1 23

Proceedings of the NationalAcademy of Sciences, India Section B:Biological Sciences ISSN 0369-8211Volume 84Number 1 Proc. Natl. Acad. Sci., India, Sect. B Biol.Sci. (2014) 84:183-192DOI 10.1007/s40011-013-0195-5

Role of 24-Epibrassinolide, Putrescine andSpermine in Salinity Stressed Adiantumcapillus-veneris Leaves

Anil Sharma, Shummu Slathia, SikanderPal Choudhary, Yash Pal Sharma &Anima Langer

1 23

Your article is protected by copyright and all

rights are held exclusively by The National

Academy of Sciences, India. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

RESEARCH ARTICLE

Role of 24-Epibrassinolide, Putrescine and Spermine in SalinityStressed Adiantum capillus-veneris Leaves

Anil Sharma • Shummu Slathia • Sikander Pal Choudhary •

Yash Pal Sharma • Anima Langer

Received: 9 January 2013 / Revised: 30 March 2013 / Accepted: 8 May 2013 / Published online: 28 June 2013

� The National Academy of Sciences, India 2013

Abstract In the present investigation, the effects of

24-epibrassinolide and polyamines (putrescine and sper-

midine) on antioxidant enzymes (superoxide dismutase,

catalase and guaiacol peroxidase), antioxidants (phenols

and proline), lipid peroxidation, proteins and photosyn-

thetic pigments in the Adiantum capillus-veneris under salt

stress were studied. 24-epibrassinolide and polyamine

treatment alone or in combination with salt stress modulate

the activities of antioxidant enzymes. Modest decrease in

proline content was observed for salt treatment alone.

However, the combination of 24-epibrassinolide and

polyamines with salt stress caused major increase in pro-

line content over salt stress alone. The combined effect of

salt with 24-epibrassinolide increased the phenolic content.

Major enhancement in phenolic content was observed for

150 mM NaCl?10-8 EBL. A remarkable decrease in

malondialdehyde content was observed in leaves treated

with 24-epibrassinolide, putrescine and spermidine with/or

without salinity stress. Proteins and photosynthetic pig-

ments did not show any remarkable change under salt

stress whereas, treatment of 24-epibrassinolide and poly-

amines enhanced the titers of protein and photosynthetic

pigments of leaves with/or without salt stress. An increase

in carotenoid content was observed with salt (150 and

300 mM) stress, which further improved remarkably by

supplementation of polyamines and 24-epibrassinolide.

Data presented here is one of the first detailed analysis of

application of EBL, Put and Spd on the antioxidant system

of Adiantum under salt stress.

Keywords Adiantum capillus-veneris �24-Epibrassinolide � Putrescine � Spermidine �Antioxidant enzymes � Proline � Lipid peroxidation

Introduction

Due to their sessile nature, plants have to endure adverse

environmental conditions such as soil salinity. Salinity is

one of the major agricultural constraints limiting plant

growth and development all over the world [1]. The over

accumulation of water soluble salts like sodium chloride

(NaCl), sodium carbonate (Na2CO3) and calcium chloride

(CaCl2) results in saline soils [2]. Salt stress severely

depresses a wide range of physiological processes such as

seed germination, seedling growth and vigour, vegetative

growth, flowering and fruit set enzyme activity and protein

synthesis [3]. High salinity also induces a decrease in

photosynthetic efficiency that is often associated with

inhibition of photosystem II [4, 5]. Additionally, salinity

stress results in ion imbalance and hyperosmotic stress in

plant system which ultimately leads to the production of

reactive oxygen species (ROS) [6]. ROS such as superox-

ide (O2•-), hydrogen peroxide (H2O2), hydroxyl radicals

(OH•) and singlet oxygen (1O2) are known to cause oxi-

dative damage to lipids, carbohydrates and DNA which

ultimately results in cell death [7, 8]. To combat ill

effects of oxidative damages caused by ROS, plants have

strong stress-protective mechanisms constituted by anti-

oxidants (ascorbic acid, proline, phenols and glutathione)

and antioxidant enzymes (superoxide dismutase (SOD),

A. Sharma (&) � S. Slathia � S. P. Choudhary �Y. P. Sharma � A. Langer

Department of Botany, University of Jammu, Jammu 180 006,

India

e-mail: asanilsharma3@gmail.com

S. P. Choudhary

Department of Horticulture, Zhejiang University, Hangzhou

310058, Zhejiang, China

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. (Jan–Mar 2014) 84(1):183–192

DOI 10.1007/s40011-013-0195-5

Author's personal copy

guaiacol peroxidase (GPOX), catalase (CAT) and gluta-

thione reductase (GR) [9, 10].

More recently, the role of phytohormones such as

abscisic acid, cytokinins, jasmonates, brassinosteroids

(BRs) and polyamines in mitigating abiotic stress [11–14]

has been widely accepted. Among plant growth regulators

(PGRs), BRs form a group of steroidal lactones implicated

in the regulation of vital physiological activities such as

cell division, elongation of hypocotyls, reproductive

growth, senescence and seed germination [15, 16]. The

ability of BRs to improve antioxidant system by elevating

the activities and levels of enzymatic and non-enzymatic

antioxidants has made them a favourite tool to increase

resistance potential against various abiotic and biotic

stresses such as salinity stress [17, 18]. Anuradha and Rao

[19] observed that BRs could significantly reduce the

inhibitory effects of salt stress in rice plants by improving

the level of nucleic acid and compatible solutes. BRs

enhanced the activities of APOX, CAT and SOD in

Brassica juncea L. under salt stressed and control condi-

tions [20]. Similarly, improved photosynthetic activity and

decreased membrane damage in B. juncea plants subjected

to NiCl2/or NaCl stress upon EBL application had also

been observed by Ali et al. [21].

Polyamines (PAs) are the low molecular weight poly-

cations found ubiquitously in all living organisms and

functioning in a wide range of biological processes such as

cell division, embryogenesis, reproductive organ develop-

ment, root growth, regulation of gene expression, transla-

tion, cell proliferation, modulation of cell signaling, and

membrane stabilization [22–26]. Capacity of PAs to

scavenge free radicals, reactive oxygen radicals and reac-

tive oxygen generated under various stresses also proves

their strong antioxidant character [27]. Exogenous appli-

cation of Put and Spd were able to ameliorate salt stress in

barley seedlings by maintaining root tonoplast integrity

[28]. Similarly Jiu Ju et al. [29] observed that exogenous

Spd application in cucumber seedlings during salinity

stress results in increased content of endogenous Spd and

spermine (Spm). Besides it also increases SOD, POD and

CAT activities and decreases ROS production rate, H2O2

and malondialdehyde (MDA) contents and electrolyte

leakage and thus significantly promotes root growth.

