Interaction of Polyamine on Oxidative Stress

8/16/2019 Interaction of Polyamine on Oxidative Stress

http://slidepdf.com/reader/full/interaction-of-polyamine-on-oxidative-stress 1/10

Theoretical and Experimental Plant Physiology, 25(3): 203-212, 2013 203

Interaction of polyamine on oxidative stressinduced by exogenously applied hydrogenperoxide in Salvinia natans Linn

Chiranjib Mandal1, Nirmalya Ghosh1, Malay Kumar Adak 1*, Narottam Dey2

1Plant Physiology and Plant Molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani, India.2Department of Biotechnology, Visva-Bharati University, Shantiniketan, India.

*Corresponding author: [email protected]

Received: 13 January 2013; Accepted: 01 August 2013

ABSTRACT: A hydroponics experiment was carried out to study the role of hydrogen peroxide (H2O

2) through changes of oxidative

stress and antioxidation activity in Salvinia natans Linn. H2O

2 is an active oxygen species, widely generated in many biological

systems, and mediates various physiological and biochemical processes in plants. In this study, we demonstrated that exogenous

H2O

2 causes the degradation of pigments and induces the oxidative stress and the antioxidation activity. These effects enhanced

with increments of applied H2O

2 concentration. Application of polyamine reduced the generation and the accumulation of H

2O

2 and

modulated the enzyme activities.

KEYWORDS: Hydrogen peroxide, reactive oxygen species, antioxidative enzymes, polyamine, Salvinia natans.

ABBREVIATIONS: H2O

2: hydrogen peroxide; Put: putrescine; ROS: reactive oxygen species; DAB: 3,3-diaminobenzidine; TCA: tricholoroacetic acid;

DTT: dithiothreitol; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GPX: guaiacol peroxidase; APX: ascorbate peroxidase; CAT: catalase;

GR: glutathione reductase; SOD: superoxide dismutase; NBT: nitro-blue tetrazolium; GSH: reduced glutathione; GSSG: oxidized glutathione.

INTRODUCTION

Hydrogen peroxide (H2O

2)

is a Reacive Oxygen Species

(ROS), bu no a ree radical, which is wo elecron reduced

produc o a molecular oxygen. e generaion o H2O

2 in plan

cells is presen in a number o cellular meabolic pahways,including phoosynhesis, phoorespiraion and oher processes

involving he elecron ranser in cellular organelle. Under

abioic sress, ROS generaion is almos mandaory (Ghosh e al.

2011). By comparing wih superoxide (O2•), hydroxyl radical

(•OH) and other ROS, H2O

2 is he mos sable compound

(Cheeseman 2007). Wihin he cellular concenraion, H2O

2

behaves as a signaling molecule and i is used or he inducion o

gene expression, helpul or physiological process (Nahues e al.

2004). H2O

2 becomes more dangerous when i reacs wih some

ransiion meals as Fe2+ and produces wo molecules o OH- by

Fenon reacion (Becana e al. 1998).

Abioic sresses in differen orms lead o he esablishmen

o an over oxidaion saus o he issue and hus resul in

oxidaive sress. H2O2 isel being a ROS is duly generaed bysome oxidaive reacions, which could be derimenal isel, and

oher subsequen produc ollowing lysis (Cheeseman 2007).

H2O

2in subsequen reacion is convered ino OH- , which

is aal o plans. In plan sysem, some o he moieies can

inerac wih H2O

2 and oher ROS o moderae he oxidaive

damages. Polyamines are one o hose moieies which have

been repored o encouner a number o abioic sresses (waer

defici, meal oxiciy, high illuminaion), inducing oxidaive

exposure (Anjum e al. 2011).

RESEARCH ARTICLE



204

Mandal et al.

Theoretical and Experimental Plant Physiology, 25(3): 203-212, 2013

Polyamines are organic polycaionic unsauraed

hydrocarbon, wih wo or more primary amino groups.

e diamine purescine, riamine spermidine and eraamine

spermine are widespread in plan sysem, which are ubiquious

in naure having low molecular weigh and sraigh chain

aliphaic amines whose concenraion flucuaes rom µM omM (Roychoudhury e al. 2011). Polyamines are involved

in a variey o undamenal cellular processes, including

ranscripion, RNA modificaion, proein synhesis and he

modulaion o enzyme aciviies (abor and abor 1999).

e simples polyamine like purescine (Pu) is derived eiher

direcly rom ornihine by ornihine decarboxylase (ODC) or

rom arginine, ollowing several seps subsequenly caalysed

by arginine decarboxylase (ADC), agmaine iminohydrolase

and N-carbamoyl purescine amidohydrolase. Pu is convered

o spermidine and spermine by successive aciviies o

spermidine synhase and spermine synhase, respecively, wihhe use o decarboxylaed S-adenosyl mehionine (dcSAM) as

an aminopropyl donor (Bagni and assoni 2001, Cona e al.

2006, Moschou e al. 2008).

In plans, polyamines are ound o be variably disribued

in differen issues. A number o physiological regulaion in

conrol o cell division, embryogenesis, roo ormaion, rui

ripening, percepion o bioic and abioic sress response is

modulaed by polyamines (Kumar e al. 1997). Moreover,

polyamines have been eviden o be effecive in up/down

expression o physiological aciviies under abioic sresses

(Alcázar e al. 2010) and polyamine applied exogenously o

downregulae he oxidaive sress is less requenly sudied.

