LIST OF PUBLICATIONS A. RESEARCH PAPERSshodhganga.inflibnet.ac.in/bitstream/10603/10220/14/14...LIST OF PUBLICATIONS A. RESEARCH PAPERS 1. Sharma, ... jasmonates and BRs play a determinant

LIST OF PUBLICATIONS

A. RESEARCH PAPERS

1. Sharma, I., Pati, P.K. and Bhardwaj, R. (2010). Regulation of Growth and

Antioxidant Enzyme Activities by 28-Homobrassinolide in Seedlings of Raphanus

sativus L. under Cadmium stress. Indian Journal of Biochemistry and Biophysics. 47:

172-177.

2. Randhawa, N., Sharma, I. and Bhardwaj, R. (2010). Effect of 24-Epibrassinolide and

28-Homobrassinolide on some Biochemical Parameters in Raphanus sativus L.

Plants under Chromium Stress. Seed Science and Biotechnology. 4: 28-32.

3. Sharma, I., Pati, P.K. and Bhardwaj, R. (2011). Effect of 24-epibrassinolide on

oxidative stress markers induced by Nickel metal in Raphanus sativus L. Acta

Physiologae Plantarum. 33: 1723-1735.

4. Sharma, I., Pati, P.K. and Bhardwaj, R. (2011). Effect of 28- homobrassinolide on

antioxidant defence system in Raphanus sativus L. under metal stress. Ecotoxicology.

13: 862-874.

5. Sharma, N., Hundal, G. S., Sharma, I. and Bhardwaj, R. (2011). Effect of 24-

Epibrassinolide on Protein content and activities of Glutathione-S-transferase and

Polyphenol oxidase in Raphanus sativus L. plants under Cadmium and Mercury metal

stress. Terrestrial and Aquatic Environmental Toxicology. (In Press).

6. Sharma, I., Pati, P.K. and Bhardwaj, R. (2011). Induction of Mercury induced

Oxidative Stress Tolerance in Raphanus sativus L. by Brassinosteroids. Journal of

Hazardous Material. (Communicated)

B. REVIEW ARTICLES

1. Bhardwaj, R., Sharma, I., Arora N., Uppal, P., Kanwar, M.K., Sharma, N and

Hundal, G.S. (2009). Regulation of oxidative stress by BRs in plants. In: Oxidative

stress: Role of antioxidants in plants (editor: Parvez Ahmad), Studium Press Pvt.

Ltd., pp. 215-231, New Delhi.

2. Bhardwaj, R., Sharma, I., Arora N., Uppal, P., Kanwar, M.K. (2010). Prospects of

brassinosteroids in medicinal applications. In: Brassinosteroids: A Class of Plant

Hormone (editor: Shamsul Hayat), Springer, pp. 452-473, The Netherlands.

3. Bhardwaj, R., Sharma, I., Kanwar, M. and Handa, N. (2011). Brassinosteroids and

human welfare. In: Practical applications of Brassinosteroids (Eds. Adaucto B.

Pereira-Netto). Bentham Sci. Publishers, UAE. (In press)

4. Bhardwaj, R., Sharma, I., Choudhary, S.P., Uppal P., Kanwar M., Handa N.,

Singh, G., Gautam, V. and Kapoor D. (2011). Cross-talk of Brassinosteroids with

other PGR's in the plants. In: Festschrift book volume to be published in the honor of

Prof. C. P. Malik. (Eds. Pravin C. Trivedi), Jaipur. (In press)

C. ABSTRACTS

1. Sharma, I., Bhardwaj, R., Pati, P.K., Choudhary, S.P. and Sharma, N. (2008).

Brassinosteroids regulated Polyamine content and activities of antioxidative enzymes

in Raphanus sativus L. seedlings under Cr metal stress. In: Golden Jubilee

Conference on “Challenges and Emerging Strategies for Improving Plant

Productivity” organized by Indian Society for Plant Physiology, Indian Agricultural

Research Institute (IARI), New Delhi. pp. 12-14.

2. Sharma, I., Arora, P., Kanwar, M.K., Pati, P.K. and Bhardwaj, R. (2009).

Regulation of Growth and Antioxidant Enzyme activities by 28 – Homobrassinolide

in seedlings of Raphanus sativus L. under cadmium stress. In: Proceedings of

International Conferences on AFRR 2009 & SFRR-India Meeting, Lucknow. pp. 72.

(Paper presentation, received Travel Grant Award)

3. Sharma, I., Pati, P.K. and Bhardwaj, R. (2010). Mitigation of Nickel metal stress in

Raphanus sativus L. seedlings by 24-Epibrassinolide. In: Proceedings of 13th Punjab

Science Congress, Panjab University, Chandigarh.

4. Sharma, I., Pati, P.K. and Bhardwaj, R. (2011). 28-Homobrassinolide mediated

amelioration of stress induced by Mercury in Raphanus sativus L. In: Proceedings of

National Seminar on Environmental Analysis and Remediation, Amritsar. pp. 102-

103.

Effect of 28-homobrassinolide on antioxidant defence system

in Raphanus sativus L. under chromium toxicity

Indu Sharma • Pratap Kumar Pati •

Renu Bhardwaj

Accepted: 14 March 2011 / Published online: 30 March 2011

Ó Springer Science+Business Media, LLC 2011

Abstract Heavy metals have emerged as major envi-

ronmental contaminants due to rapid industrialization and

urbanization. The genotoxic, mutagenic and carcinogenic

effects of heavy metal like chromium (Cr) on man, animals

and plants have been documented. In plants, accumulation

of heavy metals beyond critical levels generates oxidative

stress. This stress is generally overcome by antioxidant

defence system and stress shielding phytohormones. Thus,

the present study has been focused to analyze the effect of

one of imperative group of plant hormones, i.e., brassi-

nosteroids (BRs) which have been reported for its protec-

tive properties for wide array of environmental stresses.

Raphanus sativus L. (Pusa Chetaki) seeds pre-treated with

different concentrations of 28-homobrassinolide (28-HBL)

were raised under various concentrations of Cr(VI). It was

observed that 28-HBL treatment considerably reduced the

impact of Cr-stress on seedlings which was evinced upon

analysis of morphological and biochemical parameters of

7-days old radish seedlings. The toxic effects of Cr in terms

of reduced growth, lowered contents of chlorophyll (Chl),

protein, proline; increased malondialdehyde (MDA) con-

tent and elevated metal uptake were ameliorated by

applications of 28-HBL. Also, the activities of all the

antioxidant enzymes except guaiacol peroxidase (POD),

increased significantly when subjected to Cr stress in

combination with 28-HBL. Overall, seed pre-soaking

treatment of 28-HBL at 10-7 M was most effective in

ameliorating Cr stress. The present work emphasizes the

protective role of 28-HBL on regulation of antioxidant

enzymes and its possible link in amelioration of stress in

plants.