Adiantum is one of the important medicinal plants

mentioned in Ayurveda. It is popularly called as Hansraj or

Hanspadi in Ayurvedic system of medicine [30]. It is one

of the common species that is of a potential medicinal

importance commonly used for chest complaints, cough,

expectorant, increase lactation, cold, and to aid in kidney

function [31, 32, 33]. In the process of climate change, the

saline conditions get increased, thus only those plants are

supposed to survive, which are resistant against salinity

stress. Being a good phytoremediator with immense

medicinal importance, the present study has been under-

taken to study the effect of salt stress in Adiantum capillus-

veneris and further to study the role of brassinosteroids and

polyamines in mitigating the salinity stress.

Material and Methods

Plant Material and Growth Conditions

Adiantum capillus-veneris leaves were used for the present

investigation. Leaves were surfaced sterilized with 0.01 %

sodium hypochlorite for 1 min and then rinsed with ster-

lised distilled water three to four times. About 25 g of

leaves were dipped in petri plates in respective solution for

72 h and kept at 28 ± 2 �C in an incubator. After treatment

with different solutions they were stored in deep freezer at

low temperature for further analysis.

EBL, Put, Spd and NaCl Treatments

All the chemicals were purchased from HIMEDIA Private

Limited.

Various concentrations of EBL (10-10 and 10-8 M), Put

(1 mM), Spd (1 mM) and NaCl (75 mM, 150 mM and

300 mM) alone or in their respective combinations, were

applied to leaves by adding 50 ml of the solutions in the

Petri plates. On the fourth day, the leaves were stored at

-20 �C until further use.

Estimation of Proteins and Enzymes

Preparation of Plant Extract

Leaf tissue (0.5 g fresh weight, FW) was homogenized in

3 ml of 0.1 M potassium phosphate buffer in a pre-chilled

pestle and mortar, centrifuged at 15,000 g for 20 min. The

supernatant was used for the estimation of protein content

and antioxidant enzyme activities.

Protein Estimation

The protein content was estimated according to the method

of Lowry et al. [34]. About 0.1 ml of samples were pipetted

in test tubes and the volume was raised to 1 ml by adding

distilled water. Then 5 ml of reagent c (Reagent A = 2 %

Sodium carbonate in 0.1 N Sodium chloride, Reagent

B = 0.5 % CuSO4 in 1 % Potassium sodium tartarate,

Reagent C = 50 ml of reagent A ?1 ml of reagent B) was

added to each test tube, mixed well and allowed to stand

for 10 min, followed by addition of 0.5 ml of Folin–Cio-

calteau reagent (FC) (three times diluted) to each test tube.

After mixing, the reaction mixtures were incubated at room

184 A. Sharma et al.

123

Author's personal copy

temperature in the dark for 30 min for development of the

blue colour. The blue coloured complex was read spec-

trophotometrically at 660 nm using UV/VIS absorption

spectrophotometer.

Antioxidant Enzymes

Preparation of Plant Extract

Leaf tissue (0.5 g FW) was homogenized in 3 ml of 0.1 M

potassium phosphate buffer in a pre-chilled pestle and

mortar, centrifuged at 15,000 g for 20 min at 4 �C. The

supernatants were used for estimation of GPOX, CAT and

SOD activities.

Guaiacol Peroxidase (GPOX, EC1.11.1.7)

The GPOX activity was estimated as per method of Putter

[35]. In brief, reaction mixture consisted of 3 ml of phos-

phate buffer (0.1 M), 50 ll guaiacol solution (20 mM),

100 ll enzyme extract and 30 ll of H2O2 solution. The rate

of the formation of oxidized guaiacol product was followed

spectrophotometrically at 436 nm after 1 min.

Catalase (CAT, EC 1.11.1.6)

The activity of CAT was estimated according to the

method developed by Aebi [36]. In short, the reaction

mixture consisted of 1.5 ml of potassium phosphate buffer

(50 mM), 1.2 ml of H2O2 (150 mM) and 30 ll of enzyme

extract. The change in the absorbance was read at 240 nm

after 2 min.

Superoxide Dismutase (SOD) (EC 1.15.1.1)

SOD activity was calculated according to the method

proposed by Kono [37]. In the test tube 1.8 ml sodium

carbonate buffer, 750 ll Nitro blue tetrazolium (NBT) and

150 ll Triton x-100 (0.6 %) were taken. The reaction was

initiated by the addition of 150 ll hydroxylamine hydro-

chloride. After incubation for 2 min, about 70 ll of

enzyme extract was added. This reaction mixture was taken

in a test cuvette and inhibition in the rate of reduction of

NBT was recorded at 540 nm.

Proline Estimation

Proline content was estimated by method of Bates et al.

[38]. About 1 g F.W. of Adiantum treated leaves were

crushed in 10 ml of 3 % sulphosalicylic acid and the

homogenate was centrifuged at 16,0009g for 20 min at

room temperature. After centrifugation, supernatants were

mixed with 2.5 % ninhydrin in glacial acetic acid. This

reaction mixture was kept at 90 �C in water bath for 1 h to

develop the colour. The reaction mixtures were then cooled

immediately in ice bath followed by addition of toluene

(6 ml) to separate the chromophore. The readings were

taken spectrophotometrically at 520 nm and proline con-

tent was calculated by comparing the sample absorbance

with the standard proline curve in a concentration range of

0–25 mg/ml.

Determination of Total Phenol Content

Total phenol content was determined by the method of

Ragazzi and Veronese [39]. 0.5 g (FW) of treated leaves was

homogenized in 10 ml of distilled water. 1.5 ml of this

extract was mixed with 3 ml of FC reagent and left undis-

turbed for 30 min in the dark followed by addition of 3 ml of

sodium carbonate solution. The readings of blue coloured

solution were taken spectrophotometrically at 680 nm.

Lipid Peroxidation

Peroxidation of lipids was estimated according to the method of

Heath and Packer [40]. Adiantum leaves (0.5 g FW) supplied

with EBL and Put with/or without salinity stress were

homogenized in 3 ml of 0.1 % trichloroacetic acid (TCA).