On he oher hand, H2O

2 is well documened o change he

redox poenial o he issues (Basu e al. 2010). ereore, i

could be hypohesized ha polyamine could be ineracing

wih cellular redox in issues o such a way ha ROS-induced

oxidaive damage is minimized. Precise mechanism o such

polyamine mediaed resisance o oxidaive sress is ye o be

deciphered. Wih his background, an experimen has been

conduced o induce plan wih exogenously applied H2O

2or

developing oxidaive sress and o monior is modulaion.

In mos o he cases, angiosperms have been implicaed

in sudies abou anioxidaion pahways under various abioicsresses (Sánchez-Galván e al. 2008). Non-angiosperm plans,

like erns, are less explored in his regard. Chinese brake ern

( Peris vitaa) has been proved o be a hyper accumulaor

o heavy meals and shows a good olerance agains ROS

under oxidaive sress (Xie e al. 2009). Salvinia naans is a

small aquaic ern wih branched creeping sems, bearing

hairs bu no rue roos and are ree-floaing widely grown in

conaminaed area or waer sreams wih prouse indusrial

effluens. ese effluens essenially conain differen oxic

maerials, including oxides and hydroxides o heavy meals,

which are mainly responsible or high producion o H2O

2

in waer (Gardner and Al-Hamdani 1997, Prado e al. 2010).

Due o high diffusion o H2O

2 in waer, hose conaminaed

areas become more oxic and, as a resul, plans ge affeced.

Salvinia naans are common habias o such sringen condiionand proved is abiliy o susain wih is opimum growh

(Dhir e al. 2011). ereore, a possible relaionship in regard

o polyamine mediaed H2O

2 meabolism deserves is worh o

be sudied. us, we propose o sudy how his ern species

could reac wih H2O

2 induced oxidaive sress and modulae

heir responses wih eliciors like polyamine. is sudy was

underaken o reveal he impac o H2O

2on Salvinia naans wih

reerence o oxidaive sress and is possible ineracion

wih polyamine.

MATERIAL AND METHODS

Plant material and growth conditions: Salvinia naans

(L.), floaing aquaics wih creeping sems, was aken as he

plan maerial. Plans were colleced rom unpollued pond

or marshy land. Afer repeaed washing wih deionized waer,

hese plans were ranserred ino Murashige and Skoog media

(Murashige and Skoog 1962) and kep in a greenhouse or

7 days a 37±1°C, 70–80% o relaive humidiy and duraion

o ligh/dark 14h/10h, respecively. Afer ha ime, he plans

were incubaed wih vary ing concenraions o 0, 50, 100 µM

o H2

O2

and 100 µM o H2

O2

supplemened wih 1 mM o Pu

or 6 h in a dark chamber (Sairam and Srivasava 2000). Plans

in each se o reamens were replicaed hree imes (n=3).

Determination of H2O

2 content: e H

2O

2conen o he

issue was measured specrophoomerically (Cecil, Model

No. CE7200), according o Loreo and Velikova (2001); 1 g

o resh sample wih hree replicaions or each reamen was

homogenized in 3 mL o richloroaceic acid (CA) (1%) and

cenriuged a 10,000 x g or 10 min a 4°C (Hermle, Model

No. Z323K). Subsequenly, 0.75 mL o he supernaan was

added o he 0.75 mL o 10 mM phosphae buffer (pH 7.0)

conaining 1.5 mL o 1 M KI. e inensiy was measureda 390 nm. e H

2O

2 conen was calculaed wih molar

exincion coefficien 0.28 μmol cm-1.

Histochemical detection of H2O

2: H

2O

2 was deeced

hisochemicaly in leaves by using 3,3-diaminobenzidine (DAB)

as subsrae (Chao e al. 2010). In he presen experimen, lea issue

was dipped in DAB (1 mg mL-1) soluion (pH 4) hrough

he cu ends o ronds or 8 h under ligh a 27°C. e issues

were hen decolorized in boiling ehanol (95%) or 20 min.



205

Polyamine and oxidative stress interaction in Salvinia natans


is reamen decolorized he issues, excep or he brown

colored polymerized produc produced by DAB wih H2O

2.

Afer cooling, he issues were observed under ligh microscope

o visualize he brown spos.

Net assimilation rate (NAR): e dry weighs weremeasured beore and afer incubaion in H

2O

2 reamens.

For boh cases, 5 g o whole plan issue were aken rom each

o he reamens and he samples were dried a 80oC in an

oven o record heir dry weighs. e NAR was calculaed on

he basis o dry weighs and lea area o he plans beore and

afer he reamen; i was compued as suggesed by Base Mia

and Shamsuddin (2011) and expressed as g m-2 d-1.

Lipid peroxidation: For lipid peroxidaion in erms o

malondialdehyde (MDA) conen, 5 g o leaves rom each

reamen wih hree replicaions were exraced wih exracion buffer conaining 20% (w/v) CA and 0.5% hiobarbiuric acid

(BA) ollowed by warming a 95°C or 30 min. Reacion was

erminaed by placing he mixure in ice or 30 min ollowed by

cenriugaion a 12,000 x g or 10 min. e absorbance o he

supernaan was read a boh 532 and 600 nm. MDA conen

was derived according o Dhindsa e al. (1981).

Estimation of chlorophyll: e chlorophyll conen

was esimaed according o Ghosh e al. (2011); 5 g o resh

samples rom each reamen wih hree replicaions were

homogenized horoughly wih 80% aceone and cenriuged a

4,000 x g or 10 min a 4°C. Supernaan was aken as he source

o chlorophyll and was measured by reading he absorbance a

645 and 663 nm wih a UV–V Specrophoomeer.

