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Original Article

Accumulation of chromium and its effects onphysiological and biochemical parameters ofAlternanthera philoxeroides seedlings

Sindhujaa Vajravel*, Poornima Saravanan

Dept. of Plant Morphology and Algology, School of Biological Sciences, Madurai Kamaraj University,

Madurai 625021, India

a r t i c l e i n f o

Article history:

Received 24 April 2013

Accepted 22 July 2013

Available online 22 August 2013

Keywords:

Antioxidative enzymes

Alternanthera philoxeroides

Chromium

* Corresponding author. Tel.: þ91 9952350916E-mail address: Sindhujaapsr@gmail.com

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.07.028

a b s t r a c t

Aim: To assess the effects of different concentrations of chromium (25; 50; 100; 150 mg/l) in

the plant, Alternanthera philoxeroides.

Method: Different concentrations of chromium (25; 50; 100; 150 mg/l) were applied for 12

days and assessed by measuring changes in the growth; photosynthetic pigments activ-

ities; and antioxidative enzymes: catalase (CAT); peroxidase (POD); ascorbate peroxidase

(APX) and total soluble protein changes. Metabolic responses to chromium (Cr) exposure

and metal uptake were also experimentated.

Results: It was found that chromium was accumulated in shoots and roots of A. philoxer-

oides. The shoots accumulated 111.27 mg Cr/g of their dry weight at 150 mg/l Cr concen-

tration, while the roots accumulated 751.71 mg Cr/g. The photosynthetic pigment contents

increased with the higher concentration of Cr. Both in shoots and roots Cr could induce rise

of the activity of CAT; POD and APX. The total soluble protein contents also increased with

the increased concentration of Cr.

Conclusion: The results from the present experiments suggest that high concentrations of

Cr cause oxidative damage as evidenced by increased antioxidative enzymes, photosyn-

thetic pigments and changes in total soluble protein content. Induction of antioxidative

enzymes could the reason for tolerating higher levels of metals by A. philoxeroides plants.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction can cause liver damage; pulmonary congestion and causes

Chromium is one of the toxic metals of wide spread use. The

International Agency for Research on Cancer (IARC) has re-

ported that Cr (VI) is carcinogenic to humans and in addition it

(mobile).(S. Vajravel).2013, JPR Solutions; Publi

skin irritation resulting in ulcer formation. It is mostly used in

many industries such as wood preservation, leather

tanning, electroplating and steel productions.1,2 Phytor-

emediation is a promising cleanup technology for contami

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 6 3 3e6 3 9634

natedsoils, groundwater andwastewater that is both low-tech

and low-cost. Alternanthera philoxeroides is one of the aquatic

macrophytes which are commonly known as alligator weed. It

coexists abundantly in natural habitat all over the world.

Therefore it can be used as a convenient plant material for

heavy metal toxicity investigations.3 In many reports chro-

mium has been demonstrated to induce the formation of

reactive oxygen species (ROS) and free radicals (FR) in plants

such as hydrogen peroxide (H2O2) hydroxyl radicals (�OH) and

superoxide radicals (O2��); either by direct electron transfer

involvingmetal cations or as a consequence ofmetalmediated

inhibition of metabolic reactions.4 Free radicals can cause

oxidative damage to the biomolecules such as lipids, proteins

andnucleicacids.5 Toavoid thiskindof cellulardamage, plants

posses a complex system of antioxidative enzymes like cata-

lase, peroxidase and ascorbate peroxidase. Those play amajor

to tolerate theplantsby scavengingROSproducedunderheavy

metal stress.6

The present study was undertaken to examine Accumu-

lation of Chromium and its Effects on Physiological and

Biochemical Parameters of Alternanthera philoxeroides Seed-

lings under hydroponic systems.

Table 1 e Effect of Cr on shoots and root length of A.philoxeroides after 12 days treatment.

Cr concentration(mg/L)

Shootlength(cm)

Rootlength(cm)

IT values(%)

RWC(%)

Root Shoot

Control 25.7 � 0.72 16.8 � 0.82 0.00 0.00 66.0

25 23.5 � 0.29 12.3 � 0.14 88.3 92.7 65.8

50 22.2 � 0.47 10.8 � 0.83 85.7 75.7 65.8

100 20.3 � 0.27 11.6 � 0.81 78.6 77.0 64.5

150 20.4 � 0.54 10.2 � 0.20 80.8 64.0 64.5

2. Research methods

2.1. Plant collection and chromium treatment

Alternanthera philoxeroides were collected and then washed

several times in running tap water to wash out the soil par-

ticles from plants. Approximately same height and weights of

plants were carefully selected and transferred into plastic

container filled with full strength Hoagland Nutrient Solution

for hydroponic settings.7 The hydroponic systemwas set up in

the GreenHouse. After 12 days both the root and shoot lengths

of hydroponically growing plants were determined and

treated with Cr (potassium dichromate) in different concen-

trations 0; 25; 50; 100; 150 mg/l; while medium without these

heavy metals served as control. The physiological and

biochemical parameters were investigated after 12 days of Cr

treatment.

