flowering stage were also found to increase 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).
EBL treatments also increased drought tolerance in Arabidopsis thaliana
and Brassica napus seedlings by changing expression of drought responsive
genes (Kagale et al., 2007). EBL also helped to overcome the negative
influence of drought and high temperature in case of three winter wheat
varieties (Ebi, Estica, Samanta) thereby increasing its dry matter and yield
of grain and straw (Hnilièka et al., 2007).

Heavy metals stress

Ability of BRs to regulate cell membrane permeability and ion transport
find their applications in the areas polluted with heavy metals and
radioactive elements. Treatment of barley, sugarbeet, tomato and radish
with EBL significantly reduced the absorption of heavy metals (Khripach
et al., 1996). Barley plants (Cv. Zazersky) treated with EBL in the booted
stage at a dose of 10 mg ha–1 showed that the diminution of metal content
in the plant was 40–98% in comparison with control. Soaking tomato seeds
for 12 h in 10–8 M solution of EBL before sowing was more efficient in
decreasing the content of Zn and Cd in tomato fruits than spraying
Regulation of Oxidative Stress by Brassinosteroids in Plants 221

treatment. Khripach et al. (1997) studied the absorption of radionuclides

in barley grown on the model soil enriched with cesium (Cs) and strontium
(Sr) ions under the influence of EBL. The content of Cs and Sr in the grains
of plants treated with 0.01 ppm of EBL was diminished in comparison with
the control grown on the polluted soil. EBL (10–8 M) blocked the heavy
metals (copper, lead, cadmium and zinc) accumulation in Chlorella vulgaris
cells (Bajguz, 2000). EBL at the concentration of 10–8 M in combination
with heavy metals (in the range 10–6–10–4 M) blocked metal accumulation
in algal cells. It had been observed that BRs in combination with Pb induced
the phytochelatins synthesis in Chlorella vulgaris (Bajguz, 2002).
BRs also ameliorated the aluminium (Al) toxicity in mung bean
(Phaseolus aureus) seedlings and promoted seedling growth and chlorophyll
content under Al metal stress (Bilikisu et al., 2003). The effect of EBL on
photosystem (PS) II of winter rape plants under Cd stress examined by
Janeczko et al. (2005) revealed that EBL protected the PS II by decreasing
the accumulation of Cd in cotyledons and by diminishing the damage of
photochemical reaction centres and O2 evolving centres as well as by
maintaining efficient photosynthetic electron transport. In addition to this,
EBL pretreatment to Cucumis sativus L. seedlings also helped to alleviate
the pesticide induced depression of photosynthesis by increasing CO2
assimilation capacity and activities of antioxidant enzymes (Xia et al., 2006).
Anuradha and Rao (2007) concluded that EBL and HBL treatments
decreased Cd toxicity in adish (Raphanus sativus L.) seedlings by enhancing
seed germination, seedling growth, free proline levels, activities of
antioxidant enzymes (CAT, SOD, APOX) and reducing MDA content,
activity of POD and ascorbic acid oxidase.
28-Homobrassinolide (HBL) also increased the activities of antioxidant
enzymes (CAT, POD, SOD), nitrate content and nitrate reductase enzyme
activity, chlorophyll content and photosynthetic rate and proline content
in Brassica juncea plants under cadmium stress as compared to control
(Hayat et al., 2007). HBL protected chickpea (Cicer arietinum) from Cd
toxicity by enhancing the activities of antioxidant enzymes (SOD, CAT,
POD), proline content, nitrate reductase and carbonic anhydrase and also
by increasing the plant growth, leghemoglobin content, nodule number,
nitrogen and carbohydrates content in the nodules and leaf chlorophyll
content, which were decreased proportionately with the increasing
concentrations of Cd (Hasan et al., 2008). HBL also improved the Ni stresses
B. juncea seedling growth by increasing the net photosynthetic rate,
chlorophyll content and the activities of nitrate reductase and carbonic
anhydrase in nickel stressed B. juncea plants (Alam et al., 2007). HBL and
EBL were further explored for their heavy metal stress amelioration
property by their application on Al stressed mung bean (Vigna radiata L.)
seedlings and found to enhance the activities of antioxidant enzymes (CAT,
222 Oxidative Stress: Roll of Antioxidents in Plants