Keywords Brassinosteroids � Antioxidant enzymes �

Radish, metal stress � Lipid peroxidation � Proline content

Introduction

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). 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). Among heavy metals, Cr is widely dis-

tributed in nature because of their multifarious use in lea-

ther, refractory steel, drilling muds and electroplating

industries. 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 tri-

valent Cr(III) and hexavalent Cr(VI) species. Cr(VI) usu-

ally occurs associated with oxygen as chromate (CrO42-)

or dichromate (Cr2O72-) oxyanions. Cr(VI) is reported to

be more mobile and toxic compared to Cr(III) species (Yu

et al. 2007). Besides, Cr(VI) is a strong oxidant with high

I. Sharma � R. Bhardwaj (&)

Department of Botanical and Environmental Sciences, Guru

Nanak Dev University, Amritsar, Punjab 143005, India

e-mail: [email protected];

[email protected]

I. Sharma

e-mail: [email protected]

P. K. Pati

Department of Biotechnology, Guru Nanak Dev University,

Amritsar, Punjab 143005, India


123

Ecotoxicology (2011) 20:862–874

DOI 10.1007/s10646-011-0650-0

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). The ROS are

highly toxic and can oxidize biological macromolecules

such as nucleic acids, proteins and lipids, thereby disturb-

ing the membrane permeability (Schutzendubel and Polle

2002; Sudo et al. 2008). Thus, ROS accumulation leads to

imbalance in pro-oxidative and antioxidative defence sys-

tem resulting in oxidative stress.

Several plant hormones like abscisic acid (ABA), eth-

ylene, jasmonates and BRs play a determinant role in

implicating oxidative stress. However, BRs are unique in

their activities for not only regulating the diverse physio-

logical and morphogenetic responses in plants but also

having a significant role in amelioration of various biotic

and abiotic stresses at nanomolar to micromolar concen-

trations (Krishna 2003; Clouse and Sasse 1998). Recently

28-HBL (10-6–10-11 M) has been reported to combat salt

and heavy metals stress in Brassica juncea, Raphanus

sativus, Triticum aestivum and Zea mays (Bhardwaj et al.

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

et al. 2010). Furthermore, 28-HBL is also observed to

regulate the activities of various enzymes involved in

photosynthesis and plant defence in wheat and Indian

mustard exposed to different abiotic stresses (Hayat et al.

2007; Yusuf et al. 2010). Since BRs control several agro-

nomic traits in plants, therefore their potential in phyto-

remediation ‘‘basically a modified agronomic practice’’ is a

contentious issue from ecological viewpoint (Barbafieri

and Tassi 2011). However, development of efficient phy-

toremediation methods by increasing the efficiency of

uptake of metals/pollutants, species adaptation to particular

ecological niche requires rational scientific study. The

toxicological effects of heavy metals in plants are mediated

at several levels by different factors that also depend on

plant’s genotypic and phenotypic constitution. A number of

plant species, like B. juncea, R. sativus etc. in Brassicaceae

are known as hyperaccumulators, have adapted to envi-

ronmental stresses.

In the seeds of R. sativus, four types of BRs namely cas-

tasterone, brassinolide, 28-homoteasterone and teasterone

have been identified (Schmidt et al. 1991, 1993). In view of

the above as well as the wide occurrence, economic impor-

tance and phytoremediation perspectives of R. sativus, it is

much warranted to explore the possible role of BRs for

ameliorating specific stress in this plant. Thus, the present

study first time reports the effects of 28-HBL on morpho-

logical parameters, chlorophyll content, Cr(VI) metal

uptake, determination of superoxide anion production rate,

protein content, proline content and antioxidant enzymes

like superoxide dismutase (SOD), guaiacol peroxidase

(POD), catalase (CAT), dehydroascorbate reductase

(DHAR), monodehydroascorbate reductase (MDHAR),

glutathione reductase (GR) and ascorbate peroxidase

(APOX) and lipid peroxidation in R. sativus L. (Pusa Che-

taki) seedlings under Cr metal stress.

Materials and methods

Plant material and growth conditions

Seeds of R. sativus L. (Pusa Chetaki) were procured from

Department of Plant Breeding, Punjab Agriculture Univer-

sity, Ludhiana, India. Seeds were surface sterilized with

0.4% sodium hypochlorite for 15 min followed by repeated

rinses in sterile distilled water. Surface sterilized seeds were

given 8-h presoaking treatment in different concentrations of

28-HBL (0, 10-11, 10-9 and 10-7M). These pretreated seeds

were germinated on Whatman No. 1 filter paper lined auto-

claved glass Petri dishes (10 cm diameter, 20 seeds/dish)

each containing 4.0 ml of different concentrations of Cr (0,

0.5, 1.0 and 1.5 mM).TheCr (CrVI)was given in the formof

K2CrO4. The Petri dishes were sealed with parafilm to avoid

the desiccation and evaporation of solutions. However,

2–3 ml test solutions were added on 4th day of the experi-

ment. The experiment was conducted under controlled

conditions of light (16 h photoperiod under fluorescentwhite

light with 175 lmol m-2 s-1 intensity), temperature

(25 ± 5°C) and relative humidity (80–90%). Experiment

was repeated twice with five replicates for each treatment.

Growth analysis

On the last day of the experiment (that is, day 7 after

sowing the seeds including germination time of seedlings),

seedlings were harvested followed by separation of roots

and shoots. Seedling growth in terms of root and shoot

length was recorded. Twenty seedlings per petridish were

used for the determination of morphological parameters

(root/shoot length).

Cr(VI) uptake

Freshly harvested leaves were oven dried at 80°C for 24 h

and then ground to a fine powder. For analysis of metal

uptake dried leaf powder/sample (0.5 g) was acid digested

in H2SO4:HNO3:HClO4 (1:5:1) digestion mixture as

described by Allen et al. (1976). These digests were diluted

with double distilled water, fine filtered through Whatman

No. 1 filter paper and further diluted to make final volume

to 10 ml. The colorimetric method for analyzing the uptake

of Cr(VI) ions spectrophotometrically was followed after

Eaton et al. (1995). From 10 ml solution, 1 ml solution was

taken and diluted 50 times with 2 N H2SO4 (to adjust

Effect of 28-homobrassinolide on antioxidant defence system 863

123

pH = 1.0 ± 0.3). After 20 min, 1 ml Diphenyl carbazide

solution was added and absorbance of pink color end

product was observed at 540 nm. From the standard curve,

the uptake of Cr(VI) was calculated and expressed in terms

of mg g-1 dry biomass of leaf.

Biochemical analysis

To conduct biochemical analysis, the cotyledonary leaves

of 7-days old radish seedlings were harvested. For esti-

mation of the activities of antioxidant enzymes and the

protein content, leaf extracts were prepared by homoge-

nizing 2 g leaves in chilled 6 ml 50 mM phosphate buffer

(pH 7.0), 1 mM ethylenediaminetetraacetic acid (EDTA),

1 mM phenylmethanesulfonylfluoride (PMSF), 0.5% (v/v)

Triton X-100 and 2% (w/v) PVP-30 in a pre-chilled mortar

and pestle. In case of analysis of ascorbate peroxidase and

dehydroascorbate reductase activity, 0.5 mM ascorbate

was added to the extraction buffer. In case of monodehy-

droascorbate reductase activity, 1 g leaves were homoge-

nized in 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 fur-

ther used for biochemical analysis of antioxidant enzymes

and proteins.