Determination of anthocyanin: Among he major

anioxidans, anhocyanin rom he reaed plans was

deermined according o Eryilmaz (2006). Leaves rom

each reamen were crushed in liquid N2 ollowed by

homogenizaion horoughly in 80% mehanol–HCl (v/v) and

he homogenae was incubaed or 48 h a 4°C. e homogenae

was filered and he filrae was cenriuged a 1,000 x g or

15 min a 4°C. e absorbance was recorded a wo differen wavelenghs and he quanificaion o anhocyanin was

obained by subracing he values o absorpion in nanomeers

(i.e. A 530

-A 600

).

Determination of flavonoids: e samples were crushed

in 80% aqueous ehanol ollowed by cenriugaion a 11,000 x g

or 20 min. e supernaan was added wih a reacion mixure

conaining 5% sodium nirie (NaNO2) and 10% aluminium

chloride (AlCl3). Finally, 1M NaOH was added o he reacion

mixure and absorpion was read a 510 nm. oal flavonoid

was deermined wih a sandard soluion o quercein (Sigma)

and expressed as μmol g-1 (Ghasemi e al. 2009).

Total phenolics: e resh leaves were homogenized in 80%

ho ehanol and cenriuged a 10,000 x g or 15 min a 4ºCor deerminaion o oal phenolics. e supernaan was kep

in a waer bah o evaporae ehanol and hen dissolved in a

minimum volume o waer. An aliquo o 0.5 mL was reaed

wih Folin-Ciocaleu reagen ollowed by addiion o 20%

sodium carbonae (Na2CO

3). e mixure was incubaed in a

boiling waer bah or 1 min. e inensiy o color was recorded

a 650 nm and he phenol conen was derived wih gallic acid

as sandard, according o Mohsen and Ammar (2009).

Assay of antioxidative enzyme activity: e assay or

anooxidan enzymes o Salvinia naans plans under H2O2 reamens was moniored by sudying he guaiacol peroxidase

(GPX), ascorbae peroxidase (APX), caalase (CA),

gluahione reducase (GR) and super oxide dismuase (SOD)

ollowing appropriae proocol or exracion, purificaion and

assay reacion (Maii e al. 2012, Yang e al. 2012). For each

ype o enzyme, he leaves were crushed in liquid nirogen

ollowed by homogenizaion wih respecive exracion buffer.

e enzyme exrac was colleced ollowing cenriugaion and

purificaion. e aciviy o GPX (EC 1.11.1.7) was assayed

specrophoomerically, according o Hu e al. (2009), in a

reacion mixure conaining 0.1 M phosphae buffer, pH 6.5, 1.5

mM O-dianisidine (elecron donor), 0.2 M H2O

2(subsrae)

and equivalen amoun o enzyme exrac conaining 50 µg

o proein. e change o absorbance was read a 430 nm and

he enzyme aciviy was deermined according o Maii e

al. (2012). e aciviy o APX (EC 1.11.1.11) was assayed

in a reacion mixure conaining 100 mM phosphae buffer

(pH 7.5), 0.5 mM ascorbae and 0.2 mM H2O

2, ollowed by

adding enzyme source o equivalen amoun o proein; he

absorbance was recorded a 290 nm (Karuppanapandian

e al. 2011). For assay o CA (E.C. 1.11.1.6), he enzyme

exrac conaining 50 µg o proein was incubaed in a reacion

mixure, which was prepared by 0.5 mM poassium phosphae buffer (pH 7) and 10 mM H

2O

2.e

aciviy was deermined

by reading he decreasing absorbance a 240 nm and was

calculaed wih he exincion coefficien o H2O

2 ,

as suggesed

by Aebi (1984). Gluahione reducase (GR) (EC 1.6 .4 .2)

was assayed in a reacion mixure conaining 100 mM HEPES-

KOH buffer (pH 7.5), 10 mM magnesium chloride (MgCl2)

,

0.5 mM mehionine, 0.5 mM DT, 0.2 mM NADH and 0.5 mM

oxidized gluahione (GSSG). e enzyme exrac conaining

50 μg o enzyme proein was added o he reacion mixure.



206

Mandal et al.


e aciviy was recorded rom oxidaion o NAD(P)H wih

is exincion coefficien 6.22 mM cm-1. One uni o enzyme

was regarded as μM NADPH oxidized min-1 μg proein-1.

e aciviy o SOD (EC 1.15.1.1) was assayed in an assay

mixure conaining 50 mM sodium phosphae buffer (pH 7),

15 mM mehionine, 75 µM NB and 100 mM EDA and kepunder fluorescen ligh or 10 min, ollowed by measuring he

absorbance a 560 nm.

Statistical analysis: All he observaions were recorded

wih hree replicaions (n=3) and he daa were expressed as

mean±SE. e saisical analysis was perormed by one-way

analysis o variance (ANOVA), aking p≤0.05 as significan.

RESULTS

Inra and inercellular levels o H2O

2 increased during

environmenal sresses. From he sudy o differenphysiological parameers o Salvinia naans grown under

various H2O

2 concenraions, i was visualized ha H

2O

2 plays

a crucial role in various physiological processes. As prediced,

Pu had also been eviden as a modulaor or he response o

plans under such H2O

2 induced oxidaive sress.

e effecs o oxidaive sress and exogenous Pu applicaion

on accumulaion o H2O

2 conen are shown in Figure 1A

.

us, he H2O

2 conen was significanly upregulaed in

proporion wih doses o H2O

2. ose were 1.66 and 2.39 old

a 50 and 100 µM o H2O

2 reamen, respecively, as compared

o conrol. On he conrary, Pu minimized he H2

O2

conen

by 24.5% in comparison o he highes concenraion o H2O

2

(i.e.100 µM) (Figure 1A).