These samples were centrifuged at 10,0009g for 5 min.

Supernatants were treated with 3 ml of thiobarbituric acid

(TBA) (prepared in TCA). This solution was kept in water bath

at 95 �C. After 30 min these solutions were cooled immedi-

ately to stop the reaction. The readings were taken spectro-

photometrically at 532 and 600 nm. MDA content was

determined after subtracting the optical density for non specific

absorbance (600 nm) from the absorbance value at 532 nm.

Determination of Photosynthetic Pigments

Adiantum leaves (1 g DW) subjected to salinity stress with/

or without EBL and Put were homogenized with absolute

methanol (10 ml) to make slurry. A pinch of MgCO3 was

added to reduce pigment decomposition. These mixtures

were then kept overnight at 40 �C in a refrigerator. The

samples were centrifuged at 2,5009g. To the resultant

extract, 10 ml of methanol was added. To minimize the

photo-oxidation of pigments all the procedure was per-

formed in dim light. The readings were taken spectropho-

tometrically at 664.2, 648.6 and 470 nm, for the estimation

of Chl-a, Chl-b and carotenoid contents respectively using

the equations of Lichtenthaler [41].

Statistical Analysis

All the experiments were performed in triplicates. The data

shown are the mean of three replicate experiments along

Salinity Stressed Adiantum capillus-veneris Leaves 185

123

Author's personal copy

with standard error (n = 3). One-way analysis of variance

(ANOVA) was carried out and data were presented at

p \ 0.05. All the statistical calculations were performed

using Sigma Stat 3.5.

Result

Protein Content

Salt stressed (75 and 150 mM) leaves showed significant

enhancement in protein content when compared with

control; on the other hand, a small reduction was noticed

for 300 mM NaCl (Table 1). Major enhancement in protein

content was recorded for 10-8 and 10-10 M EBL treated

leaves over control. Put treatment also increased protein

content. No significant increase in protein content was

observed for Spd treatment (Table 1). Supplementation of

EBL to salt stressed leaves showed marked improvement in

protein content than salt stressed leaves alone. Similarly,

leaves treated with Put along with NaCl (75 and 300 mM)

showed an increase in protein content, on the contrary, with

150 mM a decrease in protein content was observed when

compared with salt stressed leaves (Table 1). Spd appli-

cation plus salt also improved protein content (Table 1).

Antioxidant Enzymes

Guaiacol Peroxidase (GPOX)

Salt stressed leaves showed significant increase in GPOX

activity at 300 mM NaCl when compared with control. A

major increase in GPOX activity was recorded alone for

leaves given 10-8 M EBL than control. Similarly, signifi-

cant increase in GPOX activity was recorded for Spd

application alone over control (Table 2). 10-10 M EBL and

Put applied to leaves caused a decline in GPOX activity

when compared with control (Table 2). Supplementation of

EBL along with NaCl enhanced the GPOX activity such

that maximum enhancement was noticed for10-10 M EBL

plus 300 mM NaCl. A small decrease in GPOX activity

was recorded for 10-10 M EBL plus 150 mM NaCl over

salt stress alone. Put and Spd application also increased the

activity of GPOX when supplied with NaCl solution over

salt stress alone, with maximum increase observed for

150 mM NaCl plus Put (Table 2).

Catalase (CAT)

A major increase in CAT activity was noticed in NaCl

stress with maximum increase observed for 300 mM NaCl

over control (Table 2). Supplementation of EBL, Put and

Spd also enhanced the CAT activity when compared with

control. Major enhancement of CAT activity was observed

for 10-8 M EBL supplemented with 300 mM NaCl solu-

tion followed by 10-10 M EBL plus 150 mM NaCl over

NaCl stress alone (Table 2). Similarly Put and Spd sup-

plementation to NaCl stressed leaves enhanced CAT

activity with maximum enhancement observed for 75 mM

NaCl plus Put when compared with 75 mM NaCl stress

alone (Table 2).

Superoxide Dismutase (SOD)

Application of NaCl stress enhanced the activity of SOD

when compared with control (Table 2). EBL, Put and Spd

application also revealed small increase in SOD activity

than in control. Supplementation of EBL to salt treated

leaves increased SOD activity but maximum and signifi-

cant increase was observed only for 10-8 M EBL plus

300 mM NaCl over control. Similarly, Put and Spd sup-

plementation to 300 mM NaCl caused significant

enhancement in SOD activity. All other combinations of

Put, Spd and NaCl stress showed a modest change in SOD

activity (Table 2).

Proline (PL)

A significant decrease in PL content was found in NaCl

treated leaves over control, with maximum decrease

observed for 150 mM NaCl (Table 3). No significant

increase in PL level was observed in leaves which under-

went Put and Spd treatment. EBL (10-8 M and 10-10 M)

treatment showed significant increase in PL, with maxi-

mum increase observed for 10-8 M EBL over control

(Table 3). Leaves applied with EBL plus NaCl were

observed with increased PL content, such that maximum

rise in PL content was recorded for 10-8 M EBL plus

300 mM NaCl when compared with control. Put and Spd

supplied to leaves which underwent salt treatment also,

enhanced PL content over salt treated leaves alone

(Table 3).

Total Phenol (TPC)

TPC level found in salt stressed seedlings was signifi-

cantly higher than that of control (Table 3). EBL, Put and

Spd treatment alone were able to enhance the TPC

level with maximum rise observed for Put application

(Table 3). No significant increase in TPC level was

observed for salt stressed leaved supplemented with Put

and Spd. On the other hand, supplementation of EBL

to NaCl stress enhanced the TPC content. Maximum

increase in TPC content was observed for 10-8 M EBL

plus 150 mM NaCl when compared to salt treated leaves

alone (Table 3).

186 A. Sharma et al.

123

Author's personal copy

Lipid Peroxidation of malondialdehyde (MDA)

Significant production of MDA under salt stress was

observed when compared with control (Table 3) with a

maximum increase observed for 300 mM NaCl solution.

No significant changes were noticed with EBL, Put and

Spd application alone to leaves when compared to control

(Table 3). Remarkable reduction of MDA content was

recorded in leaves treated with NaCl along with Put in

different combinations (Table 3). EBL supplemented with

NaCl solution was observed to reduce membrane damage

with maximum downfall in MDA concentration noted for

10-8 M EBL plus 300 mM NaCl (Table 3). Spd applica-

tion with NaCl stress also showed significant reduction in

MDA production over NaCl stress leaves (Table 3).