Chlorophyll content

Chlorophyll (Chl) content was quantified by following the

method of Arnon (1949). Freshly harvested leaves (0.2 g)

were homogenized in 3.0 ml of 80% acetone followed by

its centrifugation at 12,0009g for 15 min. Absorbance of

supernatant was observed at 645 and 663 nm against 80%

acetone as blank. The contents of total Chl, Chl A and Chl

B were calculated in terms of lg ml-1 as described by

Arnon (1949).

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 gram shoots were

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-1 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 cotyledonary leaves were homogenized in 3.5 ml of

3% sulphosalicylic acid and the homogenates were cen-

trifuged at 12,0009g for 10 min. Then, 2 ml of supernatant

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 reac-

tion 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 tem-

perature. The absorbance of red colored 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.

Determination of superoxide anion

The superoxide anion production rate was determined

spectrophotometrically by monitoring the nitrite formation

from hydroxylamine in the presence of superoxide anion

(O2•-) as described by Wu et al. (2010) with some modi-

fications. Fresh 1 g cotyledonary leaves were homogenized

in liquid N2 in pre-chilled pestle and mortar. Then, 3 ml of

50 mM phosphate buffer (pH 7.8) containing 2% (w/v)

polyvinylpyrrolidone (PVP-30) and 0.5% (v/v) Triton

X-100 was added to the homogenized tissue followed by its

centrifugation at 12,0009g for 15 min at 4°C. The super-

natant (0.5 ml) was mixed with 0.5 ml of 50 mM phos-

phate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine

hydrochloride, and then incubated at 25°C for 30 min. The

incubated solution (1 ml) was added to 1 ml of 58 mM

3-aminobenzenesulphonic acid and 1 ml of 7 mM

1-naphthylamine, and then further kept at 25°C. After

20 min, absorbance was recorded at 530 nm. To calculate

the rate of O2•- production from the reaction of O2

•- with

hydroxylamine a standard curve was used. The O2•- pro-

duction rate was expressed as nmol min-1 g -1 FW.

Protein quantification

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

and 1.5 mM) and 28-HBL (10-7, 10-9 and 10-11 M) alone

or in combinations, was quantified by following the method

of Bradford (1976) using bovine serum albumin as a

standard.

864 I. Sharma et al.

123

Antioxidant enzyme activities

Ascorbate peroxidase assay The ascorbate peroxidase

(APOX, EC 1.11.1.11) activity was determined spectro-

photometrically 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 disappear-

ance using the method of Aebi (1984). The 3.0 ml reaction

mixture contained 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 density 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.

Dehydroascorbate reductase assay Dehydroascorbate

reductase (DHAR, EC 1.8.5.1) activity was measured fol-

lowing 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 dehydroascorbate,

0.1 mM EDTA, 2.5 mM reduced glutathione (GSH) and

100 ll enzyme extract. The 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 Mannervik (1975). The 3.0 ml reaction

mixture contained 50 mM potassium phosphate buffer (pH

7.6), 1 mM oxidized glutathione (GSSG), 0.5 mM EDTA,

0.1 mM reduced nicotinamideadenine dinucleotidephos-

phate (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. The POD activity was determined

by measuring the absorbance 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 catalyzing

the oxidation of 1 lmol of guaiacol in 1 min.

Monodehydroascorbate reductase assay Monodehydro-

ascorbate reductase (MDHAR, EC 1.6.5.4) activity was

assayed using the method of Hossain et al. (1984). The

3.0 ml reaction mixture contained 50 mM Tris–HCl (pH

7.6) containing 2.5 mM Ascorbic acid, 0.1 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 is 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 coeffi-

cient 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 reduc-

tion 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

Five replicates (each containing 20 seedlings) for each

treatment were maintained. All data were subjected to two-

way analysis of variance (ANOVA) for scrutinizing inter-

actions of Cr and 28-HBL and expressed as the

mean ± standard error of five replicates. The Holm–Sidak

post hoc test was applied for the multiple comparisons

versus control using SigmaStat Version 3.5 and signifi-

cance of difference between the Cr-stress and 28-HBL

treatments was set at p B 0.05.

Results

Morphological parameters

Chromium stress imposition resulted in decreased root and

shoot length of radish seedlings (Table 1). About 4.9 times

(11.3–2.3 cm) and 2.82 times (5.967–2.117 cm) decrease

was observed at 1.5 mM Cr stress in root length and shoot

length respectively. Application of 28-HBL (10-11, 10-9


123

and 10-7 M) alone had significantly improved their root

and shoot lengths (Table 1). Further, supplementation of Cr

solutions with 28-HBL considerably reduced the inhibitory

effect of Cr on seedling length. Root length was observed

to be improved significantly by the application of 10-7 M

(16.22 cm), 10-9 M (15.37 cm) and 10-11 M (15.2 cm)

28-HBL alone (Table 1). When 10-7 M 28-HBL supple-

mented with 0.5 mM Cr solution, root length (15.65 cm)

was enhanced as compared to control (seedlings treated

with 0.5 mM Cr alone) (6.64 cm) (Table 1). A similar

trend was found when effect of 28-HBL was studied on

shoot length of the seedlings (Table 1). It was observed

that irrespective of the concentrations of 28-HBL, it has

promoting effect on root and shoot length of radish seed-

lings under Cr stress. Overall, 10-7 M 28-HBL application

was most effective in enhancing growth of seedling treated

with Cr(VI).

Uptake of Cr(VI) ions

To understand effect of 28-HBL on Cr accumulation in

radish shoots, the uptake of Cr(VI) ions was analyzed.

Observations on uptake of Cr(VI) metal ions in Cr-treated

radish seedlings revealed a significant increase in leaves

(Table 1). The content of Cr(VI) ions was highest

(0.398 mg g-1 dry matter) in 1.0 mM Cr stressed radish

leaves. But, seed-presoaking treatment of 28-HBL alone

had no significant effect on Cr(VI) uptake in comparison to

control. However, these treatments significantly lowered

uptake of Cr ions in stressed seedlings (Table 1). About

sixfolds (0.286 to 0.045 mg g-1 dry matter) decrease in

Cr(VI) uptake was observed at 0.5 mM Cr by 10-7 M

28-HBL than 0.5 mM Cr alone. Similarly, same treatment

was effective in lowering Cr(VI) ions uptake to approxi-

mately 2 times in 1.0 and 1.5 mM Cr stressed radish

seedlings. Overall among different treatments of 28-HBL,

10-7 M concentration showed maximum decrease in

Cr(VI) ions uptake in Cr treated radish leaves.