A cell ular level, he accumula ion o H2O

2 was

deeced by hisochemical mehod when he lea issues

were sained wih DAB (Figure 1B). he issue analysis

showed a signiican increase o H2O

2accumulaion wih

disinc colorizaion, according o concenraion gradien

o peroxide. On he conrary, i was less deeced in he

issue o he sample reaed wih Pu. hereore, i clearly

suggesed ha Pu had been capable o minimize he H2O

2

accumulaion in he lea issues.

Plans under varying concenraion o H2O2 showed asignifican (p≤0.05) change in growh, which was recorded by

heir oal biomass or dry mater producion by means o NAR,

which means he capaciy o he plan or acquisiion o ne

dry mater over a period o ime. I was observed a significan

(p≤0.05) decline o NAR by 30.34 and 67.79% over conrol,

whereas Pu rerieved 1.51 old in comparison o he highes

concenraion o H2O

2(Figure 1C).

Lipid peroxidaion is a reliable cellular rai or

esablishmen o oxidaive damages in plan. In he presen

experimen, plans accumulaed a significan (p≤0.05) rise o

bc

b

0

0.2

0.4

0.6

0.8

1.0

1.2

M D A c o n t e n

t ( µ m o l g - 1 F W )

a

0

0.2

0.4

0.6

0.81.0

1.2

1.4

N A R ( g m - 2 d

- 1 )

a

b

c

bc

50

150

250

350

450

0

0

0

H2O

2 concentration (µM)

H2O


H2O


H 2

O 2

c o n t e n t ( µ M g - 1 o f F W )

c

b

a

b

A

B

C

D

0

50

100

100+1 mM Put

50 100 100+1 mM Put

100+1 mM Put10050

100+1 mM Put10050

Figure 1. (A) Generation of H2O

2; (B) Tissue analysis by

3,3-diaminobenzidine (DAB) stain; (C) Total net assimilation rate(NAR); (D) Lipid peroxidation (in terms of malondialdehyde – MDAcontent) of Salvinia natans under different concentrations (0, 50and 100 µM) of H

2O

2treatment and 100 µM of H

2O

2supplemented

with 1 mM Put after 7 days of culture. The values are plotted inmeans (±Standar error) of replication (n=3); *p≤0.05).



207



MDA as a resul o lipid peroxidaion excess. I showed 1.28

old increase in MDA conen a 50 µM o H2O

2 and 1.68 old

a highes concenraion o H2O

2(i.e.100 µM)

over conrol. Pu

was eviden o downregulae he H2O

2 conen by 20.98% in

comparison o he highes concenraion o H2O

2 (Figure 1D).

On he oher hand, H2O

2reamen resuled in he

reducion o oal chlorophyll conen. e loss o chlorophyll

in Salvinia naans was eminen wih he increase o H2O

2

doses in linear ashion. us, i was ound ha he chlorophyll

conen was reduced significanly (p≤0.05) by 24.06 and

42.13% a 50 and 100 µM o H2O

2reamen, respecively, wih

respec o conrol. On he conrary, Pu alleviaed he H2O

2

effec on chlorophyll conen by 1.17 old in comparison o he

highes concenraion o H2O

2 (Figure 2A).

Anhocyanin conen had similar responses like

chlorophyll and recorded a diminishing o 30.89 and 56.54%

a 50 and 100 µM concenraion o H2O2 , respecively, incomparison o conrol. On he oher hand, Pu rerieved

1.506 old o anhocyanin conen as compared o highes

concenraion o H2O

2 reamen (Figure 2B). In addiion,

he plans were induced o accumulae flavonoids under

exogenous applicaion o H2O

2. e flavonoid conen was

significanly upregulaed (p≤0.05) and i was 1.12 and 1.31 old

under reamens o H2O

2 (i.e. 50 and 100 µM, respecively).

Pu minimized he flavonoid conen by 11.3%, as compared

o H2O

2 alone (Figure 2C). An increase in oal phenolics wih

H2O

2concenraion was anoher ineresing observaion in he

plans’ response. A consisen rend in phenol accumulaion

was significanly (p≤0.05) recorded as 1.32 and 1.51 old a 50and 100 µM o H

2O

2 reamens, respecively, as compared o

conrol. On he conrary, Pu reduced he phenolic amoun by

15.46% in comparison o he highes concenraion o H2O

2

(Figure 2D).

Antioxidative enzymes: Responses o sress are maximally

maniesed ino expression o anioxidaive enzymes. In he

presen experimen, H2O

2 , being isel a poen reacive oxygen

species, could have been able o induce some o he imporan

anioxidaive enzymes. A significan role in SOD, GPX, APX,

CA and GR aciviy had revealed some ineresing resuls.e aciviy o anioxidizing enzymes like SOD was significanly

(p≤0.05) alered under H2O

2reamens in a dose dependen

manner. I was observed 1.23 and 1.54 old a 50 and 100 µM

o H2O

2 reamen, respecively, over conrol. Ineresingly, he

applicaion o Pu had minimized he aciviy by 12.89% in

comparison o he highes concenraion o H2O

2 (Figure 3A).

Figure 2. (A) Total chlorophyll content; (B) Anthocyanin content; (C) Flavonoid content; (D) Total phenol content of Salvinia natans underdifferent concentrations (0, 50 and 100 µM) of H

2O

2treatment and 100 µM of H

2O

2supplemented with 1 mM Put after 7 days of culture.