SOD, POD) and proline content to overcome the oxidative stress generated
by Al (Ali et al., 2008a). Study carried out by Ali et al. (2008b) confirmed
the heavy metal stress ameliorative property of EBL in Brassica juncea
plants. Foliar spray of 1 µM of EBL to 15-days old B. juncea plants protected
plants against stress generated by NiCl2 (150 mM) by improving the level
of photosynthetic pigments, membrane stability index (MSI) and relative
water content. EBL improved the growth of B. juncea exposed to Ni stress
by enhancing the proline level and activities of antioxidative enzymes like
CAT, POD, SOD and GR. EBL reduced the level of MDA which was
otherwise enhanced by the Ni stress.
In our earlier studies it had been observed that EBL and HBL
treatments (presowing) improved the shoot emergence and plant biomass
production in Brassica juncea seedlings and plants under heavy metal stress
(Cu, Zn, Mn, Co and Ni). EBL and HBL have also been found to reduce 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 activities of SOD, POD, CAT, APOX and GR
antioxidative enzymes (Bhardwaj et al., 2007, Arora et al., 2008a).

Pesticides stress
Several pesticides have been developed to target specific biochemical
reactions within their target organisms. However, it is of great concern to
understand the effects of these pesticides on non-targeted crops (Matthews,
2006). BRs have been reported to be effective in reducing damage caused
by pesticides (simazine, butachlor, or pretilachor) in rice (Sasse, 2003).
The phytotoxic effect on cucumber leaves 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) has been examined (Xia et al., 2006). Plants
treated with paraquat revealed the severest phytotoxic symptoms with
the highest reduction in net photosynthetic rate (Pr), while other pesticides
except flusilazole inhibited Pr to various degrees. The inhibition of Pr by
cuproxat was accompanied by declines both in stomatal conductance and
intercellular CO2 concentartion, wheras decreased Pr for the cyazofamid
was associated with increased intercellular CO2 concentartion. For other
pesticides, inhibition of Pr was accompanied by decrease in stomatal
conductance while intercellular CO2 concentartion was increased or
unaffected. Also, inhibitions of Pr were alleviated by EBL pretreatment
except for the pesticides paraquat and flusilazole. Pesticides application
Regulation of Oxidative Stress by Brassinosteroids in Plants 223

impaires the photosynthesis of cucumber seedlings, whereas EBL

pretreatment can increase the resistance of plants to pesticides which might
be mediated by enhanced activities of CO2 assimilation enzymes.

Salt stress
Nearly 20% of the world’s cultivated area and nearly half of the irrigated
lands are affected by salinity (Akram et al., 2008). BRs have been reported
to overcome the salt stress by regulating the activities of key antioxidative
enzymes and levels of MDA and proline. In Eucalyptus camaldulensis,
treatment of seeds with EBL promoted seed germination under saline
conditions (Sasse et al., 1995). The ability of BRs to counteract the inhibitory
effects of salinity on seedling growth of groundnut was reported by Vardhini
and Rao (1997). Both EBL and HBL promoted the germination of groundnut
seeds. Further Anuradha and Rao (2003) studied the effect of BL, HBL
and EBL (3 mM) on growth, chlorophyll ‘a’ and ‘b’ content and nitrate
reductase activity of rice (Oryza sativa L.) plants grown on saline
substratum supplied with 150 mM of NaCl. BRs reduced the impact of salt
stress on growth, considerably by restoring pigment levels and enhancing
nitrate reductase activity. Further Nùñez et al. (2004) studied the influence
of polyhydroxylated spirostanic BR analogue (BB-16), on the activities of
antioxidant enzymes in rice seedlings grown in vitro culuture medium
supplemented with 75 mM of NaCl and 0.001 and 0.01 mg dm–3 BB-16.
Seedlings exposed to 0.01 mg dm–3 BB-16 showed significant increase in
activities of CAT, SOD and GR and slight increase in APOX. So BB-16,
structurally modified in lateral chain in relation to natural BRs, confer
tolerance to salt stress seedlings by changing the activities of antioxidative
enzymes.
The effects of EBL on seedlings growth, proline and soluble protein
content, lipid peroxidation and antioxidative defence system were
investigated in seedlings of the salt sensitive rice cultivar IR-28 by Ozdemir
et al. (2004) and it was observed that the seedling growth was improved by
the application of EBL to salt stressed (120 mM NaCl) seedlings by
regulating the activities of SOD, POD, CAT, APOX and GR. In addition to
this, increased proline and MDA content in NaCl stressed seedlings
decresed by the application of EBL. Similarly, Kagale et al. (2007) observed
that EBL helped to overcome a salt stress induced inhibition of seed
germination in Brassica napus. Seeds of B. napus were allowed to germinate
on a nutrient medium containing 1 or 2 µM EBL and 50, 100, 150, 200, 250
or 300 mM NaCl. Presence of EBL (2 µM) in the medium significantly
reduced the inhibitory effect of high salt concentration and promoted seed
germination and seedling growth. On count at 96 h the average germination
rate of 86, 53 and 34% were observed in treatment containing 200, 250 or
300 mM NaCl supplemented with 2 µM of EBL. Further, Zhang et al. (2007)
224 Oxidative Stress: Roll of Antioxidents in Plants