Biochemical parameters

The studies carried out on the biochemical parameters

(Tables 2, 4, 5, 6) in radish shoots revealed the consider-

able effects of 28-HBL treatments. The analyses of rate of

superoxide radical formation, activities of antioxidant

enzymes as well as contents of chlorophyll, protein and

MDA, revealed that total protein content, chlorophyll

contents and activities of antioxidant enzymes significantly

increased with increasing concentrations of 28-HBL except

for the activity of POD under Cr metal stress. Although

treatment of 28-HBL alone could not increase free proline

content significantly but 28-HBL under Cr-stress signifi-

cantly enhanced free proline content (Table 3). However, aTable

1Effectof28-H

BLonrootlength,shootlength

andCr(VI)

ionsuptakein

7-daysold

R.sativusseedlingsunder

Crstress

Rootlength

(cm)at

varioustreatm

ents

of28-H

BL

Shootlength

(cm)at

varioustreatm

ents

of28-H

BL

Cr(VI)

uptake(m

g-1g-1dry

weight)at

varioustreatm

ents

of28-H

BL

0M

10-11M

10-9M

10-7M

0M

10-11M

10-9M

10-7M

0M

10-11M

10-9M

10-7M

0mM

Cr

11.3

±0.601a,p

15.37

±0.426a,q

15.2

±0.295a,q

16.22

±0.590a,q

5.967

±0.219a,p

6.28

±0.099a,p

6.443

±0.170a,p

6.873

±0.233a,p

0.000

±0.000a,p

0.0001

±0.0021a,p

0.0007

±0.009a,p

0.0001

±0.007a,p

0.5

mM

Cr

6.64

±0.322b,p

9.68

±0.180b,q

11.87

±0.560b,r

15.65

±0.381b,s

4.5 ±0.153b,p

5.6 ±0.115b,q

4.933

±0.203b,r

6.467

±0.067b,s

0.045

±0.002b,p

0.151

±0.0018b,qr

0.129

±0.006b,r

0.286

±0.008b,r

1.0

mM

Cr

6.333

±0.580b,p

8.79

±0.120bc,q

10.24

±0.373c,r

14.31

±0.960b,s

3.233

±0.088c,p

5.533

±0.012b,q

4.567

±0.004b,r

5.467

±0.600b,s

0.221

±0.008c,p

0.215

±0.012c,q

0.289

±0.0073c,s

0.398

±0.0096c,s

1.5

mM

Cr

2.3

±0.270c,p

6.78

±0.203c,q

9.73

±0.279c,r

13.42

±0.219c,s

2.117

±0.113d,p

3.6 ±0.058c,q

4.067

±0.016b,q

5.367

±0.007c,r

0.175

±0.0027d,p

0.136

±0.003d,qs

0.213

±0.007c,s

0.204

±0.0019c,rs

Datapresentedis

mean

±SE.Differentletters(a,b,c,d)within

acolumnaresignificantlydifferent(H

olm

–Sidakposthoctest,pB

0.05)whereasdifferentletters(p,q,r,s)within

arowforasingleparam

eter

aresignificantlydifferent(H

olm

–

Sidakpost

hoctest,pB

0.05)andsignifyinteractionsofdifferentconcentrations28-H

BLwithCronrootlength,shootlength

andCr(VI)

ionsuptakerespectively


123

Table 2 Effect of 28-HBL on contents of total Chlorophyll, Chlorophyll A and Chlorophyll B in 7-days old R. sativus seedlings under Cr stress

Total chlorophyll content (lg g-1 fresh weight) at various

treatments of 28-HBL

Chlorophyll A content (lg g-1 fresh weight) at various


Chlorophyll B content (lg g-1 fresh weight) at various


0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M

0 mM Cr 278.14

± 11.219a,p276.77

± 14.099a,p257.51

± 19.170a,p285.81

± 21.23ab,p142.18

± 4.000a,p134.99

± 3.132a,p157.88

± 3.065a,p162.93

± 2.771a,p135.96

± 3.19a,p141.78

± 9.99a,q99.63

± 10.17a,r122.88

± 11.23a,s

0.5 mM Cr 271.99

± 15.300a,p263.78

± 12.390a,p297.19

± 17.21bc,r274.85

± 16.70b,pr152.18

± 5.333a,p144.79

± 4.789a,p157.88

± 4.111a,p159.99

± 3.786a,s132.77

± 15.30a,p131.79

± 11.50b,p89.46

± 9.203a,r111.65

± 6.70ab,s

1.0 mM Cr 216.75

± 18.890b,p337.73

± 12.99b,q331.89

± 13.77c,qr356.07

± 16.23c,s132.69

± 3.580b,p194.79

± 3.120b,qs199.68

± 3.730c,qrs189.99

± 2.960b,s117.47

± 8.80b,p120.29

± 9.12c,pq80.26

± 13.70b,r104.95

± 16.30b,s

1.5 mM Cr 187.95

± 14.113b,cp198.64

± 17.89c,pr219.97

± 16.77d,r241.77

± 13.79bd,r102.69

± 1.270c,p124.79

± 2.030ac,qr126.67

± 2.700d,r119.99

± 2.190c,r108.67

± 11.13b,p111.17

± 12.8c,pq66.56

± 14.60c,r88.65

± 11.71c,s

Data presented is mean ± SE. Different letters (a, b, c, d) within a column are significantly different (Holm–Sidak post hoc test, p B 0.05) whereas different letters (p, q, r, s) within a row for a single parameter are

significantly different (Holm–Sidak post hoc test, p B 0.05) and signify interactions of different concentrations 28-HBL with Cr on contents of total Chlorophyll, Chlorophyll A and Chlorophyll B respectively

Table 3 Effect of 28-HBL on contents of free proline, MDA and protein in 7-days old R. sativus seedlings under Cr stress

Free proline content (lmol g-1 fresh weight) at various


MDA content (lmol g-1 fresh weight) at various


Protein content (mg g-1 fresh weight) at various


0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M

0 mM Cr 2.249

± 0.237a,p2.279

± 0.339a,p2.247

± 0.281a,p2.334

± 0.114a,p4.984

± 0.271a,p4.799

± 0.785a,q4.587

± 0.244a,q4.349

± 0.433a,r19.49

± 0.797a,p14.90

± 1.510a,q16.52

± 3.420a,r22.36

± 3.130a,s

0.5 mM Cr 2.457

± 0.081b,p2.877

± 0.233b,q3.956

± 0.184b,r3.987

± 0.277b,r5.333

± 0.567b,p4.789

± 0.933a,q4.111

± 0.311b,r3.786

± 0.114b,s9.94

± 1.100b,p14.52

± 1.370a,q14.82

± 0.901b,q20.19

± 0.704a,q

1.0 mM Cr 2.788

± 0.197c,p2.983

± 0.171b,p4.071

± 0.157b,q4.001

± 0.133b,q5.788

± 0.733c,p4.333

± 0.111b,q3.987

± 0.293b,r3.644

± 0.189b,r9.79

± 0.420b,p16.43

± 2.472b,q17.74

± 1.732c,q15.12

± 0.399b,r

1.5 mM Cr 3.499

± 0.283d,p3.119

± 0.177b,q4.117

± 0.179b,r4.284

± 0.277b,r6.241

± 0.233d,p4.833

± 0.067a,q4.321

± 0.289bc,r4.011

± 0.242bc,r9.14

± 2.570b,p12.02

± 0.400b,q19.46

± 2.912d,r16.35

± 1.094b,s

Data presented is mean ± SE. Different letters (a, b, c, d) within a column are significantly different (Holm–Sidak post hoc test, p B 0.05) whereas different letters (p, q, r, s) within a row for a

single parameter are significantly different (Holm–Sidak post hoc test, p B 0.05) and signify interactions of different concentrations 28-HBL with Cr on contents of free proline, MDA and

protein respectively

Effect

of28-homobrassin

olid

eonantio

xidantdefen

cesystem

867

123

Table 4 Effect of 28-HBL on superoxide anion production rate, specific activities of SOD and CAT in 7-days old R. sativus seedlings under Cr stress