The values are plotted in means (±Standard error) of replication (n=3); *p≤0.05.

0

2

4

68

10

0

H2O

2concentration (µM)

0

H2O


H2O


0

H2O


C h l o r o p h y l l c o n t e n t

( m g g - 1 o

f t i s s u

e )

c bc

a

b

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

A n t h o c y a n i n c o n

t e n t

( A 5 3 0 )

b

a

b

0

0.2

0.4

0.6

0.8

1

F l a v o n o i d

c o n t

e n t

( m g g - 1 o

f f r e s h t i s s u e )

c b

a b

bc

b

0

20

40

60

80

100

120

T o t a l P h e n o l C o n t e n t

( m g g - 1 o

f t i s s u

e )

a

A B

C D

100+1 mM Put10050 100+1 mM Put10050

0 100+1 mM Put10050100+1 mM Put10050



208

Mandal et al.


e in viro aciviy o GPX was significanly (p≤0.05)

responsive under H2O

2reamen in a dose dependen manner.

I was observed 1.26 and 1.54 old in 50 and 100 µM H2O

2

reamens, respecively, in comparison o conrol. On he oher

hand, Pu reamen minimized he aciviy o GPX by 17.56%

when compared o 100 µM o H2O

2 reamen (Figure 3B).

he variaion in aciviy o APX was also dose

dependen (p≤0.05), in which 1.25 and 1.53 old a 50 and

100 µM o H2O

2 reamens were observed, respecively,

over conrol. he applicaion o Pu reduced he aciviy

by 14.78% in comparison o he highes concenraion o

H2O

2(i.e.100 µM) (Figure 3C).

On he oher hand, CA aciviy showed a differen patern

as i was decreased by 19.94 and 34.23% under 50 and 100 µM

o H2O

2reamens, respecively, over conrol. In addiion, Pu

modulaed he enzyme aciviy and recorded 1.22 old higher

over 100 µM o H2O

2reamen (Figure 3D).

Anoher anioxidaive enzyme, which is required or heseady mainenance o gluahione level, is GR. I showed an

increasing endency, which recorded 1.28 and 1.65 old a 50

and 100 µM o H2O

2 reamens, respecively, over conrol.

Pu reamen decreased he GR aciviy (Uni µg proein-1 min-1)

by 19.04% in comparison o he highes concenraion o H2O

2

(Figure 3E).

0

0.2

0.4

0.6

0.8

0

H2O

2 concentration (M)

H2O

2 concentration (M)

H2O

2 concentration (M)

H2O

2 concentration (M)

H2O

2 concentration (M)

S O D a c t i v i t y

( U m g - 1 p r o t e i n )

cbc

a b

0

1000

2000

3000

4000

5000

G P X a c t i v i t y

( U µ g - 1 p r o t e i n )

c ab

a

b

0

0.2

0.4

0.6

0.8

1

A P X a c t i v i t y

( µ m o l m i n - 1

m g - 1 p r o t e

i n )

C A T a c t i v i t y

( µ M o f H

2 O

2 u t i l i s e d

m g - 1 p r o t e i n )

c b

a

b

0

0.2

0.4

0.6

0.8

b

c b

a

00.2

0.40.6

0.8

11.21.41.6

G R A c t i v i t y

( U n i t g p r o t e i n -

1 m i n

- 1 )

b

c

bc

a

A B

C D

E

100+1 mM Put10050

0 100+1 mM Put10050

0 100+1 mM Put10050

0 100+1 mM Put10050

0 100+1 mM Put10050

Figure 3. Enzyme activities. (A) Superoxide dismutase (SOD); (B) Guaiacol peroxidase (GPX); (C) Ascorbate peroxidase (APX);(D) Catalase (CAT); (E) Glutathione reductase (GR) of Salvinia natans under different concentrations (0, 50 and 100 µM) of H

2O

2treatment

and 100 µM of H2O

2supplemented with 1 mM Put after 7 days of culture. The values are plotted in means (±Standard error) of replication

(n=3); *p≤0.05.



209



DISCUSSION

H2O

2 is no considered a ree radical like O

2- and OH-.

Moreover, wih respec o oxidaive sress, he hreshold value

o H2O

2 a he cellular level is considered an indirec sressor

or he esablishmen o he oxidaive damage (Sairam and

Saxena 2000). In he presen case, Salvinia naans recordeddisinc eaures o oxidaive damage o cellular meabolies.

us, chlorophyll conen was reduced under H2O

2 reamens

in a proporionae manner. In plans, one o he esablished

rais or oxidaive damages is he lipid peroxidaion, expressed

in erms o MDA conen. e accumulaion o MDA

conen in Salvinia plans in he presen experimen is no an

excepion. Wih he increasing concenraion o H2O

2 , he

plans exhibied an upregulaed oxidaion sae in he issues.

For various plan species under oxidaive sress, a significan

loss o chlorophyll and increase o lipid peroxidaion are

observed (Alcázar e al. 2010). In spie o his, he reducion inchlorophyll conen eiher hrough is diminished biosynheic

pahway or oxidaion is sill o be undersood (Panda 2001,

Panda and Khan 2009). e decline o phoosynhesis

hrough changes in chlorophyll fluorescence and oher relaed

phenomena have also been repored in oher species o Salvinia

under heavy meal sress (Dhir e al. 2008). Ineresingly, boh

chlorophyll and lipid peroxidaion in Salvinia naans exhibied

an ineracive benefi response wih he Pu applicaion in he

presen experimen, as i was effecive o downregulae he lipid

peroxidaion and upregulae chlorophyll conen.