2.2. Physiological parameters

2.2.1. Growth parametersBoth shoot and root lengths were measured before and after

treatment of Cr in A. philoxeroides seedlings. The biomass was

estimated by the measurement of shoot and root dry weight.

Index of tolerance (IT) for the root and shoot was calculated.

Water content of leaves was calculated, using the values ob-

tained from fresh and dry weights of Cr treated plants, ac-

cording to (FW-DW)*100/FW.8

2.2.2. Photosynthetic pigment assayA. philoxeroides leaf tissues samples (100mg) were extracted in

ice e cold pestle and mortar with 2 ml of 80% acetone (v/v) as

described by Arnon.9 Leaf extracts were centrifuged at

5000 rpm for 10 min and upper layer was collected for chlo-

rophyll a/b and carotenoid estimation. The absorbance was

measured at 470; 645; 663 nm in the UVeVisible

spectrophotometer. The cholorophyll pigments and caroten-

oids were estimated according to the standard calculations.

Chl a ¼ ½ð13:95A665 � 6:88A649Þ � 10�=100;Chl b ¼ ½ð24:96A649 � 7:32A665 � 10Þ=100�;Car ¼ ½ð1000A470 � 2:05Ca� 114:8CbÞ=245� � 10=100

2.2.3. Chromium accumulation analysis by ICP-AESThe Cr heavy metal accumulation was analysed by ICP-AES.10

2.3. Biochemical assays

2.3.1. Ascorbate peroxidase assay (APX)APX activity was determined according to the method

mentioned byNakano andAsada.11 The reactionmixture used

for this assay contained 50 mM phosphate buffer (pH 7.8);

0.5 Mm ascorbic acid 0.1 mM EDTA; 65 Mm H2O2; enzyme

extract and distilled water. The oxidation of ascorbic acid was

at 290 nm absorbance for 30 s using UVevisible spectropho-

tometer (Double Beam Spectrophotometer 2203).

2.3.2. Assay of catalase (CAT)The CAT activity was performed by Aebi method.12 The

reaction mixture used for this assay; 50 mM phosphate buffer

(pH 7.8); 75 mM H2O2, enzyme extract and distilled water. The

reaction was started by adding H2O2 and CAT activity was at

240 nm absorbance.

2.3.3. Assay of peroxidase (POD)POX activity was measured using Castillo et al, method.13 The

3 ml of reaction mixture contained; 50 mM phosphate buffer

(pH 6.1); Guaiacol (16 mM); H2O2 (2 mM); enzyme and distilled

water. POX activity was measured at 470 nm absorbance.

2.3.4. Determination of protein contentTotal soluble protein supernatant was determined according

to Bradford method14 using Bovine Serum Albumin (BSA) as

standard and was expressed in mg/g fresh weight.

3. Analysis of results

3.1. Cr toxicity on A. philoxeroides plant growth

A. philoxeroides seedlings were exposed to different con-

centrations (25; 50; 100; 150 mg/l) of Cr for 12 days. Both the

shoot and root growth were affected in all the concentra-

tions used in the experiments. Table 1 depicted the effect of

ides(m

g/g

FW

)

9day

12day

01

0.26�

0.001

0.35�

0.00

04

0.32�

0.002

0.27�

0.00

01

0.31�

0.001

0.28�

0.00

01

0.39�

0.001

0.35�

0.00

00

0.28�

0.002

0.34�

0.00

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 6 3 3e6 3 9 635

Cr on shoot and root length; index of tolerance and relative

water content between control and treated plants after 12

days treatment. Moreover; the shoot and root lengths of

plants were significantly decreased with the higher con-

centration of chromium (Fig. 1). The relative water content

and the index of tolerance revealed that both shoot and

root lengths were significantly affected with the higher

concentration of chromium. In addition; the size of the

leaves of Cr treated plants was smaller than those in the

control plant leaves.