Superoxide anion production rate (nmol min-1 g-1 fresh weight)

at various treatments of 28-HBL

Specific activity of SOD (mol UA mg fresh protein-1)


Specific activity of CAT (mol UA mg fresh protein-1)


0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M

0 mM Cr 7.999

± 0.399a,p7.129

± 0.899a,q7.899

± 0.977a,pqr8.999

± 1.333a,s4.400

± 0.189a,p5.602

± 0.055a,q5.490

± 0.094a,q3.814

± 0.530a,q54.46

± 4.000a,p39.82

± 3.132a,q42.16

± 3.065a,q41.73

± 0.370a,q

0.5 mM Cr 8.599

± 0.257a,p9.667

± 0.957b,q10.999

± 0.703b,r9.467

± 0.999a,qs8.690

± 0.087b,p9.870

± 0.123b,q11.376

± 0.0048b,r12.169

± 0.312b,r31.29

± 1.420b,p44.46

± 3.180b,q57.35

± 2.760b,r62.38

± 1.780b,s

1.0 mM Cr 11.199

± 1.577b,p11.537

± 0.999c,pq12.367

± 0.378c,r15.367

± 0.699c,s8.720

± 0.360b,p9.210

± 0.090b,pq9.760

± 0.210c,q10.330

± 0.390c,r38.76

± 1.540c,p42.21

± 1.765ab,p49.73

± 1.450a,q59.61

± 2.030bc,r

1.5 mM Cr 12.267

± 0.967bc,p13.699

± 1.367d,q15.167

± 0.667d,r14.367

± 0.277c,qrs2.590

± 0.390c,p4.553

± 0.760c,qr4.857

± 0.180d,rs4.027

± 0.300d,s41.12

± 1.300c,p40.24

± 1.680a,rp43.32

± 1.320c,prs45.18

± 1.817d,qs


significantly different (Holm–Sidak post hoc test, p B 0.05) and signify interactions of different concentrations 28-HBL with Cr on superoxide anion production rate, specific activity of SOD and CAT respectively

Table 5 Effect of 28-HBL on specific activities of GR, APOX and POD in 7-days old R. sativus seedlings under Cr stress

Specific activity of GR (mol UA mg fresh protein-1)


Specific activity of APOX (mol UA mg fresh protein-1)


Specific activity of POD (mol UA mg fresh protein-1)


0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M 0 M 10-11 M 10-9 M 10-7 M

0 mM Cr 0.012

± 0.001a,p0.002

± 0.002a,q0.003

± 0.0001a,q0.002

± 0.0005a,q0.044

± 0.004a,p0.041

± 0.0012a,p0.035

± 0.0001a,rq0.037

± 0.0003a,r0.280

± 0.002a,p0.081

± 0.004a,q0.059

± 0.011a,r0.059

± 0.0025a,r

0.5 mM Cr 0.001

± 0.0001b,p0.019

± 0.0001b,q0.024

± 0.001b,r0.037

± 0.0007b,s0.003

± 0.0001b,p0.018

± 0.0001b,q0.098

± 0.0004b,r0.116

± 0.0008b,s0.330

± 0.013b,p0.047

± 0.002b,q0.049

± 0.002ab,q0.280

± 0.008b,r

1.0 mM Cr 0.002

± 0.0001b,p0.0179

± 0.0001b,q0.0201

± 0.0007c,r0.035

± 0.0009b,s0.005

± 0.0001b,p0.017

± 0.0001b,q0.092

± 0.005b,r0.115

± 0.0018b,s0.244

± 0.012c,p0.064

± 0.004c,q0.039

± 0.007bc,r0.200

± 0.015c,s

1.5 mM Cr 0.001

± 0.0001b,p0.025

± 0.0012c,q0.021

± 0.0008c,r0.023

± 0.0011c,s0.007

± 0.0000b,p0.033

± 0.0013c,q0.015

± 0.0001c,r0.011

± 0.0002c,rs0.159

± 0.006d,p0.063

± 0.008c,q0.034

± 0.009c,q0.062

± 0.018d,q


significantly different (Holm–Sidak post hoc test, p B 0.05) and signify interactions of different concentrations 28-HBL with Cr on specific activities of GR, APOX and POD respectively

868

I.Sharm

aet

al.

123

remarkable reduction in content of MDA (Table 3) and rate

of superoxide radical formation (Table 4) was observed in

28-HBL treated seedlings grown under Cr toxicity.

Chlorophyll content

Phytotoxicity of Cr(VI) ions is due to its high mobility

across biological membranes and its interference with

electron transport in respiration and photosynthesis (Yu

et al. 2007). Thus, to determine toxicity of Cr(VI) ions,

chlorophyll (Chl) content was measured. The toxicity

induced by Cr resulted in significant decrease in contents of

total chlorophyll (Chl), Chl A and B (Table 2). About 1.5

times (from 278.18 to 187.95 lg g-1 FW) decrease in total

Chl content was decreased in 1.5 mM Cr stress than control

(Table 2). Similarly, 1.4 and 1.25 folds decline in contents

of Chl A (Table 2) and B (Table 2) was observed under

1.5 mM Cr treated seedlings than untreated. Seed presoa-

king treatments of 28-HBL alone had no significant effect

on total Chl and Chl A content. However, treatments of

28-HBL alone had significant enhancing effect on content

of Chl B (Table 2). Furthermore, 28-HBL treatment had

significantly increased Chl A and total Chl content in Cr

metal treated seedlings. Seed presoaking treatments at 10-7

M 28-HBL had resulted in 1.7 times increase in total Chl

content in 1.0 mM Cr stressed seedlings in comparison to

1.0 mM Cr alone (Table 2). A similar increasing effect of

28-HBL treatment was followed in Chl A content. While

28-HBL treatments significantly decreased the contents of

Chl B as compared to control.

Free proline content

The free proline content (Table 3) was observed to increase

significantly in radish seedlings grown under Cr toxicity

(2.457–3.499 lmol g-1 FW) when compared to control

(2.249 lmol g-1 FW) (Table 3). However, applications of

28-HBL alone could vaguely enhance free proline contents

and maximum enhancement at 10-7 M concentration

(2.344 lmol g-1 FW) as compared to untreated seedlings

(2.249 lmol g-1 FW) (Table 3). But seeds pretreated with

28-HBL under Cr metal stress, significantly enhanced free

proline levels than only Cr metal treated radish seedlings

(Table 3). 10-7 M of 28-HBL significantly increased pro-

line levels under Cr stress, with 1.224-fold increase at

1.5 mM Cr in conjunction with 10-7 M 28-HBL

(4.284 lmol g-1 FW) as compared to 1.5 mM Cr treat-

ment alone (3.499 lmol g-1 FW).