Polyamines like spermidine, spermine and cadaverine ac

as alleviaors o he oxidaive sress in oher plan species like

Oryza saiva, Triicum aesivum , Arabidopsis halianaand Aropa

belladona (Roy e al. 2005). A he cellular level, polyamine

could miigae he lipid peroxidaion by downregulaing he

NAD(P)H-oxidase/NAD(P)H peroxidase aciviy (Gill and

ueja 2010). NAD(P)H oxidase is regarded as a poenial

source o O2

- and H2O

2 generaion, hus causing oxidaive

injuries in plan issues (Sharma e al. 2012). In he presen

experimen, a significan increase in H2O

2 conen migh be

an indirec indicaion o he NAD(P)H-oxidase aciviy as

he accumulaion o H2O

2 in he issues o he Salvinia naans

was observed wih DAB saining. Pu reduced he H2O2

accumulaion in he affeced issue ha was clearly observed

hrough he changes in he DAB saining.

Plans cope wih oxidaive sress wih he implemenaion o

anioxidaive pahways by enzymaic and non-enzymaic atribues

(Mandal e al. 2013). Among non-enzymaic atribues, differen

secondary meabolies are involved in quenching he energy o

ROS (Karuppanapandian e al. 2011). Salvinia naans exhibied

an elevaion o flavonoids and oal phenolics wih respec o

H2O

2 concenraion. e acivaion and increase o phenolics

become correlaed as a useul rai or plans under oxidaive

sress (Ghosh e al. 2011). e higher aciviy o hese major

anioxidans supposedly renders he Salvinia naans o

minimize he H2O

2 induced damages. For insance, flavonoids

are considered as a low molecular weigh anioxidan ha could

poenially scavenge he ROS and ree radical aciviy o escapehe lipid peroxidaion (Basu e al. 2010). us, anhocyanin

and phenolics are wo anioxidans ound o be poenial agens

in scavenging aciviy o ROS. However, anhocyanin recorded

a declining rend according o increasing concenraion o

H2O

2. In respec o non-enzymaic anioxidaion in Salvinia

naans , as recorded in he presen invesigaion, Pu modulaed

or changes in concenraions o flavonoids and anhocyanin

in he issue reaed wih H2O

2. Similar observaion was also

recorded in oher plans under condiion inducing oxidaive

sress (Roychoudhury e al. 2012). us, oxidaive sress

is characerized by abundance o phenolics in many planspecies, which migh be useul as an index or adapive naure

o plans. Polyamine has effecively supplemened in dose

dependen manner in ineracion o phenolics in quenching o

ROS, as repored earlier (Farooq e al. 2009). Simulaneously,

plans are able o cope wih he oxidaive damages by using

some enzymaic pahways, eiher hydrolyzing he ROS o

a sae o lower energy or downregulaing is biosynhesis.

Wihin he cellular hreshold, H2O

2can ac as an inercellular

messenger o induce some enzymes like SOD, GPX, APX,

CA, GR ec. (Shah and Nahakpam 2012, Mandal e al.

2013). Salvinia naans exhibied a concomian acivaion o

hose enzymes according o H2O

2 exposure as well as under

supplemenaion wih Pu. SOD, GPX, APX and GR were

significanly upregulaed wih H2O

2 reamen whereas CA

showed a considerable deacivaion. SOD underakes an

irreversible conversion o superoxide (O2

-) ino H2O

2 , which,

in urn, is reduced ino H2O and O

2 by various phenolics as

elecron donors (Upadhyaya e al. 2007).

Plans can induce a number o peroxidases in dieren

cellular comparmens (Achary e al. 2012). hus,

peroxidase, apar rom lysis o H2O

2 , can induce he

polymerizaion o phenolic residues in he cell wall, i.e.

in he process o ligniicaion reacion, OH- and oherree radicals ac as a conjugan o phenolic monomers.

Peroxidases like GPX and APX require guaiacol and

ascorbae as heir elecron donors, respecively. In he

presen invesigaion, heir aciviies were induced under

H2O

2 reamen and hereaer diminished by applicaion

o Pu. In general, plans conain a pool o peroxidases

ha are expressed according o he issue speciiciy and

even wih he sress inensiy, in discriminaing manner.

Any change or deviaion o anioxidaive enzy mes may



210

Mandal et al.


perurb he cellular redox o plans under abioic sresses

(Gill and ueja 2010). hereore, he deacivaion o

CA under H2O

2 reamen is deviaing rom he endency

o upregulaing he anioxidaive enzymes, as recorded in

presen ex perimen wih Salvinia naans. In ac, a signiican

decline in CA aciviy was also repored in oher planslike angiosperms (Ghosh e al. 2012). he deacivaion

o he CA by abioic sresses migh be undersood as

eiher reardaion o enzyme synhesis or is inacivaion

(Roychoudhury e al. 2011). Irrespecive o he cases, i

could be ascerained as a shorall o lysis o H2O

2 by he

enzyme. he oher possibiliy may arise rom he ac ha

cellular level o H2O

2 could be oxic enough o inhibi CA

aciviy (Basu e al. 2010). Salvinia naans could be able o

rerieve he CA aciviy by applicaion o Pu. Probably,

Pu migh have diminished he H2O

2impac

ha could

inerere wih CA aciviy (Shah and Nahakpam 2011).e changes in GR aciviy under H

2O

2 reamen and

is modulaion by Pu are indicaive o he alered oxidaive

saus in he issues o Salvinia naans. e increase in GR

along wih H2O

2 exposure hus indicaes he recycling

o GSH (reduced orm) rom GSSG (oxidized orm)

sae. I is also repored ha GSH is more effecive in

mainaining he adequae oxidaion sae in plans under

oxidaive sress (Ghosh e al. 2012). GSH, on he oher

hand, could supplemen he ascorbae rom is dehydraed

sae as a subsrae or ascorbae peroxidase. ereore, he

complemenaion o gluahione and ascorbae is essenial ormainaining he cellular equilibrium under sress condiions.

