Effect of 24-epibrassinolide on oxidative stress markers induced by nickel-ion in Raphanus sativus L

ORIGINAL PAPER

Effect of 24-epibrassinolide on oxidative stress markers inducedby nickel-ion in Raphanus sativus L.

Indu Sharma • Pratap Kumar Pati •

Renu Bhardwaj

Received: 20 October 2010 / Revised: 23 December 2010 / Accepted: 30 December 2010 / Published online: 21 January 2011

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2011

Abstract The present study illustrates the effect of

24-epibrassinolide (24-EBL) on morphological and bio-

chemical parameters in radish (Raphanus sativus L.) seed-

lings grown under nickel (Ni) ion stress. The radish seeds

pre-soaked in different concentrations of 24-EBL were sown

in petridishes containing various concentrations of heavy

metal (Ni).Observations were made on root/shoot length,

fresh biomass, activities of antioxidant enzymes (ascorbate

peroxidase, superoxide dismutase, catalase, monodehydro-

ascorbate reductase, dehydroascorbate reductase, guaiacol

peroxidase and glutathione reductase), lipid peroxidation,

proline and protein content in 7-day-old Ni-stressed radish

seedlings. Results indicate that seeds presoaked with

24-EBL reduced the impact of Ni-stress which was evident

by assessing the morphological parameters, protein content

and antioxidant enzyme activities. It was also observed that

24-EBL reduced the toxicity of heavy metal by influencing

proline and malondialdehyde (MDA) content. The present

study lays a foundation for understanding the role of 24-EBL

in heavy metal stress amelioration, particularly in food crop.

Analysis of behaviour of antioxidant enzymes will play a

critical role in understanding the stress networking, further

filling the knowledge gap on the subject.

Keywords Antioxidant enzymes � Radish � Nickel ion

stress � 24-epibrassinolide � Lipid peroxidation

Introduction

Brassinosteroids (BRs) are an emerging group of steroidal

phytohormones which are essential for plant growth and

development (Pinol and Simon 2009). Recently, several

BR-regulated genes associated with diverse physiological

responses, such as cell division and expansion, differenti-

ation, programmed cell death, stomatal development and

functions, homeostasis and gene expression have been

isolated by genome-wide microarray analysis (Divi and

Krishna 2009; Tanaka et al. 2009). In addition to their

growth regulatory activities, BRs have also been reported

to play pivotal potential for their implication in both stress-

protection and stress-amelioration (Krishna 2003; Bajguz

and Hayat 2009). Furthermore, BRs are tested for anti-

genotoxicity by employing Allium cepa chromosomal

aberration bioassay, to ensure their safe use in agricultural

practices (Sondhi et al. 2008). In Arabidopsis, Li et al.

(2007) isolated a gene HSD1 encoding a protein with

homology to animal 11-b-hydroxysteroid dehydrogenase

(HSD). Divi and Krishna (2009) observed that overex-

pression of AtHSD1 in Arabidopsis led to BR-responsive

gene expression and in Brassica napus enhanced stress

tolerance. Divi and Krishna (2009) suggested that crop

yields and stress tolerance in plants could be achieved by

manipulating the genes involved in the BRs biosynthetic

and signalling pathways.

Communicated by S. Lewak.

I. Sharma � R. Bhardwaj (&)

Department of Botanical and Environmental Sciences,

Guru Nanak Dev University, Amritsar 143005, Punjab, India

e-mail: [email protected]

I. Sharma


P. K. Pati

Department of Biotechnology, Guru Nanak Dev University,

Amritsar 143005, Punjab, India


123

Acta Physiol Plant (2011) 33:1723–1735

DOI 10.1007/s11738-010-0709-1

Heavy metal stress is one major stress faced by the

agricultural crops. Some metals like Fe, Se, Mn, Co, Zn,

Mo and Ni, are essential micronutrient, but when ‘‘certain

trace levels’’ exceed, they are highly toxic to plants (Eskew

et al. 1983; Hall and Williams 2003). Also, being persistent

in nature, these heavy metals get accumulated in soils and

plants. Nickel (Ni) is an essential micronutrient and func-

tions as an active centre of the enzyme urease required for

the hydrolysis of urea and nitrogen metabolism in higher

plants (Brown et al. 1987; Gerendas et al. 1999). But at

higher levels, Ni produces toxic symptoms like stunting

growth, leaf chlorosis, mitotic inhibition, vein necrosis, etc.

in plants (Seregin and Kozhevnikova 2006; Llamas et al.