Lipid peroxidation

Chromium metal stress significantly enhanced the MDA

content (5.333–6.421 lmol g-1 FW) as compared toTable

6Effectof28-H

BLonspecificactivitiesofDHARandMDHARin

7-daysold

R.sativusseedlingsunder

Crstress

SpecificactivityofDHAR

(molUA

mgfreshprotein-1)at

varioustreatm

ents

of28-H

BL

SpecificactivityofMDHAR

(molUA

mgfreshprotein-1)at

varioustreatm

ents

of28-H

BL

0M

10-11M

10-9M

10-7M

0M

10-11M

10-9M

10-7M

0mM

Cr

0.0387±

0.003a,p

0.0321±

0.002a,q

0.0397±

0.0027a,r

0.041±

0.0029a,r

0.377±

0.033a,p

0.372±

0.043a,p

0.367±

0.013a,p

0.383±

0.019a,p

0.5

mM

Cr

0.0344±

0.0013b,p

0.0256±

0.0015b,q

0.0323±

0.002b,r

0.0392±

0.0028a,s

0.327±

0.017b,p

0.357±

0.019ba,q

0.379±

0.025ab,r

0.407±

0.018ab,s

1.0

mM

Cr

0.0367±

0.0012b,p

0.0287±

0.004b,q

0.0396±

0.0047c,r

0.0421±

0.0015a,s

0.254±

0.028c,p

0.334±

0.037bc,q

0.389±

0.041a,r

0.423±

0.026b,s

1.5

mM

Cr

0.0247±

0.006c,p

0.0264±

0.0048b,q

0.0347±

0.0064b,r

0.0407±

0.0018a,s

0.237±

0.019c,p

0.317±

0.037c,q

0.351±

0.029b,r

0.389±

0.029a,s

Datapresentedismean±

SE.Differentletters(a,b,c,d)within

acolumnaresignificantlydifferent(H

olm

–Sidakposthoctest,pB

0.05)whereasdifferentletters(p,q,r,s)within

arowfora

single

param

eter

aresignificantlydifferent(H

olm

–Sidakpost

hoctest,pB

0.05)andsignifyinteractionsofdifferentconcentrations28-H

BLwithCronspecificactivitiesofDHAR

and

MDHAR

respectively


123

control (4.984 lmol g-1 FW) in radish seedlings

(Table 3). The maximum increase in MDA content was

observed at 1.5 mM Cr (6.421 lmol g-1 FW) and this

increase in MDA content was further reduced significantly

by 28-HBL presoaking treatments (Table 3). The maxi-

mum reduction in MDA content was recorded in seedlings

treated with 10-7 M 28-HBL under 1.0 mM Cr metal stress

(3.644 lmol g-1 FW) (Table 3). It was also observed that

seedlings treated with 28-HBL alone could lower MDA

levels than control (Table 3). The most effective 28-HBL

concentration which reduced MDA level to the lowest level

was 10-7 M (4.349 lmol g-1 FW) (Table 3).

Total protein content

The observations on Cr stressed shoots revealed that pro-

tein content decreased with increased concentrations of Cr

metal (Table 3). Minimal protein content was observed in

case of shoots treated with 1.5 mM Cr (9.14 mg g-1 FW)

as compared to control (19.49 mg g-1 FW), where only

distilled water was given (Table 3). Shoots treated with

28-HBL alone showed significant increase in soluble pro-

tein content (Table 3) in comparison to untreated shoots.

The treatment of shoots with 10-7 M of 28-HBL resulted in

significantly enhanced protein content (22.36 mg g-1 FW)

when compared to the control (19.49 mg g-1 FW). The

protein content (Table 3) was significantly higher in the

shoots treated with Cr along with 28-HBL than Cr alone.

Superoxide (O2•-) anion production

Cr-induced oxidative stress is mediated via generation,

accumulation and imbalance of a steady-state level of ROS

in stressed plants. To analyze effect of 28-HBL in amelio-

ration of oxidative stress, the generation of O2•- anion was

determined (Table 4). In radish shoots, the production of

O2•- anion increased about 1.53 and 1.4 times respectively

under 1.5 and 1.0 mM Cr stress than control. Moreover,

28-HBL treatments alone had significantly increased O2•-

content as compared to control. Furthermore, 28-HBL had

also remarkably enhanced O2•- anion production under Cr-

metal toxicity. Seed presoaking 28-HBL treatment at 10-7M

had resulted in 1.37 folds (11.199–15.367 nmol min-1 g-1

FW) and 1.92 folds (7.999–15.367 nmol min-1 g-1 FW)

increase in O2•- production in 1.0 mM Cr when compared to

1.0 mM Cr alone and control respectively. Overall, 10-7 M

28-HBL treatment was most effective in enhancing the

generation of O2•- anion during Cr metal stress (Table 4).

Activities of antioxidant enzymes

Activity of SOD (Table 4) increased significantly under

0.5 mM (8.69 mol Unit Activity mg protein-1) and

1.0 mM (8.72 mol UA mg protein-1) Cr metal stress in

radish shoots as compared to control (4.4 mol UA mg

protein-1). Seed presoaking treatments of 28-HBL alone

was not able to alleviate the decreased levels of SOD

activity. However, when Cr metal solutions were supple-

mented alongwith 28-HBL, it resulted in increased levels

of SOD activity (Table 4). Maximum activity of SOD was

reported in case of radish shoots treated with 10-7 M

28-HBL supplemented with 0.5 mM Cr (12.169 mol UA

mg protein-1) when compared to 0.5 mM Cr alone

(8.69 mol UA mg protein-1) (Table 4). Activities of CAT

(Table 4) and GR (Table 5) were decreased under Cr stress

than control. There activities were significantly enhanced

in Cr treated shoots supplemented with 28-HBL in com-

parison to Cr metal alone. Activity of CAT (Table 4) was

maximum (62.38 mol UA mg protein-1) in shoots treated

with 10-7 M 28-HBL supplemented with 0.5 mM of Cr

solution (31.29 mol UA mg protein-1). Similarly, GR

activity (Table 5) was also increased significantly by

applications of different concentrations of 28-HBL under

Cr stress. Maximum enhancement in the activity of GR

(0.037 mol UA mg protein-1) was observed in case of

shoots, treated with 0.5 mM of Cr solution supplemented

with 10-7 M 28-HBL (Table 5). Activity of APOX was

increased with increased concentrations of Cr as compared

to control (Table 5). It was further enhanced by applica-

tions of different concentrations of 28-HBL under Cr stress

(Table 5). The maximum APOX activity (Table 5) was

observed in case of shoots, treated with 0.5 mM of Cr

solution supplemented with 10-7 M 28-HBL (0.116 mol

UA mg protein-1).

Application of 28-HBL significantly decreased the POD

activity (Table 5) and it was also decreased under metal

stress except 0.5 mM Cr stress. Supplementation of 10-9

and 10-11 M 28-HBL along with Cr metal significantly

decreased POD activity in the shoots (Table 5). The

activities of DHAR (Table 6) and MDHAR (Table 6) got

decreased from 0.0387 mol UA mg protein-1 and

0.377 mol UA mg protein-1 respectively in control to

0.0247 mol UA mg protein-1 (DHAR) and 0.237 mol UA

mg protein-1 (MDHAR) in Cr metal treated seedlings.