Similar endency has also been inerpreed in oher plan

species where involvemen o GR becomes a sensible rai or

assessing he oxidaive sress (Ding e al. 2012).

Conclusively, he resuls o he presen invesigaion clearly

reveal he cellular responses o Salvinia naans under H2O

2

induced oxidaive sress resuling in various physiological,

enzymaic and non-enzymaic changes. Pu is effecively

capable o miigae hose oxidaive damages. Salvinia naans ,

being a non-angiosperm, peridophyic plan species, is ound

o inerac wih polyamine a cellular level as revealed by hepresen experimen.

ACKNOWLEDGMENTS

is work was financially suppored by DS-PURSE

program, India.

REFERENCES

Achary VMM, Patnaik AR, Panda BB (2012) Oxidative biomarkers

in leaf tissue of barley seedlings in response to aluminium stress.Ecotoxicology and Environmental Safety 75:16-26.

Aebi H (1984) Catalase in vitro. Methods in Enzymology105:121-126.

Alcázar R, Altabella T, Marco F, Bortolotti C, ReymondM, Koncz C, et al (2010) Polyamines: molecules withregulatory functions in plant abiotic stress tolerance. Planta231:1237-1249.

Anjum SA, Xie X, Wang L, Saleem MF, Man C, Lei W (2011)Morphological, physiological and biochemical responses ofplants to drought stress. African Journal of Agricultural Research6:2026-2032.

Bagni N, Tassoni A (2001) Biosynthesis, oxidation andconjugation of aliphatic polyamines in higher plants. AminoAcids 20:301-317.

Baset Mia MA, Shamsuddin ZH (2011) Physio-morphologicalappraisal of aromatic fine rice (Oryza sativa L.) in relation to yieldpotential. International Journal of Botany 7:223-229.

Basu S, Roychowdhury A, Saha P, Sengupta DN (2010) Differentialantioxidative responses of indica rice cultivars to drought stress.Plant Growth Regulation 60:51-59.

Becana M, Moran JF, Iturbe-Ormaetxe I (1998) Iron-dependent

oxygen free radical generation in plants subjected toenvironmental stress: toxicity and antioxidant protection. Plantand Soil 201:137-147.

Chao YY, Chen CY, Huang WD, Kao CH (2010) Salicylic acid-mediated hydrogen peroxide accumulation and protectionagainst Cd toxicity in rice leaves. Plant and Soil 329:327-337.

Cheeseman JM (2007) Hydrogen peroxide and plant stress: achallenging relationship. Plant Stress 1:4-15.

Cona A, Rea G, Angelini R, Federico R, Tavladoraki P (2006)Functions of amine oxidases in plant development and defence.Trends in Plant Science 11:80-88.

Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence:correlated with increased levels of membrane permeability andlipid peroxidation, and decreased levels of superoxide dismutaseand catalase. Journal of Experimental Botany 32:93-101.

Dhir B, Sharmila P, Pardha Saradhi P (2008) Photosyntheticperformance of Salvinia natans exposed to chromium and zincrich wastewater. Brazilian Journal of Plant Physiology 20:61-70.

Dhir B, Sharmila P, Pardha Saradhi P, Sharma S, Kumar R, Mehta D(2011) Heavy metal induced physiological alterations in Salvinianatans. Ecotoxicology and Environmental Safety 74:1678-1684.



211



Ding S, Lei M, Lu Q, Zhang A, Yin Y, Wen X, et al (2012) Enhancedsensitivity and characterization of photosystem II in transgenictobacco plants with decreased chloroplast glutathione reductaseunder chilling stress. Biochimica et Biophysica Acta 1817:1979-1991.

Eryılmaz F (2006) The relationships between salt stressand anthocyanin content in higher plants. Biotechnology &

Biotechnological Equipment 20:47-52.

Farooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA (2009)Plant drought stress: effects, mechanisms and management.Agronomy for Sustainable Development 29:185-212.

Gardner JL, Al-Hamdani S (1997) Interactive effects of aluminumand humic substances on Salvinia. Journal of Aquatic PlantManagement 35:30-34.

Ghasemi K, Ghasemi Y, Ebrahimzadeh MA (2009) Antioxidantactivity, phenol and flavonoid contents of 13 citrus species peels andtissues. Pakistan Journal of Pharmaceutical Sciences 22:277-281.

Ghosh N, Adak MK, Ghosh PD, Gupta S, Sengupta DN, Mandal C

(2011) Differential responses of two rice varieties to salt stress.Plant Biotechnology Reports 5:89-103.

Ghosh N, Das SP, Mandal C, Gupta S, Das K, Dey N, et al (2012)Variations of antioxidative responses in two rice cultivars withpolyamine treatment under salinity stress. Physiology andMolecular Biology of Plants 18:301-313.

Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidantmachinery in abiotic stress tolerance in crop plants. PlantPhysiology and Biochemistry 48:909-930.

Hu Y, Ge Y, Zang C, Zu T, Cheng W (2009) Cd toxicity andtranslocation in rice seedlings are reduced by hydrogen peroxidetreatments. Plant Growth Regulation 59:51-61.