2008). Anthropogenic activities, for example effluent dis-

posal, metal mining, smelting, electroplating, sewage

sludge and fertilizer application result in Ni pollution

(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 ion 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). The

imbalance between ROS production and amelioration leads

to oxidative stress causing phytotoxicity (Sudo et al. 2008).

To overcome this stress, the plants possess enzymatic and

non-enzymatic antioxidant defence system (Mittler 2002;

Skorzynska-Polit et al. 2010). Also, the antioxidant defence

system of plants is regulated by certain phytohormones like

Abscisic acid, Jasmonates and BRs under stress (Bari and

Jones 2009). But, the role of BRs in this direction is yet to

be studied in Raphanus sativus plants under heavy metal

stress.

In view of the above as well as the wide occurrence and

economic importance of R. sativus (radish), the present

study was carried out to explore the possible role of BRs in

ameliorating specific stress. The effects of 24-EBL have

been focused on the present piece of work to study the

morphological parameters, activities of antioxidant

enzymes, lipid peroxidation, proline and protein content in

Raphanus sativus L. (Pusa Chetaki) seedlings under Nickel

(Ni) ion stress.

Materials and methods

Plant material and growth conditions

Seeds of Raphanus sativus L. (Pusa Chetaki) were pro-

cured from Department of Plant Breeding, Punjab Agri-

culture University, Ludhiana, India. Seeds were surface

sterilized with 0.4% sodium hypochlorite for 15 min fol-

lowed by repeated rinses in sterile distilled water. Surface-

sterilized seeds were given 8-h presoaking treatment in

different concentrations of 24-EBL (0, 10-11, 10-9 and

10-7 M). These pretreated seeds were germinated on

Whatman No. 1 filter paper lined autoclaved glass Petri

dishes, each containing various concentrations of Ni (0,

0.5, 1.0 and 1.5 mM). The Ni (II) ion stress was given in

the form of NiSO4�7H2O. The experiment was conducted

under controlled conditions of light (16 h photoperiod

under fluorescent white light with 175 lmol m-2 s-1

intensity), temperature (25 ± 5�C) and relative humidity

(80–90%). The experiment was repeated twice with five

replications (each containing 20 seedlings) for each

treatment.

Growth analysis

Seven-day-old seedlings were harvested and roots and

shoots were separated. Seedling growth in terms of root

and shoot length was recorded. Twenty seedlings per pet-

ridish were used for the determination of morphological

parameters (root/shoot length), fresh biomass and per-

centage germination. The seedlings were oven-dried at

80�C for 24 h to determine their dry weights.

Biochemical analysis

Lipid peroxidation

Lipid peroxidation was determined by measuring the con-

tent of Malondialdehyde (MDA), a secondary end product

of the oxidation of polyunsaturated fatty acids, by the

method of Hodges et al. (1999). One gramme of shoots was

homogenized in 5 ml of 80% ethanol and then centrifuged

at 12,0009g for 5 min. Experiment was conducted using

20.0% (w/v) TCA (trichloroacetic acid), i.e. (-) TBA

(thiobarbituric acid) solution and 0.65% (w/v) TBA in

20.0% (w/v) TCA, i.e. (?) TBA solution. One ml aliquot of

supernatant was added to two different test tubes, one

containing 1 ml (–) TBA solution and other test tube

containing (?) TBA solution. Samples were then mixed

vigorously, kept in water bath at 95�C for 30 min and then

cooled quickly on ice bath. Then, samples were centrifuged

at 12,0009g for 5 min. Absorbance of red adduct was

observed at 440, 532, and 600 nm and Malondialdehyde

equivalents/g fresh weight (nmol ml-1) were calculated as

described by Hodges et al. (1999).

Proline content

The free proline content was estimated spectrophotomet-

rically following the method of Bates et al. (1973). Fresh

1 g of cotyledonary leaves was homogenized in 3.5 ml of

3% sulphosalicylic acid and the homogenates were

1724 Acta Physiol Plant (2011) 33:1723–1735

123

centrifuged at 12,0009g for 10 min. Then, 2 ml of super-

natant was reacted with 2 ml of acid ninhydrin and 2 ml of

glacial acetic acid in test tubes for 1 h at 100�C. The

reaction was terminated by putting the test tubes in ice

bath. The reaction mixture was extracted with 4 ml of

toluene and mixed vigorously by shaking for 15–20 s.