Pretreatments of 28-HBL alone significantly enhanced

DHAR activity in radish seedlings than control

(0.0387 mol UA mg protein-1), with maximum increase at

10-7 M 28-HBL alone (0.041 mol UA mg protein-1)

(Table 6). However, there was insignificant change in

MDHAR activity under the seed presoaking treatments of

28-HBL alone (Table 6). Yet, 28-HBL treatments under

metal stress could restore the activity of MDHAR and the

maximum enhancement was recorded at 10-7 M 28-HBL

along with 1.0 mM Cr (0.423 mol UA mg protein-1)

(Table 6). Also, in Cr stressed seedlings maximum activity

of DHAR (0.0421 mol UA mg-1 protein) was observed at


123

10-7 M 28-HBL in combination with 1.0 mM Cr metal

(Table 6). It was observed that 28-HBL helped in con-

quering the stress in radish plants by regulating the activ-

ities of antioxidant enzymes. The Present study thus

indicates a possible regulation of antioxidant enzymes by

BRs in amelioration of heavy metals stress in plants

(Fig. 1).

Discussion

Phytotoxicity induced by Cr have been explored in many

important crop plants and also in lower plants (Panda and

Choudhury 2005; Shanker et al. 2005; Yu et al. 2007).

Chromium causes wilting and plasmolysis in root cells and

thus affects root growth of plants (Panda and Choudhury

2005). Total root length of wheat was affected by 20 mg

Cr(VI) kg-1 soil (Chen et al. 2001). Rout et al. (1997)

reported adverse effects of Cr on plant height and shoot

growth. Growth inhibition by Cr can be due to chromo-

somal aberrations which lead to inhibition of cell division

(Liu et al. 1993). In present study, decrease in root length

(Table 1); shoot length (Table 1); Chl A, Chl B and total

Chl contents (Table 2) has been observed under Cr stress.

A similar declining trend for Chl content was observed in

Glycine max under water deficits which was improved by

brassinolide treatments (Zhang et al. 2008). The present

work reveals that seed-presoaking treatments of 28-HBL

improved the seedling growth and contents of total Chl

(Table 2) and Chl A (Table 2) under Cr stress. Besides,

increased uptake of Cr(VI) ions was remarkably lowered

by 28-HBL treatments thereby resulted in mitigation of Cr

metal toxicity in radish seedlings (Table 1). These results

are in coherence with the reports of Sharma et al. (2007). In

B. juncea, Zn metal ions uptake was significantly lowered

by 28-HBL treatments. Thus, applications of 28-HBL may

increase cell division, reduces chromosomal aberrations

and alters the membrane permeability (Fig. 1). Besides,

stress protective effects of 28-HBL on plant growth, pho-

tosynthesis, activities of carbonic anhydrase, nitate reduc-

tase and antioxidant enzymes in B. juncea plants under Cd

stress were studied by Hayat et al. (2007). They observed

that the toxic effects generated by Cd were reduced with

28-HBL spraying treatment due to increase in plant growth

and enhanced enzyme activities. Also, BRs have been

reported to increase the ATPase activity in azuki bean

epicotyls and maize roots, leading to proton extrusion, and

induced cell wall relaxation (Cerana et al. 1984; Haubrick

and Assmann 2006). Further, Bajguz (2000) stated that

BRs increased DNA, RNA and protein contents of Chlo-

rella vulgaris. Similarly, in the current study protein con-

tent was found to be significantly improved in Cr stressed

shoots treated with 28-HBL (Table 3).

Heavy-metal detoxification and tolerance is mediated

via metal-binding cystein-rich compounds like phytochel-

atins (PC) which facilitates the chelation of Cr ions in

cytosol or their compartmentalization in vacuoles (Fig. 1)

(Shanker et al. 2005). However, in higher concentrations

Fig. 1 Possible mechanism of brassinosteroids regulated heavy metal

stress tolerance in plants. Dotted line represents the proposed possible

mechanisms, which remains to be explored. ROS reactive oxygen

species, PC phytochelatins, BR brassinosteroid, BRI1 brassinosteroid

insensitive 1, BAK1 BRI1-associated kinase 1, P phosphate, BIN2

brassinosteroid insensitive 2, BZR1 brassinozole-resistant 1, BES1

BRI1-ems-suppressor 1, V-ATPase vacuolar H?-ATPase, APOX

ascorbate peroxidase, CAT catalase, POD guaiacol peroxidase, DHAR

dehydroascorbate reductase, MDHAR monodehydroascorbate redu-

catse, GR glutathione reductase


123

Cr(VI) generates ROS like superoxide radicals (O2•-),

hydrogen peroxide (H2O2), hydroxyl radicals (HO-), per-

oxy radicals (ROO•), alkoxy radicals (RO•), singlet oxygen

(O21), etc. (Bhattacharjee 2005; Shanker et al. 2005). Cr

induced toxicity leads to over-production of ROS and thus

causes oxidative damage leading to membrane destruction

which in turn affects the levels of antioxidants and metal-

lozymes or antioxidant enzymes (Pandey et al. 2005;

Shanker et al. 2005). Similarly, in present study, generation

of O2•- anion had significantly increased under Cr stress

(Table 4). Thus, Cr stress may enhance accumulation of

ROS resulting in disturbance in its detoxification. The

balance between ROS production and detoxification is

maintained by ROS-scavenging antioxidant enzymes such

as SOD, CAT, APOX, GR, DHAR, MDHAR and POD etc.

(Fig. 1) (Mittler 2002). Superoxide dismutase is a family of

metallozymes which catalyze the disproportionation of

superoxide radicals (O2•-) to O2 and H2O2 (Fig. 1). The

H2O2 is further removed by CAT in the peroxisomes or by

APOX in the chloroplast or by membrane bounded POD

(Foyer et al. 1997). Glutathione reductase is a member of

flavoenzyme family which catalyzes the NADPH depen-

dent reduction of glutathione disulphide (GSSG) to gluta-

thione (GSH) and it maintains glutathione in the reduced

state (Fig. 1), which in turn reduces dehydroascorbate to

ascorbate (Noctor and Foyer 1998). Besides, ascorbic acid

in its reduced form is essential for ROS scavenging and its

cellular pool is maintained by DHAR and MDHAR using

NADPH as reducing power (Mittler 2002). This may lead

to altered redox potential (Fig. 1) of the cells ensuing

membrane destabilization under stress (Bhattacharjee

2005; Scandalios 2005; Skorzynska-Polit et al. 2010).

Recently, Liu et al. (2010) reported that two mitogen-

activated protein kinases (MAPKs), MPK3 and MPK6, are

triggered in response to Cd via the accumulation of ROS in

Arabidopsis. Also, in response to both abiotic and biotic

stresses, Calcium-dependent protein kinases (CDPKs),

calmodulins, calmodulin-like proteins or calcineurin B-like

proteins are activated as important sensors of Ca2? in

plants (Ludwig et al. 2004). The role of stress on antioxi-

dant enzymes has been overemphasized in literature (Mit-

tler 2002; Scandalios 2005). However, the effect of BRs

and its implications against stress is not well understood.