Caro

teno

3day

6day

01

0.16�

0.001

0.33�

0.0

02

0.27�

0.004

0.22�

0.0

01

0.29�

0.005

0.24�

0.0

01

0.31�

0.003

0.35�

0.0

02

0.33�

0.002

0.33�

0.0

3.2. Cr effect on photosynthetic pigments activity

The effects of chromium on photosynthetic pigments are

chlorophyll a; chlorophyll b and carotenoides of plant leaves

is presented in Table 2. Different concentrations of chro-

mium on different exposure periods significantly increased

the contents of chlorophyll a, chlorophyll b and carotenoides

in comparison with the untreated plants (Figs. 2e4).

hyllb(m

g/gFW

)

9day

12day

02

0.26�

0.002

0.32�

0.0

03

0.29�

0.004

0.25�

0.0

02

0.29�

0.004

0.24�

0.0

03

0.37�

0.002

0.31�

0.0

02

0.28�

0.002

0.32�

0.0

3.3. Cr accumulation

The Cr concentration in A. philoxeroides increased with

increasing Cr levels in the nutrient solution. The highest Cr

concentrations accumulated in shoots and roots were 111.27

and 751.71 mg g�1 DW respectively; when plants were

treated with 150 mg l�1 Cr in the solution. The Cr concen-

trations in roots were much higher than that in shoots.

ca

rotenoidesofA.philox

eroides

leaves.

gFW

)Chloro

p

day

12day

3day

6day

�0.02e

1.11�

0.00a

0.13�

0.001

0.21�

0.0

�0.006

0.85�

0.004

0.24�

0.001

0.18�

0.0

�0.005

0.79�

0.004

0.33�

0.007

0.20�

0.0

�0.003

1.06�

0.002

0.27�

0.003

0.31�

0.0

�0.008

1.03�

0.003

0.29�

0.004

0.30�

0.0

3.4. Effect of Cr treatment on antioxidative enzymeactivities in leaves tissues of A. philoxeroides

3.4.1. Cr effect on CAT activityTable 3 depictes the effects of chromium on catalase activity

(U/g FW) of leaves of A. philoxeroides at different concentra-

tions and exposure periods. The activity of catalase was

significantly increased in A. philoxeroides seedlings with

metal treatments and also catalase activities differed with

increasing concentrations of metals as well as different

exposure periods (Fig. 5). The increased trend of catalase

activity (1.634 U/g FW)was observed at 100mg/l Cr treatment

and there was slight decrease in (1.097 U/g FW) at 150mg/l Cr

treatment.

Table

2e

Effect

ofCronch

loro

phylla/b

and

Crco

nc.

(mg/L)

Chloro

phylla(m

g/

3day

6day

9

Control

0.454�

0.002

0.716�

0.004

0.697

25

0.804�

0.000

0.659�

0.003

0.83

50

0.866�

0.002

0.71�

0.004

0.82

100

1.012�

0.003

1.081�

0.002

1.06

150

0.969�

0.001

1.012�

0.005

1.03

Fig. 1 e Effect of Cr on growth responses of A. philoxeroides.

3.4.2.

Creffect

onAPX

activ

ityThech

angesoccu

rredin

APXactiv

itiesare

depicted

inTable

3.TheAPX

activ

ityin

leaveswasgradually

incre

ase

din

A.

philoxeroid

esse

edlin

gsatth

ehigherco

nce

ntra

tionofCr.

But

theactiv

itywasslig

htly

decre

ase

d(3.356U

mg �

1pro

tein)at

thehigherco

nce

ntra

tionof150m

g/lC

r;however,th

eactiv

ity

(1.24U

mg �

1pro

teins)

incre

ase

dsig

nifica

ntly

(p<

0.05)in

all

Crtre

atm

ents

use

dasco

mparedto

theco

ntro

l(Fig.6).

Fig.3e

Effe

ctofCron

chloro

phyllbofA.philox

eroides

leaves.

Fig.4

eEffe

ctofCron

caro

tenoidesofA.philox

eroides

leaves.

Fig.2e

Effe

ctofCron

chloro

phyllaofA.philox

eroides

leaves.

journalofpharmacy

research

7(2

013)

636

Table 3 e Effect of Cr on CAT activity (U/g FW), APX activity (U/g FW) and POD of leaves of plants at different concentrations and exposure periods. Data are means ± SE(n [ 3), significantly different ( p < 0.05) to control plants.