reported that seed priming of three varieties of lucerne (Medicago sativa

L.) (Victoria, Golden Empress, and Victor) with BL (5 µM l–1) enhanced
their percentage germination and seedling growth under a high level of
salt stress (13.6 dS m–1 NaCl). BL treatment also significantly enhanced
the activities of antioxidant enzymes (POD, SOD, CAT) and reduced the
MDA content of seedlings expose to salt stress thereby suggesting that
priming of lucerne seed with BL at a suitable concentration could ameliorate
salt stress.
Study carried out by Shahbaz et al. (2008) and Ali et al. (2008c) further
confirmed the salt stress ameliorative property of EBL in Triticum aestivum
and Brassica juncea plants. Foliar application of EBL (0.0125, 0.025 and
0.0375 mg l–1) to 41 days old wheat plants grown in Hoagland’s nutrient
medium (supplemented with 150 mM of NaCl) successfully ameliorated
the salinity induced growth inhibition by enhancing net photosynthetic
rate and activities of antioxidative enzymes (POD and CAT). Foliar spray
of 1 µM of EBL to 15-days old B. juncea plants protected the plants against
stress generated by salinity (150 mM NaCl) by improving the level of
photosynthetic pigments, membrane stability index (MSI) and relative
water content. EBL further improved the growth of B. juncea exposed to
salinity by enhancing the proline level and activities of antioxidative
enzymes like CAT, POD, SOD and GR. Our earlier studies also showed
that HBL ameliorated the salinity stress in Zea mays seedlings and plants
by increasing the activities of antioxidative enzymes (CAT, POD, SOD, GR
and APOX) and by reducing the level of lipid peroxidation (Arora et al.,
2008a).

Tempertaure stress
BRs have also been reported to show growth promoting effects on eggplant,
cucumber, and maize under chilling stress (Mandava, 1988; He et al., 1991).
Similarly, cucumber seedlings germinated from seeds soaked in BRs
solution showed an enhanced growth as compared to controls under cold
conditions (5oC for 3 days). In rice, BRs increased seedling resistance to
chilling injury (Wang and Zeng, 1993) and increased the morphological
parameters (height, root length, root biomass, and total biomass) of rice
under low temperature conditions (Kim and Sa, 1989; Hirai et al., 1991).
Kamuro and Takatsuto (1991) observed higher fruit settings in tomato
plants sprayed with BRs under winter conditions. The study on the effects
of BRs-treated wheat leaves at 43o C by Kulaeva et al. (1991) showed that
protein synthesis was maintained similar to those at 23oC, whereas in
untreated leaves it decreased 2.5 fold at 43oC as compared to control
temperature.
Wang and Zeng (1993) also reported that treatment with EBL reduced
the MDA content, slowed the decrease in SOD activity, and increased the
Regulation of Oxidative Stress by Brassinosteroids in Plants 225