Then toluene layer was separated from aqueous phase and

warmed to room temperature. The absorbance of red col-

oured Proline-ninhydrin product was measured in toluene

layer at 520 nm. Proline concentration was calculated from

a standard curve using 0–500 lM concentrations of

L-proline.

Preparation of leaf extracts

Leaf extracts were prepared to estimate the activities of

antioxidant enzymes and the protein content by homoge-

nizing 2 g cotyledonary leaves of 7-day-old seedlings in

chilled 6 ml 50 mM phosphate buffer (pH 7.0), 1 mM

ethylenediaminetetraacetic acid (EDTA), 1 mM phe-

nylmethanesulfonylfluoride (PMSF), 0.5% (v/v) Triton

X-100 and 2% (w/v) polyvinylpyrrolidone (PVP-30) in a

pre-chilled mortar and pestle. For estimation of ascorbate

peroxidase and dehydroascorbate reductase 0.5 mM

ascorbate was added to the extraction buffer. In case of

monodehydroascorbate reductase activity, 1 g of leaves

was homogenized in 3 ml of 50 mM Tris–HCl buffer (pH-

7.6) containing 2.5 mM Ascorbic acid. The homogenates

were centrifuged at 12,0009g for 20 min at 4�C. The

supernatant was further used for biochemical analysis.

Protein quantification

Total protein content of different samples of Ni (0, 0.5, 1.0

and 1.5 mM) and 24-EBL (0, 10-11, 10-9 and 10-7 M)

alone or in combinations, was quantified by following the

method of Bradford (1976) using bovine serum albumin as

a standard.

Ascorbate peroxidase assay

The ascorbate peroxidase (APOX, EC 1.11.1.11) activity

was determined spectrophotometrically as described by

Nakano and Asada (1981). The 3.0 ml reaction mixture

contained 50 mM Potassium phosphate buffer (pH 7.0),

0.5 mM ascorbate, 1.0 mM H2O2 and 100 ll enzyme

extract. The H2O2-dependent oxidation of ascorbate was

followed by monitoring the decrease in absorbance at

290 nm using the extinction coefficient 2.8 mM-1 cm-1.

The reaction was carried out for 3 min at 25�C. One unit of

APOX activity is defined as the amount of enzyme that can

oxidize 1 lmol of ascorbate per minute.

Catalase assay

Catalase (CAT, EC 1.11.1.6) activity was assayed by

measuring the initial rate of H2O2 disappearance using the

method of Aebi (1984). The 3.0 ml reaction mixture con-

tained 50 mM Potassium phosphate buffer (pH 7.0),

15 mM H2O2 and 100 ll enzyme extract The decrease in

hydrogen peroxide was followed as decline in optical den-

sity at 240 nm for 30 s at 25�C. The enzyme activity was

calculated using an extinction coefficient 39.4 mM-1 cm-1

for H2O2. One unit of enzyme activity is defined as the

decomposition of 1 mmol H2O2 per minute/g fresh

biomass.

Dehydroascorbate reductase assay

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity

was measured following the method given by Dalton et al.

(1986). The 3.0 ml reaction mixture contained 50 mM

Potassium phosphate buffer (pH 7.0), 0.2 mM dehydro-

ascorbate, 0.1 mM EDTA, 2.5 mM reduced glutathione

and 100 ll enzyme extract. DHAR activity was measured

by following the increase in absorbance at 265 nm due to

ascorbate formation at 265 nm using extinction coefficient

of 14 mM-1 cm-1.

Glutathione reductase assay

Glutathione reductase (GR, EC 1.6.4.2) activity was

determined by using the method of Carlberg and Manner-

vik (1975). Three ml of reaction mixture contained 50 mM

Potassium phosphate buffer (pH 7.6), 1 mM oxidized

glutathione (GSSG), 0.5 mM EDTA, 0.1 mM reduced ni-

cotinamideadenine dinucleotidephosphate (NADPH) and

100 ll enzymes extract. The reaction was initiated by

addition of 0.1 mM NADPH at 25�C. The GR activity was

determined by the oxidation of NADPH at 340 nm with

extinction coefficient of 6.22 mM-1 cm-1.