Conc of Cr(mg/L)

CAT activity (U/g FW) time (days) APX activity (U/g FW) POD activity (U/g FW)

3 days 6 days 9 days 12 days 3 days 6 days 9 days 12 days 3 days 6 days 9 days 12 days

Control 0.194 � 0.039 0.867 � 0.0115 1.029 � 0.008 0.250 � 0.002 3.277 � 0.294 3.260 � 0.065 3.351 � 0.030 3.091 � 0.054 3.517 � 0.233 3.774 � 0.088 2.143 � 0.039 4.278 � 0.002

25 0.291 � 0.019 0.750 � 0.014 1.242 � 0.035 0.465 � 0.019 3.430 � 0.052 3.551 � 0.231 3.649 � 0.040 3.333 � 0.027 4.486 � 0.489 4.186 � 0.128 5.973 � 0.149 5.283 � 0.019

50 0.408 � 0.017 0.954 � 0.039 1.257 � 0.024 0.470 � 0.034 3.857 � 0.034 3.684 � 0.154 3.757 � 0.062 3.360 � 0.056 5.565 � 0.218 7.591 � 0.347 6.621 � 0.022 8.550 � 0.034

100 0.328 � 0.013 1.106 � 0.022 1.260 � 0.023 0.609 � 0.025 3.702 � 0.067 3.721 � 0.147 3.745 � 0.062 3.574 � 0.102 8.480 � 0.125 9.490 � 0.087 6.767 � 0.179 8.925 � 0.025

150 0.210 � 0.000 1.044 � 0.052 1.634 � 0.007 1.097 � 0.186 3.277 � 0.294 3.892 � 0.099 3.812 � 0.108 3.356 � 0.081 7.194 � 0.212 9.909 � 0.240 7.758 � 0.449 10.04 � 0.186

633e639

Fig. 5 e Effect of Cr on CAT activity.

Fig. 7 e Effect of Cr on POD activity.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 6 3 3e6 3 9 637

3.4.3. Cr effect on POD activityThe effects of Cr on POX are illustrated in Table 3. Plants

exposed to Cr showed an increase in the POX activity in all

concentrations used in the present study when compared to

the control. However, a significant increase in the activity of

POX (10 U mg�1 protein) was observed at 150 mg/l Cr treat-

ment (Fig. 7). Therefore, it seems that a low concentration of

Cr (25 mg/l) in the medium was sufficient to activate the

antioxidant system which aims to protect plants from heavy

metal stress.

3.4.4. Effect of Cr treatment on antioxidative enzymeactivities in root tissues of A. philoxeroidesTable 4 shows the effect of chromium on catalase, peroxidase

and ascorbate peroxidase activity (U/g FW) of root tissues of

A. philoxeroides at different concentrations after 12 days

treatment. The activity of catalase, peroxidase and ascorbate

peroxidase significantly increased in the roots of A. philoxer-

oides with increasing metal treatments (Fig. 8). However the

catalase, peroxidase and ascorbate peroxidase activities

differed with concentrations. But in the chromium treated

plants the highest increase in POD activity was noticed when

compared to other enzyme activities.

Fig. 6 e Effect of Cr on APX activity.

3.5. Cr effect on total soluble protein content

TreatmentwithdifferentCrconcentrationsshowedasignificant

effect on the total soluble content (Fig. 9). Accumulation of total

solubleproteincontent level in leavesshowed increasedtrendin

all the concentrations used, however the significant level of

protein accumulation noticed was 11.91 and 11.77mg protein/g

fresh wt. with 100 and 150 mg/l Cr treatments, respectively

(Table 5). This result indicates that the plant is experiencing

heavy metal stress at higher Cr concentrations that triggers

various antioxidant enzymes as consequence.

4. Discussion

In the present study, the effect of chromium heavy metal treat-

ment on A. philoxeroides under hydroponics system was

observed. The obtained results showed that the growth of A.

philoxeroides seedlings were significantly affected in general but

shoot growth was highly affected than root at higher concen-

trations of chromium (Fig. 1). Reduction of shoot growth at

higher concentration of Cr may be correlated to hyper accumu-

lation of Crmetal byA. philoxeroides. Similar growth responses of

A. philoxeroides in the presence of Cr were also reported in Ses-

bania drummondii plants treated with Pb; Cu; Ni and Zn.15

Although there was a growth inhibition in Cr seedlings, the

rate of growth reductionwasnot statistically significant at lower

concentrations in roots compared to the control, while the

growth reduction in shoot suggests that the plant was accumu-

lating more Cr ions in their aerial parts as consequence. When

increased the concentrationsofCr in themedium, theshoot and

root lengths of the seedlings were decreased gradually.