Photosynthetic Pigments

Chlorophyll-a (Chl-a)

An increase in Chl-a was found in leaves treated with 75 and

300 mM NaCl stress over control; on the other hand, a

decrease was observed for 150 mM NaCl (Table 4). EBL, Put

and Spd treatment also significantly enhanced the Chl-a level

when compared to control with maximum enhancement

observed for EBL treatment. Put and Spd supplementation to

75 and 150 mM NaCl stressed leaves enhanced the Chl-a

content over control. On the contrary, a small decrease in

Chl-a content was observed with 300 mM NaCl over salt

stressed leaves alone (Table 4). Application of EBL to salt

stressed leaves also enhanced the Chl-a content with maxi-

mum increase recorded for 10-8 M EBL plus 300 mM NaCl

(Table 4).

Chlorophyll-b (Chl-b)

Leaves subjected to NaCl stress showed a significant rise in

Chl-b when compared with control. EBL applied alone was

observed to enhance Chl-b level with maximum rise found

at 10-10 M EBL than control. Application of Put caused a

small rise in Chl-b. On the other hand, Spd caused a sig-

nificant and major increase in Chl-b when compared with

control (Table 4). Supplementation of EBL to NaCl stress

was observed to significantly increase Chl-b level than

control, with a maximum increase observed for 10-10 M

EBL plus 300 mM NaCl (Table 4). Put and Spd along with

NaCl stress also enhanced the Chl-b content but significant

enhancement was observed for 75 mM NaCl plus Put and

300 mM NaCl plus Spd (Table 4).

Carotenoids (Crt)

A decline in Car was observed at 75 mM NaCl, on the

other hand an increase in Car was observed for 150 and

300 mM NaCl over control. EBL, Put and Spd also

revealed enhancing effects on Crt content as compared

with control (Table 4). For the leaves treated with EBL

along with NaCl recorded an enhancement in Car content

than NaCl treated leaves alone (Table 4). Supplementation

of Put and Spd showed no significant rise in Crt content

over salt treatment alone (Table 4).

Discussion

Ubiquitously, no toxic substance restricts plant growth

more than salt. General symptoms of damage by salt stress

are accelerated development, growth inhibition and

senescence and ultimate death after prolonged exposure.

Growth inhibition is the primary injury that leads to other

symptoms although programmed cell death may also occur

under severe salinity shock. Salt stress induces the syn-

thesis of abscisic acid which is responsible for stomata

closure. Consequently, photosynthesis gets affected and

photo inhibition and oxidative stress occur [42]. Moreover,

ROS are also produced under stress conditions. Although

ROS are produced under normal conditions in chloroplasts

and peroxisomes through photorespiration in light and in

Table 1 Effect of EBL, Put and Spd on protein content of Adiantum

capillus-veneris leaves treated with/or without salt stress

Treatment Protein content

(mg/g F.W.)

CN 28.22 ± 1.41

75 mM NaCl 38.38 ± 0.98a

150 mM NaCl 35.06 ± 2.33

300 mM NaCl 25.09 ± 1.38

10-8 MEBL 62.02 ± 1.70a

10-10 MEBL 72.06 ± 2.9a

Put 61.30 ± 4.45a

Spd 26.86 ± 2.99

75 mM NaCl?10-8 EBL 59.89 ± 1.14b,c

75 mM NaCl?10-10 EBL 63.22 ± 1.97b,c

75 mM NaCl?Put 43.34 ± 3.45

75 mM NaCl?Spd 45.34 ± 6.78

150 mM NaCl?10-8 EBL 72.88 ± 2.34b,c

150 mM NaCl?10-10 EBL 54.12 ± 4.55b,c

150 mM NaCl?Put 26.67 ± 1.12

150 mM NaCl?Spd 46.45 ± 2.32

300 mM NaCl?10-8 EBL 56.78 ± 4.33b,d

300 mM NaCl?10-10EBL 67.87 ± 2.98d

300 mM NaCl?Put 34.444 ± 6.72

300 mM NaCl?Spd 23.29 ± 2.39

a,b,c,d Indicate statistically significant differences from control, 75,

150 and 300 mM NaCl stress respectively at p B 0.05

Salinity Stressed Adiantum capillus-veneris Leaves 187

123

Author's personal copy

mitochondria during darkness, but during stressful condi-

tions, their concentrations gets increased manifolds which

causes harmful effects [43, 44]. Among phytohormones, BRs

and PAs have been widely used to confer salt stress tolerance

in plants [45]. Keeping this in mind, the present study was

therefore, aimed to understand the possible ameliorative

action of BRs and PAs in NaCl stress mitigation.

In the present investigation, NaCl treated leaves at a

concentration of 75 and 150 mM showed an enhancement

in protein content as compared to control, on the contrary a

decrease in protein content was observed for leaves treated

with 300 mM NaCl (Table 1). The protein in leaves treated

with NaCl further got improved by application of EBL, Put

and Spd with/or without salt stress. The results may further

be supported by the observations of Maity and Bera, [46]

who also observed enhanced titers of protein content by

exogenous application of brassinolide in green gram. Sol-

uble protein content was increased by 24-EBL application

in rice plants grown under salinity stress [47]. Moreover,

Arora et al. [48] also observed that BR application could

enhance protein content in Zea mays seedlings under

salinity stress.

NaCl stress has been found to enhance the activities of

antioxidant enzymes (GPOX, CAT and SOD) in Adiantum

leaves, which further got improved upon application of

EBL, Put and Spd alone or in various combinations with/or

without salinity stress (Table 2). Enhanced activities of

these enzymes in turn suggest the involvement of these

enzymes in the removal of H2O2 [49]. Various antioxidant

enzymes involved in oxidative stress management are

CAT, SOD, GPOX, POD, GR, and APOX [50]. The

present findings could be supported by the observations of

Sirhindi et al. [20] that BRs enhanced the activities of

APOX, CAT and GR in B. juncea under salt stress con-

ditions. Similarly, application of putrescine had also been

shown to ameliorate NaCl stress in chickpea plants through

elevating the activities of CAT, GPOX, GR and SOD [51].