Guaiacol peroxidase assay

Guaiacol peroxidase (POD, EC 1.11.1.7) activity was

assayed using the method of Sanchez et al. (1995) with

some modifications. The 3.0 ml reaction mixture contained

50 mM Potassium phosphate buffer (pH 7.0), 20 mM

guaiacol, 12.3 mM H2O2 and 100 ll enzyme extract.

Activity of POD was determined by measuring the absor-

bance at 436 nm and using an extinction coefficient of

26.6 mM-1 cm-1. One unit of POD activity represents the

amount of enzyme catalysing the oxidation of 1 lmol of

guaiacol in 1 min.

Acta Physiol Plant (2011) 33:1723–1735 1725

123

Monodehydroascorbate reductase assay

Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4)

activity was assayed using the method of Hossain et al.

(1984). Three ml of reaction mixture contained 50 mM

Tris–HCl (pH 7.6) containing 2.5 mM Ascorbic acid,

0.1 mM mM reduced nicotinamideadenine dinucleotide

(NADH), 0.14 units of Ascorbic acid oxidase and 100 ll of

enzyme extract. Reaction was started by adding ascorbic

acid oxidase, and the enzyme activity was measured by

following the decrease in absorbance due to the oxidation

of NADH at 340 nm. This decrease in absorbance was

measured for 1 min and enzyme activity was determined

using extinction coefficient of 6.2 mM-1 cm-1.

Superoxide dismutase assay

Superoxide dismutase (SOD, EC 1.15.1.1) activity was

assayed by measuring the ability of the enzyme extract to

inhibit the photochemical reduction of nitrobluetetrazolium

(NBT) (Kono 1978). For total SOD assay, 3.0 ml reaction

mixture contained 50 mM sodium carbonate (pH 10.2),

24 lM NBT, 0.1 mM EDTA, 1 mM hydroxylamine,

0.03% (v/v) Triton X-100 and 70 ll enzyme extract. The

absorbance was recorded at 560 nm for 2 min. One unit of

SOD activity was defined as the amount of enzyme

required that caused 50% of NBT reduction at 25�C.

Statistical analysis

The data were subjected to two-way analysis of variance

(ANOVA) for analysing the interactions of various doses

of Ni and treatments of 24-EBL and expressed as the

mean ± standard error of five replicates. The significance

of difference between the control and treatments was set at

p B 0.05. Holm-Sidak post hoc test was applied for the

multiple comparisons versus control using SigmaStat

Version 3.5 and significance of difference between the Ni-

stress and 24-EBL treatments was set at p B 0.05.

Results and discussion

Morphological parameters

The present research showed that seed presoaked with

24-EBL improved the seedling growth, biomass and per

cent germination under Ni stress. Treatment of 24-EBL

increased root/shoot length (Figs. 1, 2), fresh biomass

(Fig. 3) and per cent germination (Fig. 4) of radish seed-

lings as compared with seedlings raised without 24-EBL

presoaking treatment under Ni stress. The root length

(Fig. 1) is decreased significantly with increased concen-

trations of Ni ion as compared with seedlings grown under

distilled water (Fig. 1). Root length was observed to be

minimum at 1.5 mM Ni (1.313 cm) as compared with

0 mM Ni (11.3 cm). However, no significant change was

observed in seedlings raised after 24-EBL presoaking

treatment only as compared with control. Further, presoa-

king treatments were found to be effective in alleviating

Ni-stressed radish seedlings (Figs. 1, 2, 3, 4). Furthermore,

the root length of seedlings (Fig. 1) treated with 10-7 M

24-EBL supplemented with 0.5 mM Ni solution

(14.31 cm) was maximum as compared with the seedlings

treated with Ni alone (5.187 cm). Also, the shoot length

was observed to decrease under Ni stress and increased

Fig. 1 Effect of 24-EBL on root length in 7-day-old Raphanussativus seedlings under Ni metal stress. Bar represents the SE