Karuppanapandian T, Moon JC, Kim C, Manoharan K, Kim W(2011) Reactive oxygen species in plants: their generation, signaltransduction, and scavenging mechanisms. Australian Journal ofCrop Science 5:709-725.

Kumar A, Altabella T, Taylor MA, Tiburcio AF (1997) Recentadvances in polyamine research. Trends in Plant Science2:124-130.

Loreto F, Velikova V (2001) Isoprene produced by leaves protectsthe photosynthetic apparatus against ozone damage, quenchesozone products, and reduces lipid peroxidation of cellularmembranes. Plant Physiology 127:1781-1787.

Maiti S, Ghosh N, Mandal C, Das K, Dey N, Adak MK (2012) Responses

of the maize plant to chromium stress with reference to antioxidationactivity. Brazilian Journal of Plant Physiology 24:203-212.

Mandal C, Ghosh N, Maiti S, Das K, Gupta S, Dey N, et al (2013)Antioxidative responses of Salvinia (Salvinia natans Linn.) toaluminium stress and it’s modulation by polyamine. Physiologyand Molecular Biology of Plants 19:91-103.

Mohsen S, Ammar ASM (2009) Total phenolic contents andantioxidant activity of corn tassel extracts. Food Chemistry112:595-598.

Moschou PN, Paschalidis KA, Roubelakis-Angelakis KA (2008)Plant polyamine catabolism: the state of the art. Plant Signaling& Behavior 3:1061-1066.

Murashige T, Skoog F (1962) A revised medium for rapid growthand bio assays with tobacco tissue cultures. PhysiologiaPlantarum 15:473-497.

Nathues E, Joshi S, Tenberge KB, von den Driesch M, OeserB, Bäumer N, et al (2004) CPTF1, a CREB-like transcriptionfactor, is involved in the oxidative stress response in thephytopathogen Claviceps purpurea and modulates ROS level inits host Secale cereale. Molecular Plant-Microbe Interactions17:383-93.

Panda SK (2001) The biology of oxidative stress in green cells:a review. In: Panda SK. Advances in Stress Physiology of Plants.India: Scientific Publishers. pp.1-13.

Panda SK, Khan MH (2009) Growth, oxidative damage andantioxidant responses in greengram (Vigna radiata L.) undershort-term salinity stress and its recovery. Journal of Agronomy

and Crop Science 195:442-454.

Prado C, Rodríguez-Montelongo L, González JA, Pagano EA, HilalM, Prado FE (2010) Uptake of chromium by Salvinia minima: effecton plant growth, leaf respiration and carbohydrate metabolism.Journal of Hazardous Materials 177:546-553.

Roy PK, Niyogi K, SenGupta DN, Ghosh B (2005) Spermidinetreatment to rice seedlings recovers salinity stress-induceddamage of plasma membrane and PM-bound H+-ATPase insalt-tolerant and salt-sensitive rice cultivars. Plant Science168:583-591.

Roychoudhury A, Basu S, Sengupta DN (2011) Amelioration ofsalinity stress by exogenously applied spermidine or spermine

in three varieties of indica rice differing in their level of salttolerance. Journal of Plant Physiology 168:317-328.

Roychoudhury A, Basu S, Sengupta DN (2012) Antioxidantsand stress-related metabolites in the seedlings of twoindica rice varieties exposed to cadmium chloride toxicity. ActaPhysiologiae Plantarum 34:835-847.

Sairam RK, Saxena DC (2000) Oxidative stress and antioxidants inwheat genotypes: possible mechanism of water stress tolerance.Journal of Agronomy and Crop Science 184:55-61.

Sairam RK, Srivastava GC (2000) Induction of oxidative stressand antioxidant activity by hydrogen peroxide treatment intolerant and susceptible wheat genotypes. Biologia Plantarum

43:381-386.

Sánchez-Galván G, Monroy O, Gómez J, Olguín EJ (2008)Assessment of the hyperaccumulating lead capacity of Salvinia minima using bioadsorption and intracellular accumulationfactors. Water, Air, & Soil Pollution 194:77-90.

Shah K, Nahakpam S (2012) Heat exposure alters the expressionof SOD, POD, APX and CAT isozymes and mitigates low cadmiumtoxicity in seedlings of sensitive and tolerant rice cultivars. PlantPhysiology and Biochemistry 57:106-113.



212

Mandal et al.


Shah K, Nahakpam S (2011) Heat stress and cadmiumtoxicity in higher plants: an overview. In: Hemantranjan A. Advances in Plant Physiology . Jodhpur: Scientific Publishers.pp.243-280.

Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactiveoxygen species, oxidative damage, and antioxidative defense

mechanism in plants under stressful conditions. Journal ofBotany 2012:1-26.

Tabor CW, Tabor H (1999) It all started on a streetcar in Boston.Annual Review of Biochemistry 68:1-32.

Upadhyaya H, Khan MH, Panda SK (2007) Hydrogen peroxideinduces oxidative stress in detached leaves of Oryza sativa L.General and Applied Plant Physiology 33:83-95.

Xie QE, Yan XL, Liao XY, Li X (2009) The ArsenicHyperaccumulator Fern Pteris vittata L. Environmental Science &Technology 43:8488-8495.

Yang ZB, Eticha D, Albacete A, Rao IM, Roitsch T, Horst WJ (2012)Physiological and molecular analysis of the interaction betweenaluminium toxicity and drought stress in common bean (Phaseolusvulgaris). Journal of Experimental Botany 63:3109-3125.

Documents

Interaction of Polyamine on Oxidative Stress