Besides antioxidants and antioxidant enzymes, certain

compatible solutes such as proline, sorbitol and glycinebe-

tains also get accumulated that are actively involved in NaCl

stress amelioration [52]. In the present investigation, a

decrease in proline content was observed in salt stressed

leaves. Supplementation of EBL, Put and Spd alone or with

different combinations of NaCl enhanced titers of proline.

The results may further be supported by the observations of

Anuradha and Rao [53] which observed that EBL application

could enhance free proline levels in radish seedlings under Cd

stress. Similarly, Ozturk and Demir [54], observed enhanced

titers of proline in spinach leaves treated with putrescine and

ethephon under salt stress.

In the present study, the authors noticed a significant

production of total phenols in NaCl stressed leaves and

their enhancement upon EBL, Put and Spd alone or in

combinations (Table 3). The results could be supported by

Table 2 Effect of EBL, Put and

Spd on activity of antioxidant

enzymes (GPOX, CAT and

SOD) content of Adiantum

capillus-veneris leaves treated

with/or without salt stress

a,b,c,d Indicate statistically

significant differences from

control, 75, 150 and 300 mM

NaCl stress respectively at

p B 0.05

Treatment GPOX (unit

activity/mg

Prot. g-1 F.W.)

CAT (unit

activity/mg

Prot. g-1 F.W.)

SOD (unit

activity/mg

Prot. g-1 F.W.)

CN 0.020 ± 0.0047 0.033 ± 0.00153 0.20 ± 0.004

75 mM NaCl 0.022 ± 0.0016 0.104 ± 0.0016a 0.423 ± 0.005a

150 mM NaCl 0.038 ± 0.0093a 0.30 ± 0.021a 0.512 ± 0.002a

300 mM NaCl 0.033 ± 0.00093a 0.45 ± 0.0081a 0.429 ± 0.007

10-8 MEBL 0.063 ± 0.00047a 0.69 ± 0.0089 0.623 ± 0.0025

10-10 MEBL 0.025 ± 0.0033 4.45 ± 0.024a 0.923 ± 0.0065a

Put 0.016 ± 0.0045 0.092 ± 0.024 0.824 ± 0.0028

Spd 0.036 ± 0.0012 1.25 ± 0.0065a 0.562 ± 0.0087

75 mM NaCl?10-8 EBL 0.073 ± 0.0012b,d 2.72 ± 0.016b,c,d 1.12 ± 0.0056b,d

75 mM NaCl?10-10 EBL 0.023 ± 0.0093 1.40 ± 0.026 0.98 ± 0.0034

75 mM NaCl?Put 0.045 ± 0.0029 0.11 ± 0.0065 1.59 ± 0.009b

75 mM NaCl?Spd 0.031 ± 0.0019 4.02 ± 0.052b,c 1.23 ± 0.0012

150 mM NaCl?10-8 EBL 0.046 ± 0.010 2 ± 0.0044c 0.732 ± 0.0023

150 mM NaCl?10-10 EBL 0.049 ± 0.0020 1.40 ± 0.026 0.653 ± 0.022

150 mM NaCl?Put 0.076 ± 0.0031b,c 0.87 ± 0.037 1.32 ± 0.0039c

150 mM NaCl?Spd 0.047 ± 0.0021 2.18 ± 0.0122b,c 0.56 ± 0.0049

300 mM NaCl?10-8 EBL 0.032 ± 0.0012 5.84 ± 0.037d 3.56 ± 0.0057c,d

300 mM NaCl?10-10EBL 0.067 ± 0.0067 1.07 ± 0.023d 1.52 ± 0.0019

300 mM NaCl?Put 0.059 ± 0.0034 0.32 ± 0.0081 0.982 ± 0.0033d

300 mM NaCl?Spd 0.0992 ± 0.0023d,b 1.54 ± 0.045 2.12 ± 0.0054b,c,d

188 A. Sharma et al.

123

Author's personal copy

the observations of Abdel Wahed et al. [55] that Spd

treatments significantly increased phenolic content of

chamornile leaves as compared to control.

Enhanced MDA concentration recorded in NaCl stres-

sed leaves indicated severe damage to the lipid membranes

of the leaves caused by ROS. However, reduced MDA

content was observed in leaves treated with EBL, Put and

Spd with/or without salinity stress (Table 3). Decline in

MDA content brought by EBL, Put and Spd can be

attributed to its ability to enhance activity of CAT, GPOX

and SOD enzymes which in turn scavenge ROS and

thereby reducing its impact on lipids. The present obser-

vations are supported by the findings of Ali et al. [21] that

EBL application could significantly reduce membrane

damage in B. juncea L. plants subjected to NiCl2/NaCl

stress. Similar observations for Put were also recorded by

Sheokand et al. [51], which showed significant reduction in

MDA production in chickpea plants under NaCl stress

when treated with Put.

Differential response for photosynthetic pigments were

observed on NaCl stressed leaves than in control. Chl-a got

decreased at 150 mM NaCl, while increased at 75 and

300 mM NaCl stress. On the other hand, Chl-b got

enhanced at all concentrations. Crt enhanced at 150 and

300 mM NaCl stress, while a slight decrease was noticed at

75 mM NaCl. Celik and Atak [56] found Chl-a and Chl-b

to be decreased with increasing NaCl concentrations. For

Crt, it got enhanced at 50, 100 and 150 mM NaCl and then

starts decreasing at 200, 250, 300 and 300 mM NaCl stress

in tobacco plants. The various combinations of EBL, Put

and Spd with/or without salt stress showed an enhanced

level of photosynthetic pigments (Table 4). The results

may further be supported by the observations of Anuradha

and Rao [57], who also observed that BRs could signifi-

cantly reduce the inhibitory effects of salt stress in rice

plants by improving the pigment level and nitrate reductase

activity. Similarly, Houmili et al. [58] also highlighted that

spraying of EBL along with salinity stress in pepper

resulted in significant rise in chlorophyll values. Moreover,

exogenous application of Spd and Spm reduced the dele-

terious effect of salt stress in rice plants by reducing

chlorophyll loss [59]. Similarly, Anjum [60] also observed

that exogenously applied Spd improved the chlorophyll

content in citrus plants under saline conditions.