(n = 100). Different letters (a, b, c, d) within various concentrations

of Cr (0, 0.5, 1.0 and 1.5 mM) are significantly different (Holm-Sidak

post hoc test, p B 0.05), whereas different letters (p, q, r, s) within

various treatments of 28-HBL (0, 10-11, 10-9 and 10-7 M) are

significantly different (Holm-Sidak post hoc test, p B 0.05) and

signify interactions of different concentrations 28-HBL with Cr on

root length


123

when 24-EBL presoaking treatments were given as com-

pared with controlled seedlings (Fig. 2). Similar trends

were observed in case of fresh biomass (Fig. 3) and per

cent germination (Fig. 4) of radish seedlings. These results

are in coherence with the prior report of Vardhini and Rao

(2003) that BRs application alleviated osmotic stress in

three varieties of Sorghum vulgare by enhancing the

seedling length, seedling fresh and dry biomass. Also, in

rice 24-EBL alleviated the inhibition of percent seed ger-

mination, seedling growth and prevented the photosyn-

thetic pigment loss induced by salinity stress (Anuradha

and Rao 2001, 2003). Earlier it has been reported that BRs

induce the plant growth via cell elongation and cell

division (Clouse and Sasse 1998; Haubrick and Assmann

2006).

Biochemical parameters

Protein content

Total soluble protein content of seedlings alleviated

significantly in all treatments of 24-EBL as compared

with control (Fig. 5). In seedlings treated with 1.5 mM

Ni, protein content was remarkably higher (26.77 mg

g-1FW) as compared with control (19.96 mg g-1FW).

Moreover, the protein content was increased, when 10-7

Fig. 2 Effect of 24-EBL on shoot length in 7-day-old Raphanussativus seedlings under Ni metal stress. Bar represents the SE







shoot length

Fig. 3 Effect of 24-EBL on fresh biomass in 7-day-old Raphanussativus seedlings under Ni metal stress. Bar represents the SE







fresh biomass


123

M 24-EBL treated seeds were grown under 1.5 mM Ni

stress (28.99 mg g-1FW) as compared with untreated

seeds (1.5 mM Ni alone, 26.77 mg g-1FW). Similar work

was proposed by Cag et al. (2007) reported EBL

(0.001 lM) to be effective in enhancing the protein

content of excised Brassica oleraceae cotyledons. This

increase in protein content may be attributed to 24-EBL-

induced denovo polypeptide synthesis as earlier proposed

by Kulaeva et al. (1991) in wheat leaves under thermal

stress.

MDA content

Membrane destabilization is generally attributed to lipid

peroxidation (enhanced accumulation of MDA), due to

an increased ROS production under stressed condition

(Skorzynska-Polit et al. 2010). Presently, as a conse-

quence of heavy metal stress in radish seedlings, MDA

content had increased with increasing concentrations of

Ni but decreased with 24-EBL applications (Fig. 6).

Minimum content of MDA (2.123 l mol gFW-1) was

Fig. 4 Effect of 24-EBL on percent germination in 7-day-old

Raphanus sativus seedlings under Ni metal stress. Bar represents

the SE (n = 100). Different letters (a, b, c, d) within various

concentrations of Cr (0, 0.5, 1.0 and 1.5 mM) are significantly

different (Holm-Sidak post hoc test, p B 0.05), whereas different

letters (p, q, r, s) within various treatments of 28-HBL (0, 10-11, 10-9

and 10-7 M) are significantly different (Holm-Sidak post hoc test,

p B 0.05) and signify interactions of different concentrations 28-HBL

with Cr on per cent germination

Fig. 5 Effect of 24-EBL on protein content in 7-day-old Raphanussativus seedlings under Ni metal stress. Bar represents the SE

(n = 10). Different letters (a, b, c, d) within various concentrations of

Cr (0, 0.5, 1.0 and 1.5 mM) are significantly different (Holm-Sidak





protein content


123

observed in 10-9 M presoaked seeds 24-EBL grown

under 0.5 mM Ni stress as compared with control

(0.5 mM Ni alone, 4.036 l mol gFW-1) (Fig. 6).

24-EBL regulated decrease in MDA content under Ni

stress may be credited to the effective ROS scavenging

by 24-EBL than the seedlings treated with metal

alone. These observations are consistent with preceding

report that 24-EBL significantly lowered the salinity-

induced MDA content in rice seedlings (Ozdemir et al.

2004).