McCord (2000) reported that 28-HBL treated seedlings

might be scavenging ROS more effectively than the seed-

lings treated with metal alone. It may be possible that

28-HBL maintained the altered redox status of cells by

regulating activities of antioxidant enzymes viz. SOD,

CAT, APOX, GR, DHAR, MDHAR etc. (Fig. 1). Further,

redox potential may be re-established by reducing lipid

peroxidation of phosho-lipids in cell membranes and

accumulation of certain osmoprotectants like proline or

betaine (Fig. 1). Previous reports also showed that

exogenous application of BRs modified antioxidant

enzyme activity in stressed maize, mustard, radish and

wheat plants (Bhardwaj et al. 2007; Sharma et al. 2007,

2010, 2011; Yusuf et al. 2010). BRs (modified Sterols) can

modulate the activity of proteins and other enzymes within

the membrane by affecting either protein conformation

(functionality) or protein activity by direct protein-sterol

interactions (Lindsey et al. 2003). Li et al. (2002) reported

that BRs are perceived by a protein complex that includes

the leucine-rich repeat receptor-like protein kinase (LRR-

RLK) brassinosteroid-insensitive 1 (BRI1) (Fig. 1). Wang

and He (2004) suggested the possibility that in plants BRI1

plays a role in defence by perceiving peptide signals.

Under stress these generated signals may regulate defence

responses. Recently, Dhaubhadel and Krishna (2008)

identified the six differentially expressed genes in 24-epi-

brassinolide treated heat stressed Brassica napus seedlings

which signify that BRs regulated genes are involved in

diverse physiological responses in stress conditions. Vari-

ous stress responsive genes regulated by BRs may code for

the phytochelatins, organic acids, osmolytes, metallothio-

nines and stress protective proteins (Late Embryogenesis

Abundant i.e., LEA proteins and Heat Shock Proteins, i.e.

HSPs) (Gendron and Wang 2007; McSteen and Zhao 2008)

(Fig. 1).

Over-production of ROS during Cr-stress might increase

oxidative degradation of phospholipids, ensuing in dis-

ruption of the permeability of cell membranes which can

not be re-stored by antioxidant enzymes (Sudo et al. 2008;

Skorzynska-Polit et al. 2010). However, it’s feasible that

treatments of BRs contribute in inhibition of lipid degra-

dation as well as stimulation of activities of antioxidant

enzymes under stress (Fig. 1). In present study, similar

increased expression of GR (Table 5), DHAR (Table 6)

and MDHAR (Table 6) in presence of 28-HBL enhances

tolerance to oxidative stress generated by Cr has been

observed. This information is in accordance to the previous

reports where exogenous application of BRs modifies

antioxidant enzyme activity. Here, POD activity (Table 5)

is decreased in Cr treated seedlings (except 0.5 mM Cr)

and further decreased by 28-HBL treatments, suggesting no

protective role for 28-HBL on POD activity. However,

28-HBL enhanced APOX activities in seedlings grown

under Cr metal stress (Table 5). Both POD and APOX are

involved in removal of H2O2. Thus, it can be concluded

that 28-HBL enhanced the activities of antioxidant

enzymes which might be owed to the up-regulation of the

Brassinosteroids-mediated gene expression during metal

stress (Fig. 1). This aligns with the prior reports of

Bhardwaj et al. (2007) and Hayat et al. (2007) in B. juncea

and Z. mays respectively. So, the alteration of antioxidant

enzyme activities may suggest that 28-HBL treated seed-

lings were less affected by Cr metal than the untreated


123

seedlings. Therefore, improvement in seedling growth,

contents of proline, protein, total Chl and Chl A, lowered

Cr(VI) ions uptake, lowered MDA content and activities of

antioxidant enzymes recommends the stress protective

effects of BRs.

Intensifying environmental pollution and shrinking

habitats has obligated to trail new, scientific and eco-

friendly techniques for rehabilitating and restoring polluted/

toxic sites for social use. Being economic, environment-

friendly and plant-based natural technique, phytoremedia-

tion offers a promising prospect for addressing ecological

concerns of a range of pollutants. Since plants with high and

rapid rate of biomass production and hyperaccumulation

potential are appropriate for phytoremediation, subse-

quently recent studies are focused to improve their effi-

ciency by use of additives for their enhanced applicability in

phytoremediation. Assisted-phytoremediation exploits the

use of chemical additives like chelators, organic acids etc.

to soil for increasing the bioavailability of heavy metals for

plant uptake. Recently, a few studies have addressed the use

of phytohormones as additives to plants for phytoremedia-

tion purpose (Barbafieri and Tassi 2011). Since, BRs con-

trol several agronomic traits in plants; therefore these are of

great concern for its implications in a modified agronomic

practice i.e., phytoremediation. Thus, studies on effects of

BRs could provide positive interactions with plants for

more efficient assisted-phytoremediation techniques to

reduce the risks associated with the exposure of human and

environment to sites contaminated with heavy metals.

Conclusions

In recent years, BRs have been reported for increasing

plant productivity and plant protection against biotic as

well as abiotic stress. The stress-amelioration might be

mediating at different levels of metal toxicity viz., metal

binding and its translocation, accumulation and detoxifi-

cation of ROS. Besides metal toxicity in plants also

depends on its uptake and bioaccumulation which is

influenced by the bioavailability, route of uptake, storage,

degradation, immobilization, excretion, and avoidance/

tolerance mechanisms. These characteristics are important

for enhancing the potential of assisted-phytoremediation in

hyperaccumulator plants to decontaminate the polluted

sites. Also, the role of phytohormones in assisted-phytor-

emediation technologies may be exploited to decontami-

nate the habitats contaminated with heavy metals.

However, the acquisition of knowledge at the molecular

level and its advancement will further give an insight into

understanding the anti-stress effect of 28-HBL in greater

detail and facilitate in developing suitable strategies for

plant protection against heavy metals stresses. Further

investigation on interactive effects of BRs with other

phytohormones can be focused to sort out the intricacies of

phytohormones signaling involved in various defence

mechanisms.

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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1 23

Acta Physiologiae Plantarum

ISSN 0137-5881

Volume 33

Number 5

Acta Physiol Plant (2011)

33:1723-1735

DOI 10.1007/s11738-010-0709-1

Effect of 24-epibrassinolide on oxidativestress markers induced by nickel-ion inRaphanus sativus L.Indu Sharma, Pratap Kumar Pati & RenuBhardwaj

1 23

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ORIGINAL PAPER

Effect of 24-epibrassinolide on oxidative stress markers induced

by 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

Ó 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-EBLwere 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


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

Author's personal copy

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

personal copy

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

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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 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

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 Raphanus








shoot length

Fig. 3 Effect of 24-EBL on fresh biomass in 7-day-old Raphanus








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 Raphanus


(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 Raphanus


(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 l

mol 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 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 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 mMNi (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

personal copy

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 bothDHARandMDHARunder Ni stress. These

enhanced activities ofDHARandMDHARare inevitable for

the scavenging of overproduced ROS during Ni ion-induced

oxidative burst (Mittler 2002). During redox regulation and

tomaintain a highGSH/GSSG ratio for the protection of cells

against photoinhibition, GR is prerequisite (Vyas et al.

2007). In the present report, Ni stress reduced theGR 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). 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 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 mMNi-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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LIST OF PUBLICATIONS A. RESEARCH PAPERSshodhganga.inflibnet.ac.in/bitstream/10603/10220/14/14...LIST OF PUBLICATIONS A. RESEARCH PAPERS 1. Sharma, ... jasmonates and BRs play a determinant