Conclusion

To the best of authors’ knowledge, this is the first report

dealing with the application of EBL, Put and Spd on

antioxidant potential of Adiantum under salt stress. The

present findings clearly suggest ameliorative role of EBL,

Put and Spd on salinity stress by enhancing enzyme

activity, increasing the amount of antioxidants and

decreasing lipid peroxidation. Out of various EBL, Put and

Spd combinations, 10-8 and 10-10 M EBL with/or without

Table 3 Effect of EBL, Put and

Spd on proline, total phenols

and lipid peroxidation (MDA)

content of Adiantum capillus-

veneris leaves treated with/or

without salt stress

a,b,c,d Indicate statistically

significant differences from

control, 75, 150 and 300 mM

NaCl stress respectively at

p B 0.05

Treatment Proline

(mg/g F.W.)

Total phenols

(mg/g F.W.)

Lipid peroxidation

(lmol/g F.W.)

CN 5.67 ± 0.037 10.61 ± 0.012 0.998 ± 0.02

75 mM NaCl 3.21 ± 0.012 12.19 ± 0.032 4.13 ± 0.056a

150 mM NaCl 2.91 ± 0.0163a 17.43 ± 0.047a 2.93 ± 0.098a

300 mM NaCl 4.7 ± 0.0123 13.27 ± 0.012 5.93 ± 0.014a

10-8 MEBL 9.22 ± 0.0169a 13.71 ± 0.026 0.923 ± 0.023

10-10 MEBL 8.11 ± 0.0125a 14.55 ± 0.034 0.829 ± 0.032

Put 6.91 ± 0.0077 13.27 ± 0.020 0.728 ± 0.048

Spd 5.93 ± 0.0012 16.29 ± 0.012 0.629 ± 0.039

75 mM NaCl?10-8 EBL 10.24 ± 0.0125b 21.26 ± 0.038b,c,d 2.32 ± 0.023b

75 mM NaCl?10-10 EBL 7.27 ± 0.0308 16.92 ± 0.024b 0.928 ± 0.098

75 mM NaCl?Put 8.93 ± 0.0122b,c 10.27 ± 0.026 1.23 ± 0.056b

75 mM NaCl?Spd 5.91 ± 0.0170b,c 8.46 ± 0.024 1.29 ± 0.058b

150 mM NaCl?10-8 EBL 9.61 ± 0.016b,c 23.20 ± 0.067b,c,d 0.92 ± 0.092b,c

150 mM NaCl?10-10 EBL 8.96 ± 0.033b,c 15.55 ± 0.045 0.42 ± 0.032b,c

150 mM NaCl?Put 7.65 ± 0.016b,c 12.30 ± 0.043 1.20 ± 0.093b,c

150 mM NaCl?Spd 8.26 ± 0.019b,c 7.82 ± 0.022 1.30 ± 0.089

300 mM NaCl?10-8 EBL 11.64 ± 0.030b,c,d 18.92 ± 0.004 0.52 ± 0.043b,c,d

300 mM NaCl?10-10EBL 9.93 ± 0.040b,c,d 22.66 ± 0.0023b,c,d 0.59 ± 0.02b,c,d

300 mM NaCl?Put 9.63 ± 0.012 10.59 ± 0.0029 0.56 ± 0.01b,c,d

300 mM NaCl?Spd 8.65 ± 0.028 20.13 ± 0.005c,d 0.67 ± 0.014b,c,d

Salinity Stressed Adiantum capillus-veneris Leaves 189

123

Author's personal copy

salt stress showed better improvement in all parameters

(antioxidant enzymes, antioxidants, membrane damage,

protein content and photosynthetic pigments) studied.

Among antioxidant enzymes, best results were obtained for

GPOX and SOD. The results obtained could be used to

further investigate the application of brassinosteroids and

polyamines in important horticultural crops to negate the

effects of various abiotic stresses, including salinity stress.

Acknowledgments The authors are indebted to Prof. Geeta Sumbali

Head, Department of Botany, University of Jammu, for providing

necessary facilities in the lab. They wish to express their sincere

thanks to Assistant Prof. Vijay Shivgotra from Department of Sta-

tistics for his efficient support in the statistical analysis.

References

1. Gao S, Ouyang C, Wang S, Xu Y, Tang L, Chen F (2008) Effects

of salt stress on growth, antioxidant enzyme and phenylalanine

ammonia-lyase activities in Jatropha curcas L. seedlings. Plant

Soil Environ 54:373–381

2. Nawaz K, Hussain K, Majeed A, Farah K, Afghan S, Ali K

(2010) Fatality of salt stress to plants: morphological physio-

logical and biochemical aspects. Afr J Biotech 9:5475–5480

3. Munns R, Tester M (2008) Mechanisms of salinity tolerance.

Annu Rev Plant Biol 59:651–681

4. Kalaji HM, Bosa GK, Koscielniak J, _Zuk-Gołaszewska K (2010)

Effects of salt stress on photosystem II efficiency and CO2

assimilation of two Syrian barley landraces. Environ Exp Bot.

doi:10.1016/j.envexpbot

5. Xia JR, Li YJ, Zou DH (2004) Effects of salinity stress on PSII in

Ulva lactuca as probed by chlorophyll fluorescence measure-

ments. Aquat Bot 80:129–137

6. Blokhina O, Virolainen E, Gagerstedt KV (2003) Antioxidants,

oxidative damage and oxygen deprivation stress: a review. Ann

Bot (Lond) 91:179–194

7. McCord LM (2000) The evolution of free radicals and oxidative

stress. Am J Med 108:652–659

8. Wang W, Vincour B, Altman A (2003) Plant responses to

drought, salinity and extreme temperatures: towards genetic

engineering for stress tolerance. Planta 218:1–14

9. Ahmad P, Sarwat M, Sharma S (2008) Reactive oxygen species,

antioxidants and signallling in plants. J Plant Biol 5:167–173

10. Andre CM, Yvan L, Daniele E (2010) Dietary antioxidants and

oxidative stress from a human and plant perspective: a review.

Curr Nutr sci 6:2–12

11. Clouse SD (2011) Brassinosteroid signal transduction: from

receptor kinase activation to transcriptional networks regulating

plant development. Rev Plant Cell 23:1219–1230

12. Divi VK, Rahman T, Krishna P (2010) Brassinosteroids mediated

stress tolerance in Arabidopsis shown interactions with ascorbic

acid, ethylene and salicylic acid pathways. BMC Plant Biol

10:151

13. Hussain K, Nisar MF, Majeed A, Nawaz K, Bhatii KH, Afghan S,

Shahazad A, Zia-ul-Hussnian S (2010) What molecular mecha-

nism is adapted by plants during salt stress tolerance. Afr J

Biotech 9:416–422

14. Choudhary SP, Kanwar M, Bhardwaj R, Yu J-Q, Tran L-S (2012)

Chromium stress mitigation by polyamine-brassinosteroid appli-

cation involves phytohormonal and physiological strategies

in Raphanus sativus L. PLoS ONE. doi:10.1371/journal.pone.