Free proline content

Proline is accumulated in many plant species under stress

and its accumulation is dependent on the expression of

enzymes (D1pyrroline-5-carboxylate synthase and proline

dehydrogenase), which catalyse the rate-limiting steps of

proline biosynthesis and degradation. In present investi-

gation, exogenous applications of 24-EBL alone had no

significant change in free proline content in radish seed-

lings as compared with untreated control (Fig. 7). Even

Fig. 6 Effect of 24-EBL on MDA content in 7-day-old Raphanussativus seedlings under Ni metal stress. Bar represents the SE

(n = 10). Different letters (a, b, c, d) within various concentrations of

Cr (0, 0.5, 1.0 and 1.5 mM) are significantly different (Holm-Sidak





MDA content

Fig. 7 Effect of 24-EBL on free proline content in 7-day-old

Raphanus sativus seedlings under Ni metal stress. Bar represents

the SE (n = 10). Different letters (a, b, c, d) within various

concentrations of Cr (0, 0.5, 1.0 and 1.5 mM) are significantly





with Cr on free proline content


123

though, the free proline content was observed to increase

under heavy metal stress which was further increased under

24-EBL presoaking treatments (Fig. 7). The increase in

free proline was maximum (4.189 l mol gFW-1) in 10-7

M 24-EBL alongwith 1.0 mM Ni as compared with radish

seedlings under 1.0 mM Ni stress alone (2.967 lmol gFW-1) as well as untreated control seedlings

(2.388 l mol gFW-1) (Fig. 7). This increase in proline

content may be attributed to the stimulation of D1pyrroline-

5-carboxylate synthase responsible for proline synthesis

under stressed conditions. Also, proline is a protective

osmolyte, membrane stabilizer and ROS scavenger

(Bandurska 2001; Hartzendorf and Rolletschek 2001). This

is in agreement with Fariduddin et al. (2009) who observed

an increase in proline content in B. juncea leaves under

both drought stress and 28-homobrassinolide treatments,

whereas their interaction had an additive effect on proline

accumulation.

Antioxidant enzyme activities

Heavy metal stress enhances ROS production and imbal-

ance between pro-oxidant and antioxidant system leads to

oxidative stress, thereby, altering the activities of antioxi-

dant enzymes (Sudo et al. 2008; Triantaphylides and

Havaux 2009). Recently, BRs had been reported to regulate

the activities of antioxidant enzymes to ameliorate various

biotic/abiotic stresses in plants (Bhardwaj et al. 2007;

Hayat et al. 2007; Arora et al. 2008; Bajguz and Hayat

2009). In present study it had been observed that presoa-

king treatments of 24-EBL ameliorated the Ni ion stress in

radish seedlings by regulating the activities of antioxidant

enzymes (Figs. 8, 9, 10, 11, 12, 13, 14). Nickel stress

significantly lowered the activities of CAT, DHAR, GR,

POD whereas it also significantly increased the activities of

APOX and SOD. A similar increase in protein content and

activities of antioxidant enzymes were observed under Ni

stress in Nasturtium officinale (Duman and Ozturk 2010).

However, the activities of antioxidant enzymes (APOX,

CAT, GR, DHAR, MDHAR and SOD) were significantly

enhanced with 24-EBL treatments except POD (Fig. 12)

under Ni stress.

Activities of ROS-scavenging-antioxidant enzymes SOD

act as first line of defence against ROS, dismutating O2- to

H2O2. Subsequently CAT, APOX, POD detoxify the H2O2

to H2O (Mittler 2002). In radish seedlings, APOX activity

got significantly increased with increase in the concentra-

tions of Ni ion in comparison to control (Fig. 8). Also,

APOX activity was observed to increase with increasing

24-EBL concentration. The activity of APOX was

observed maximum at 10-7 M 24-EBL alone (0.0942

activities per unit) when compared with untreated seedlings

(0.085 activities per unit). Seed presoaking treatment at a

concentration of 10-9 M of 24-EBL showed maximum

increment in APOX activity (0.159 activities per unit)