Furthermore; IT values and RWC in the plants under Cr stress

were increased in the lower higher concentration and it is

decreased in higher concentration after 12 days of exposure

(Table1).Basedon these traits; it is suggested thatA.philoxeroides

seedlingshavetheability inhyperaccumulationofCr; since they

toleratemetal toxicity which is crucial characteristic feature for

hyper accumulators. Excessive Cr accumulation in plant tissue

can be toxic to the plants, affecting several physiological and

biochemical processes and growth.

Table 4e Effect of total soluble protein content in leaves of A. philoxeoides after 12 days of Cr treatment. Data aremeans ± SE(n [ 3), significantly different (P < 0.05) to control plant.

Cr concentration mg/l 3rd day (mg/g F. Wt) 6th day (mg/g F. Wt) 9th day (mg/g F. Wt) 12th day (mg/g F. Wt)

Control 7.99 � 0.208 12.38 � 0.083 12.10 � 0.208 10.61 � 0.168

25 10.3 � 0.05 16.02 � 0.083 12.57 � 1.154 10.89 � 0.227

50 9.18 � 0.084 15.70 � 0.682 12.95 � 0.168 11.56 � 0.168

100 9.12 � 0.239 16.31 � 0.207 11.56 � 0.082 11.91 � 0.114

150 8.19 � 0.531 17.01 � 0.054 11.46 � 0.193 11.77 � 0.207

Fig. 8 e Effect of Cr treatment on antioxidative enzyme

activities in root tissues.

Fig. 9 e Effect of total soluble protein content in leaves.

Table 5 e Effect of Cr treatment on antioxidative enzymeactivities in root tissues of A. philoxeroides. Data aremeans ± SE (n [ 3) significantly different (P < 0.05) tocontrol plants.

Cr concentration(mg/L)

CAT(U/g F. Wt)

POD(U/g F. Wt)

APX(U/g F. Wt)

Control 0.255 � 0.005 2.602 � 0.126 1.407 � 0.124

25 0.293 � 0.002 3.056 � 0.149 2.418 � 0.085

50 0.338 � 0.009 3.190 � 0.257 2.496 � 0.015

100 0.335 � 0.002 2.880 � 0.137 2.695 � 0.078

150 0.105 � 0.005 2.771 � 0.117 2.771 � 0.014

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 6 3 3e6 3 9638

Cr metal accumulation in A. philoxeroides seedlings was

positively correlated with the induction of antioxidative en-

zymes. The enzyme CAT is one of the key enzymes for detoxi-

fication ofH2O2 via two electron transfer.16 In the present study,

increasedCATactivity inboth leaves and roots ofA. philoxeroides

was observed (Figs. 5 and 8). The maintenance of high CAT ac-

tivity in A. philoxeroides seedlings Cr stress represents an

important feature of metal accumulator tolerance under Cr

toxicity. APX showed highest sensitivity reaching maximal ac-

tivity inA. philoxeroides (Figs 6 and 8). This result suggests that Cr

triggered antioxidant level responsible for the removal of

excessive H2O2. Similar results were also reported by earlier re-

sults. The increased APX activity suggests its role in the detoxi-

ficationofH2O2 intowaterusingascorbateas theelectrondonor;

resulting in the formation of dehydroascorbate. It is recycled

back to ascorbate using reduced GSH as an electron donor and

the oxidized glutathione reductase.17

POD catalyses H2O2 dependent oxidation of substrate. Figs. 7

and 8 shows A. philoxeroides seedlings exposed to different Cr

concentrations and there was a significant difference in POD

activity. The increased POD activity had also been reported in

rice 18; Thus increased POD activity might be associated with

elevated ROS levels in A. philoxeroides seedlings under Cr stress.

Total soluble protein contents in the leaves of A. philoxeroides

seedlings in response to Cr exposure are also shown in (Fig. 9).

Since the soluble protein content in the leaf tissueswere slightly

higher inCr treated plants than in control plants in the 12 day of

the experiment; it is likely thatCr induced stress over the course

of the treatment and that antioxidative enzymes activitieswere

consequently same. It is reported that heavy metal stress has

beenshown to induceavarietyofproteins resulting inanoverall

increase in protein content.19 However the additional experi-

ment is necessary to confirm the tolerance of these plants to

heavymetal stress.

5. Conclusion

The results of the present study indicated that A. philoxeroides

accumulates high amounts of Cr in roots than shoots. A.

philoxeroides is a fast growing plant and has the ability to

tolerate high Cr (150 mg/l Cr) concentrations. Thus it can be

used for phytoremediation.

Conflicts of interest

All authors have none to declare.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 6 3 3e6 3 9 639

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