0033210

Table 4 Effect of EBL, Put and

Spd on photosynthetic pigments

of Adiantum capillus-veneris

leaves treated with/or without

salt stress

a,b,c,d Indicate statistically

significant differences from

control, 75, 150 and 300 mM

NaCl stress respectively at

p B 0.05

Treatment Chlorophyll a

(mg/g F.W.)

Chlorophyll b

(mg/g F.W.)

Carotenoids

(mg/g F.W.)

CN 0.114 ± 0.0093 0.052 ± 0.0032 0.028 ± 0.0047

75 mM NaCl 0.170 ± 0.0093a 0.080 ± 0.0081 0.02 ± 0.0081

150 mM NaCl 0.092 ± 0.0047 0.160 ± 0.0094a 0.059 ± 0.0028a

300 mM NaCl 0.159 ± 0.0034a 0.073 ± 0.0045 0.045 ± 0.0024

10-8 MEBL 0.246 ± 0.0016a 0.11 ± 0.0012 0.073 ± 0.0026a

10-10 MEBL 0.267 ± 0.0078a 0.208 ± 0.005a 0.053 ± 0.0029a

Put 0.142 ± 0.0034a 0.060 ± 0.0024 0.0429 ± 0.0016

Spd 0.159 ± 0.0024a 0.109 ± 0.0038a 0.066 ± 0.0023a

75 mM NaCl?10-8 EBL 0.186 ± 0.0044 0.143 ± 0.0034 0.088 ± 0.0024

75 mM NaCl?10-10 EBL 0.308 ± 0.0081b,c,d 0.187 ± 0.0022 0.104 ± 0.0044b,c,d

75 mM NaCl?Put 0.186 ± 0.0030 0.238 ± 0.0045 0.025 ± 0.0032

75 mM NaCl?Spd 0.347 ± 0.0020b,c,d 0.163 ± 0.0016b,d 0.096 ± 0.0029b

150 mM NaCl?10-8 EBL 0.178 ± 0.0057b 0.212 ± 0.0028b,c,d 0.061 ± 0.0012

150 mM NaCl?10-10 EBL 0.384 ± 0.0052b,c,d 0.227 ± 0.0021b,c,d 0.075 ± 0.0030

150 mM NaCl?Put 0.257 ± 0.0015b,c,d 0.098 ± 0.0081 0.034 ± 0.0017

150 mM NaCl?Spd 0.130 ± 0.0018 0.109 ± 0.0012 0.053 ± 0.0093

300 mM NaCl?10-8 EBL 0.420 ± 0.0045 0.249 ± 0.0094b,c,d 0.95 ± 0.0019b,d

300 mM NaCl?10-10EBL 0.187 ± 0.0056 0.233 ± 0.0077b,c,d 0.089 ± 0.005b,d

300 mM NaCl?Put 0.126 ± 0.0021 0.127 ± 0.0081 0.056 ± 0.0087

300 mM NaCl?Spd 0.142 ± 0.0043 0.132 ± 0.0032 0.071 ± 0.0035b,d

190 A. Sharma et al.

123

Author's personal copy

15. Clouse SD (1996) Molecular genetic studies confirm the role of

brassinosteroids growth and development. Plant J 10:1–8

16. Fathutdinova RA, Shakirova FM, Chemeris AV, Sabirzhanov BE,

Vakhitov VA (2002) NOR (nucleolar organizer region) reactivity

in wheat with ploidy levels treated with phytohormones. Russ J

Genet 38:1335–1338

17. Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007)

Brassinosteroid confers tolerance in Arabidopsis thaliana and

Brassica napus to a range of abiotic stresses. Planta 225:353–364

18. Slathia S, Sharma A, Choudhary SP (2012) Influence of exoge-

nously applied epibrassinolide and putrescine on protein content,

antioxidant enzymes and lipid peroxidation in Lycopersicon es-

culentum under salinity Stress. Am J Plant Sci 3:714–720

19. Anuradha S, Rao SSR (2001) Effect of brassinosteroids on

salinity stress induced inhibition of seed germination and seedling

growth of rice (Oryza sativa L.). Plant Growth Regul 33:151–153

20. Sirhindi G, Kumar S, Bhardwaj R, Kumar M (2009) Effects of

24-epibrassinolide and 28-homobrassinolide on the growth and

antioxidant enzyme activities in the seedlings of Brassica juncea

L. Physiol Mol Biol Plants 15:335–341

21. Ali B, Hayat S, Fariduddin Q, Ahmad A (2008) 24-epibrassino-

lide protects against the stress generated by salinity and nickel in

Brassica juncea. Chemosphere 72:1387–1392

22. Cohen S (1998) A guide to the polyamines. Oxford University

Press, Oxford

23. Igarashi K, Kashiwagi W (2000) Polyamines: mysterious mod-

ulators of cellular functions. Biochem Biophys Res Commun

271:559–664

24. Sawhney RK, Tiburcio AF, Altabella T, Galston AW (2003)

Polyamines in plants: an overview. J Cell Mol Biol 2:1–12

25. Takahashi T, Kakehi JI (2009) Polyamines: ubiquitous polyca-

tions with unique roles in growth and stress responses. Ann Bot

105:1–6

26. Giridhar P, Mahendranath G, Venugopalan A, Ravishankar GA

(2012) Enhanced yield of food colourant annatto from seeds of

Bixia orellana L. The efficacy of polyamines floral spray. Proc

Natl Acad Sci India Sect B Biol Sci 8:553–556

27. Kuznetsov V, Radyukina NL, Shevyakova NI (2006) Polyamines

and stress: biological role, metabolism and regulation. Russ J

Plant Physiol 53:583–604

28. Zhao FG, Qin P (2004) Protective role of exogenous polyamines

on root tonoplast function against salt stress in barley seedlings.