under 1.5 mM Ni stress in comparison to untreated stressed

seedlings (Fig. 8). An analogous trend was followed in

case of SOD activity (Fig. 14) and was observed maximum

at 1.5 mM Ni alone (7.578 activities per unit) with respect

to untreated radish seedlings (4.036 activities per unit). To

overcome Ni-stress, maximum activity of SOD was

Fig. 8 Effect of 24-EBL on specific activity in ascorbate peroxidase

(APOX) of 7-day-old Raphanus sativus seedlings under Ni metal

stress. Bar represents the SE (n = 10). Different letters (a, b, c,

d) within various concentrations of Cr (0, 0.5, 1.0 and 1.5 mM) are

significantly different (Holm-Sidak post hoc test, p B 0.05), whereas

different letters (p, q, r, s) within various treatments of 28-HBL (0,

10-11, 10-9 and 10-7 M) are significantly different (Holm-Sidak post

hoc test, p B 0.05) and signify interactions of different concentrations

28-HBL with Cr on specific activity of APOX


123

observed at 10-7 M 24-EBL in combination with 1.0 mM

Ni (11.334 activities per unit) as compared with 1.0 mM

Ni-treated plants (6.014 activities per unit) (Fig. 14).On the

contrary, the activities of hydrogen peroxide detoxifying

antioxidant enzymes such as CAT (Fig. 9) and POD

(Fig. 12) were decreased significantly with increasing Ni-

concentrations in radish seedlings. The activity of POD

remarkably decreased under the influence of 24-EBL as

compared with control. Minimum POD activity (0.985

activities per unit) was observed in seedlings treated with

1.5 mM Ni alone as compared with untreated seedlings

(1.574 activities per unit) (Fig. 12). Although in Ni-stress

the 24-EBL treatments were not able to elevate POD

activity significantly, On the other hand, the CAT activity

(Fig. 9) was enhanced significantly under 24-EBL presoa-

king treatments in comparison with control. CAT showed

maximum activity (57.608 activities per unit) in case of

seedlings treated with 10-9 M 24-EBL along with 1.0 mM

Ni as compared with 1.0 mM Ni-treated seedlings alone

(41.864 activities per unit) (Fig. 9). The present study is in

consistence with the studies carried out by Arora et al.

(2008); Hayat et al. (2007) and Hasan et al. (2008)

reporting the heavy metal stress amelioration by BRs in

Zea mays, Brassica juncea and Cicer arietinum seedlings

by increasing the activities of APOX, CAT, GR, POD and

SOD.

Fig. 9 Effect of 24-EBL on

specific activity in catalase

(CAT) of 7-day-old Raphanussativus seedlings under Ni metal

stress. Bar represents the SE

(n = 10). Different letters (a, b,

c, d) within various

concentrations of Cr (0, 0.5, 1.0

and 1.5 mM) are significantly

different (Holm-Sidak post hoc

test, p B 0.05), whereas

different letters (p, q, r,

s) within various treatments of

28-HBL (0, 10-11, 10-9 and

10-7 M) are significantly

different (Holm-Sidak post hoc

test, p B 0.05) and signify

interactions of different

concentrations 28-HBL with Cr

on specific activity of CAT

Fig. 10 Effect of 24-EBL on specific activity in dehydroascorbate

reductase (DHAR) of 7-day-old Raphanus sativus seedlings under Ni

metal stress. Bar represents the SE (n = 10). Different letters (a, b, c,






28-HBL with Cr on Specific activity of DHAR


123

Activities of antioxidant-regenerating-antioxidant enzymes

In plant cells, in addition to ROS-scavenging enzymes, the

antioxidant (ascorbate and glutathione) regenerating

enzymes function simultaneously to balance the uncon-

trolled redox reactions. APOX detoxify the H2O2 to H2O by

oxidizing ascorbate into monodehydroascorbate (MDHA),

which is reverted back to ascorbate by MDHAR (Mittler

2002). The monodehydroascorbate can be spontaneously

converted into dehydroascorbate (DHA) and DHAR is

required for ascorbate regeneration (Mittler 2002). In the

present investigation, DHAR and MDHAR showed

remarkably enhanced activities in Ni-stressed seedlings over

untreated seedlings (Figs. 10, 13, respectively). DHAR

(Fig. 10) and MDHAR (Fig. 13) activities were observed

minimum at 1.5 mM Ni (0.0152 and 0.247 activities per unit,

respectively) with respect to untreated seedlings (0.0281 and

Fig. 11 Effect of 24-EBL on specific activity in Glutathione

reductase (GR) of 7-day-old Raphanus sativus seedlings under Ni

metal stress. Bar represents the SE (n = 10). Different letters (a, b, c,






28-HBL with Cr on specific activity of GR

Fig. 12 Effect of 24-EBL on specific activity in Guaiacol peroxidase

(POD) of 7-day-old Raphanus sativus seedlings under Ni metal stress.