proline content of rice under chilling stress. In brome grass epibrassinolide

treatment enhanced tolerance to high temperature (Wilen et al., 1995).
Dhaubhadel et al. (1999) reported that EBL treatment increased the basic
thermotolerance of Brassica napus and tomato seedlings grown under heat
stress. 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. The higher accumulation of HSPs in EBL-treated
seedlings raises the possibility that HSPs contribute, at least in part, to
thermotolerance in EBL-treated seedlings. In 2002, Dhaubhadel et al.,
conducted both in vivo as well as in vitro experiments so as to understand
mechanisms controlling hsp synthesis in EBL-treated and untreated
seedlings and assessed the levels of components of the translational
machinery in these seedlings. It was 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.
Further in the same course, Rao et al. (2002) reported that application
of 24-epibrassinolide improved the resistance against chilling stress
(1–5oC) in rice and this tolerance was coupled with increased ATP, proline
level and SOD activity. The ultra structure of tomato leaf discs treated
with BB6 (BRs analogue with spirostanic structure as active ingredient)
under high temperature increased the rate of production of heat shock
proteins, which protected mRNA from stress-induced degradation (Sam
et al., 2001). Mazorra et al. (2002) studied the effect of EBL and MH5
(polyhydroxylated spirostanic analogue of BRs) was analyzed on CAT,
peroxidase and SOD activity in tomato leaf discs at 25–40 oC. Both
concentrations (10.60 and 2.12 nM) of BB6 and MH5 stimulated SOD
activity at 25 and 40oC, the MH5-stimulated increase of this enzyme activity
was greater. The altered activities of these enzymes by BRs, suggests a
possible role of EBL and MH5 in the reduction of cell damage produced by
heat stress. Mussig et al. (2002) identified the oxidative stress related genes
encoding monodehydroascorbate reductase 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 (38oC). They observed that tomato plants
treated with EBL were more tolerant to high temperature than untreated
226 Oxidative Stress: Roll of Antioxidents in Plants

plants. To determine, if BRs are vital for heat shock proteins synthesis,
Kagale et al. (2007) further extended the studies of Dhaubhadel et al. (2002)
to A. thaliana seedlings and BRs-deficient mutant seedlings. The treatment
of EBL enhanced the basic thermotolerance of A. thaliana seedlings under
heat stress, similar to its effect on B. napus. It was further found by Kagale
et al. (2007) that EBL increased the transcripts of three cold responsive
structural genes-rd29a, a BN115 homolog and COR47 in A. thaliana
seedlings as compared with untreated seedlings.
Janeczko et al. (2007) observed that EBL exerts opposite effects on
cell membranes of oilseed rape at different temperatures; at 20oC, it
increased, while at 2oC, it decreased the membrane permeability. Janeczko
et al. (2009) studied the relationship between the temperature of oilseed
rape growth and the influence of EBL on biochemical changes in cells. Effect
of EBL on fatty acids composition and sugar content in winter oilseed rape
callus cultured at 20oC and 5oC has been observed and it has been concluded
that EBL-action is highly temperature-dependent.

Water stress
Several studies have been investigated on the effects of BRs on water-
stressed plants. Schilling et al. (1991) reported that HBL increased tap-
root weight, sucrose content, and yield of sugar beets grown under drought
stress. They exposed sugar beet plants to drought stress which lead to a
reduction in taproot mass in proportion to stress severity and increase in
root growth in BR-treated plants versus untreated plants was seen only
under water stress conditions. Similarly, Sairam (1994) found that HBL
significantly increased the relative water content, chlorophyll a content,
photosynthetic rate, leaf area and biomass production of wheat under
moisture stress. The water stressed plants of Jackpine, treated with
homobrassinolide evolved more ethylene (Rajasekaran and Blake, 1999).
In a study with cucumber plants it was also demonstrated that BRs
treatment improves resistance to desiccation and high-temperature stress.
The content of free amino acids and amides in the leaves of treated plants
was higher as compared to untreated plants (Pustovoitova et al., 2001).
Thus, we can say that treatment with Brassinosteroids may be a useful
management tool for agricultural applications in arid and semiarid areas.
Li et al. (2008) found that treatment with 0.2 mg/l BL decreased the
transpiration rate, stomatal conductance and malondialdehyde (MDA)
content of Robinia seedlings growing under moderate or severe water stress
compared to untreated seedlings. Also, 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. Jager et al. (2008) studied the
endogenous level of BR and ABA in wild type (WT) and BR-deficient mutant
Regulation of Oxidative Stress by Brassinosteroids in Plants 227

(lkb) and BR-perception mutant (lka) pea plants exposed to water stress.
There was enhancement in level of ABA in water stressed plants but no
alteration in level of BR observed. It proposed that change in endogenous
BR levels was not normally part of the plant’s response to water stress.

Mechanism of Brassinosteroids 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 EBL treated heat stressed Brassica 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, EBL 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-
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).

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