Plant Growth Regul 42:97–103

29. Jiu-ju D, Shi-Rong G, Yun-Yan K, Yan-Sheng J (2007) Effects of

exogenous spermidine on polyamine content and antioxidant

system in roots of cucumber under salinity stress. J Ecol Rural

Environ 4:11–17

30. Limaye AS, Deore GB, Shinde BH, Laware SL (2010) Assese-

ment of Adiantum trapeziforme L for antioxidant activities. Asian

J Exp Biol Sci 1:75–80

31. Ahmad P, Jallel CA, Arooz MM, Nabi G (2009) Generation of

ROS and non enzymatic antioxidants during abiotic stress in

plants. Bot Res Int 2:11–20

32. Li X-W, Lei M, Chen T-B, Wan X-M (2009) Roles of sulfur in

the arsenic tolerant plant Adiantum capillus-veneris and the

hyperaccumulator Pteris vittata. J Korean Soc Appl Biol Chem

52:498–502

33. Singh N, Raj A, Khare PB, Tripathi RD, Jamil S (2010) Arsenic

accumulation pattern in 12 Indian ferns and assessing the

potential of Adiantum capillus-veneris, in comparison to Pteris

vittata, as arsenic hyper accumulator. Bioresour Technol 101:

8960–8968

34. Lowry OH, Rosebrough NJ, Farr AL (1951) Protein measurement

with the folin phenol reagent. J Biol Chem 193:265–275

35. Putter J (1974) Peroxidase. In: Bergmeyer HU (ed) Methods of

enzymatic analysis. Verlag Chemie, Weinhan, pp 685–690

36. Aebi HE (1974) Catalase. In: Bergmeyer HU (ed) Methods of

enzymatic analysis, vol 4. Verlag Chemie, Weinhan, pp 273–282

37. Kono Y (1978) Generation of superoxide radical during autoxi-

dation of hydroxylamine and an assay for superoxide dismutase.

Arch Biochem Biophys 186:189–195

38. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of

free proline for water stress studies. Plant Soil 39:205–208

39. Ragazzi E, Veronese G (1973) Quantitative analysis of phenolics

compounds after thin-layer chromatography separation. J Chro-

matogr 77:369–375

40. Heath RL, Packer L (1968) Photoperoxidation in isolated chlo-

roplasts. I. Kinetics and stoichiometry of fatty acid peroxidation.

Arch Biochem Biophys 125:189–198

41. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of

photosynthetic biomembranes. Methods Enzymol 148:350–382

42. Zhu JK (2007) Plant salt stress. In: Encyclopedia of Life Science,

Wiley, Chichester

43. Ahmad P, Jallel CA, Arooz MM, Nabi G (2009) Generation of

ROS and non enzymatic antioxidants during abiotic stress in

plants. Bot Res Int 21:11–20

44. Scandalios JG (1993) Oxygen stress and superoxide dismutase.

Plant Physiol 101:7–12

45. Chai YY, Jiang CD, Shi L, Shi TS, Gu WB (2010) Effect of

exogenous spermine on sweet sorghum during germination under

salinity. Biol Planta 54:145–148

46. Maity U, Bera AK (2009) Effect of exogenous application of

brassinolide and salicylic acid on certain physiological and bio-

chemical aspects of green gram (Vigna radita L. wilczek). Indian

J Agric Res 43:194–199

47. Ozdemir FO, Bor M, Demiral, T Tuurkan (2004) Effects of

24-epibrassinolide on seed germination, seedling growth, lipid

peroxidation, proline content and antioxidative system of rice

(Oryza sativa L.) under salinity stress. Plant Growth Regul 42:

203–211

48. Arora N, Bhardwaj SharmaP, Arora HK (2008) Effect of

28-homobrassinolide on growth, lipid peroxidation and antioxi-

dative enzyme activities in seedlings of Zea mays L. under

salinity stress. Acta Physiol Planta 30:833–839

49. Chen S, Schopfer P (1999) Hydroxyl-radical production in

physiological reactions. Eur J Biochem 260:726–745

50. Scandalios JG (1997) Molecular genetics of SOD in plants. In:

Scandalios JG (ed) Oxidative stress and the molecular biology

ofantioxidant defense. Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, pp 527–568

51. Sheokand S, Kumari A, Sawhney V (2008) Effect of nitric oxide

and putrescine on antioxidative responses under NaCl stress in

chickpea plants. Physiol Mol Biol Plants 14:355–362

52. Sairam RK, Tyagi A (2004) Physiology and molecular biology of

salinity stress tolerance in plants. Curr Sci 86:407–421

53. Anuradha S, Rao SSR (2007) The effect of brassinosteroids on

radish (Raphanus sativus) seedlings under cadmium stress. Plant

Soil Environ 53:465–472

54. Ozturk L, Demir Y (2003) Effects of putrescine and ethephon on

some oxidative stress enzyme activities and proline content in salt

stressed spinach leaves. Plant Growth Regul 40:89–95

55. Abd El-Wahed MS, Gamal El din KM (2004) Stimulation effect

of spermidine and stigmasterol on growth, flowering, chemical

constituents and essential oil in chamomile plants (Chamomila

recutital L. Rausch) Bull. J Plant Physiol 30:48–60

56. Celik O, Atak C (2012) The effect of salt stress on antioxidative

enzymes and proline content of two Turkish tobacco varieties.

Turk J Biol 36:339–356

57. Anuradha S, Rao SSR (2003) Application of brassinosteroids to

rice seed (Oryza sativa) reduce the impact of salt stress on

growth, prevented photosynthetic pigment loss and increased

nitrate reductase activity. Plant Growth Regul 40:29–32

Salinity Stressed Adiantum capillus-veneris Leaves 191

123

Author's personal copy

58. Houimli SIM, Denden M, Mouhandes BD (2010) Effect of

24-epibrassinolide on the growth, chlorophyll, electrolyte leakage

and proline by pepper plants under NaCl stress. Eur Asian J

Biosci 4:96–104

59. Chattopadhayay MK, Tiwari BS, Chattopadhayay G, Sengupta

DN, Ghosh B (2002) Protective role of exogenous polyamines on

the salinity stressed rice (Oryza sativa) plants. Physiol Planta

116:192–199

60. Anjum MA (2011) Effect of exogenously applied spermidine on

growth and physiology of citrus rootstock Troyer citrange under

saline conditions. Turk J Agric For 35:43–53

192 A. Sharma et al.

123

Author's personal copy