Bar represents the SE (n = 10). Different letters (a, b, c, d) within

various concentrations of Cr (0, 0.5, 1.0 and 1.5 mM) are significantly





with Cr on Specific activity of POD


123

0.347 activities per unit, respectively). Though under

24-EBL presoaking treatments alone, activity of MDHAR

(Fig. 13) did not reveal a considerable increase, but DHAR

activity (Fig. 10) was enhanced significantly as compared

with the control. However, 24-EBL treatments increased the

activities of both DHAR and MDHAR under Ni stress. These

enhanced activities of DHAR and MDHAR are inevitable for

the scavenging of overproduced ROS during Ni ion-induced

oxidative burst (Mittler 2002). During redox regulation and

to maintain a high GSH/GSSG ratio for the protection of cells

against photoinhibition, GR is prerequisite (Vyas et al.

2007). In the present report, Ni stress reduced the GR activity

and 24-EBL treatments alone elevated its activity (Fig. 11).

Minimum GR activity was observed at 1.5 mM Ni alone

(0.0089 activity per unit) as compared with untreated seed-

lings (0.0167 activities per unit). Further, 24-EBL soaking

treatments in combinations with Ni-stressed seedlings

enhanced GR activity as compared with Ni concentrations

Fig. 13 Effect of 24-EBL on specific activity in Monodehydroascor-

bate peroxidase (MDHAR) of 7-day-old Raphanus sativus seedlings

under Ni metal stress. Bar represents the SE (n = 10). Differentletters (a, b, c, d) within various concentrations of Cr (0, 0.5, 1.0 and

1.5 mM) are significantly different (Holm-Sidak post hoc test,

p B 0.05) whereas different letters (p, q, r, s) within various

treatments of 28-HBL (0, 10-11, 10-9 and 10-7 M) are significantly

different (Holm-Sidak post hoc test, p B 0.05) and signify interac-

tions of different concentrations 28-HBL with Cr on Specific activity

of MDHAR

Fig. 14 Effect of 24-EBL on specific activity in Superoxide dismu-

tase (SOD) of 7-day-old Raphanus sativus seedlings under Ni metal

stress. Bar represents the SE (n = 10). Different letters (a, b, c,






28-HBL with Cr on Specific activity of SOD


123

alone. Furthermore, 10-7 M 24-EBL treatments in combi-

nation with 1.0 mM Ni (0.0267 activities per unit) showed

maximum GR activity as compared with 1.0 mM Ni-treated

plants (0.0129 activities per unit) in radish seedlings. Hence,

24-EBL pretreatments might help in maintaining the novel

pool of glutathione in the reduced state by enhancing GR

activity under metal-stressed conditions.

Recently, Dhaubhadel and Krishna (2008) identified six

differentially expressed genes in 24-EBL-treated heat-

stressed Brassica napus seedlings. BRs have been specu-

lated to act via receptor/ligand complex that binds to nuclear

or cytoplasmic sites to regulate the expression of specific

stress-related genes. Stress-responsive genes activated by

BRs may code for the PCs, organic acids, osmolytes and

stress-protective proteins (LEA proteins and HSPs) (Gen-

dron and Wang 2007; McSteen and Zhao 2008). Also,

Bajguz (2002) reported that BRs acted as phytochelatin

stimulator in Chlorella vulgaris to ameliorate lead metal

toxicity. Thus, the present study specifies the effect of

24-EBL by analysing the behaviour of antioxidant enzymes

in heavy metal stress amelioration in a food crop (radish).

Further, detailed studies in validating the synergistic role of

BRs as well as other phytohormones under stress will play a

critical role in understanding the stress networking in plants.

Conclusions

The seed presoaking treatments of 24-EBL improved radish

seedling growth, germination, antioxidant enzyme activi-

ties, proline and MDA content under both stressed and non-

stressed conditions. Under Ni-stress, 24-EBL enhanced

antioxidant enzyme activities more significantly, suggesting

the amelioration of metal-induced ROS. Thus, 24-EBL

mediated Ni-stress amelioration might be mediated through

the modification of antioxidant defence system of plant.

Acknowledgments Financial assistance from Department of Sci-

ence and Technology (DST), Ministry of Science & Technology,

Government of India, New Delhi, India, is duly acknowledged.

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Effect of 24-epibrassinolide on oxidative stress markers induced by nickel-ion in Raphanus sativus L

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