BIOTIC AND ABIOTIC STRESS

Cadmium induced oxidative stress and changes in solubleand ionically bound cell wall peroxidase activities in rootsof seedling and 3–4 leaf stage plants of Brassica juncea (L.) czern

Kusum Verma Æ G. S. Shekhawat Æ Astha Sharma ÆS. K. Mehta Æ V. Sharma

Received: 27 January 2008 / Revised: 5 April 2008 / Accepted: 17 April 2008 / Published online: 1 May 2008

� Springer-Verlag 2008

Abstract Metabolic adaptations to heavy metal toxicity

in plants are thought to be related with developmental

growth stage and the type of metal by which plant is

affected. In the present study, changes in ionically bound

CWP, soluble peroxidase activity, H2O2 level and Malon-

aldehyde content in roots of cadmium and copper stressed

seedlings and cadmium stressed 3–4 leaf stage plants of

Brassica juncea were investigated. Cadmium inhibits root

growth and reduces fresh biomass. The reduction in root

growth and fresh biomass is correlated with increased lipid

peroxidation and reduced tolerance. Treatment with cad-

mium resulted in an increase in ionically bound CWP

activity in roots of seedlings but no significant change in its

activity was found in roots of 3–4 leaf stage plants.

Increased level of H2O2 in roots of cadmium and copper

treated seedlings, show a direct correlation with increased

activity of ionically bound CWP. H2O2 level in 3–4 leaf

stage plant roots was found to be very low. Soluble per-

oxidase activity decreased in cadmium (50 and 100 lM)

treated seedlings but it was ineffective to cause any change

in its activity in 3–4 leaf stage plants. Copper treated

seedlings showed an increase in ionically bound CWP

activity, H2O2 level and MDA content. Ascorbic acid

(50 mM) pretreated seedlings shows significant decrease in

ionically bound CWP activity when exposed to 50 lM

cadmium. Hence, it is concluded that inhibition of root

growth in Brassica juncea seedlings by cadmium, is

associated with CWP catalyzed H2O2 dependent reactions

which are involved in metabolic adaptations to heavy-

metal stress.

Keywords Ionically bound cell wall peroxidase �Cadmium � Peroxidase � Brassica juncea L. �Oxidative stress

Abbreviations

CWP Cell wall peroxidase

MDA Malonaldehyde

ROS Reactive oxygen species

H2O2 Hydrogen peroxide

TMB 3, 30, 5, 50-tetramethyl benzidine

OFW Original fresh weight

FW Fresh weight

Introduction

Defensive responses in plants to abiotic stresses like heavy

metals have become a major part of the research in plant

sciences which mainly concentrate on the elucidation of

mechanisms playing role during plant–metal interactions.

This type of interactions mainly involves the roots of the

plant and their interaction with rhizosphere which is con-

taminated with heavy metal. Elevated levels of heavy

metals and various other environmental stress factors

accelerate the formation of reactive oxygen species (ROS)

in plants. There are many potential sources of ROS which

exist in plant cells that include reactions occurring in

normal metabolism and to stress. Sources producing ROS

Communicated by J. Zou.

K. Verma � G. S. Shekhawat (&) � A. Sharma � V. Sharma

Department of Bioscience and Biotechnology,

Banasthali University, Banasthali 304022, Rajasthan, India

e-mail: gyans34@yahoo.com

S. K. Mehta

Department of Botany, Mizoram University,

Aizawl 796001, Mizoram, India

123

Plant Cell Rep (2008) 27:1261–1269

DOI 10.1007/s00299-008-0552-7

are localized in almost every cell compartment, including

chloroplast, mitochondria, peroxisomes, apoplasm, plasma

membrane and cell walls (Mittler 2002). Among these

heavy metals cadmium is generally considered as a non-

essential heavy metal that causes oxidative stress via

induction of reactive oxygen species in plants and reduces

overall plant growth (Chaoui and El Ferjani 2005; Briat

2002; Stohs and Bagchi 1995). Phytotoxic symptoms of

cadmium toxicity include chlorosis, growth inhibition,

water imbalance, phosphorous and nitrogen deficiency

reduced manganese transport and accelerated senescence

(Benavides et al. 2005).

Plants have a range of potential mechanisms at the

cellular level that might be involved in the detoxification

and thus tolerances to heavy metal stress (Hall 2002). Cell

wall is sometimes considered as the site of primary action

of plant peroxidases. These peroxidases are known to be

involved in growth regulation and different biochemical

pathways. Increased rate of peroxidase activity has been

reported in many plant systems in response to reduced

growth rate (Lee and Lin 1995; Chen and Kao 1995).

Therefore, peroxidases are believed to be a putative wall

rigidification enzyme (Cosgrove 1997) and are directly

involved in lignin biosynthesis. Monomeric precursors of

lignin are enzymatically dehydrogenated in the cell wall to

phenoxy radicals (Lee et al. 2007). These radicals poly-

merize spontaneously, yielding a complex net of cross-

linking among monolignols, proteins and polysaccharides

(Iiyama et al. 1994). Peroxidases have been implicated in

these cross-linking reactions (Lewis and Yamamoto 1990;

Polle et al. 1994).

It has been well documented that biotic and abiotic

stresses are responsible for the increase in cell wall lignifi-

cation (Chazen and Neumann 1994; Katerji et al. 1997)

which would be associated with decreased plant growth and

nutrient content (Guenni et al. 2002). This is so because cell

wall peroxidases (CWP) are associated with the generation

of hydroxyl radicals in cell walls mediating extension

growth (Liszkay et al. 2003). An increase in ionically bound

cell wall peroxidase activity is associated with growth

inhibition of rice seedling roots caused by NaCl has been

reported (Lin and Kao 1999, 2001). At the same time, Chen

et al. (2000) reported an increase in cell wall peroxidase

activity in response to copper stress. However, little has been

known about changes in ionically bound CWP activity

related to growth responses under heavy metal stress con-

ditions. Consequently, to test the hypothesis that an increase

in ionically bound CWP activity is associated with growth

inhibition and plays some role in tolerance to heavy metal

stress in plants a considerable research on peroxidases bound

to the cell wall is required.

The present investigation has been designed to study the

metabolic responses of ionically bound CWP and soluble

peroxidase activities of seedling roots and 3–4 leaf stage

plant roots of Brassica juncea encountering cadmium

stress. Here we have hypothesized that the induction rate of

peroxidases that are ionically bound to the cell wall

depends on the growth stage of plant and the type of heavy

metal stress. Much of the work has been published on

ionically bound CWP but the effect of heavy metal toxicity

on ionically bound CWP activity in Brassica juncea at

different growth stages is yet unpublished.

Materials and methods

Plant growth, culture and metal treatment

Mustard seeds (Brassica juncea cv ‘‘Varuna’’ T-59) were

surface sterilized and germinated in a petridish (10 cm)

containing filter paper moistened with 10 ml of distilled

water. Each petridish contained 30 seeds. These seeds were

allowed to germinate at 30�C in dark. After 3 days of

incubation, germinated seedlings were exposed to light.

These seedlings were grown in thermostatically controlled

culture room maintained at 25 ± 2�C temperature under

white light of 500 lmol-2 s-1 photosynthetic photon flux

density by a combination of fluorescent tubes and tungsten

lamps for 16 h daily. After 5 days, the seedlings were

transferred to Hoagland’s nutrient solution having pH 6.4.

The nutrient solution was changed every third day. Sub-

sequent to day 15, seedlings were subjected to different

concentrations of cadmium (0, 5, 25, 50, 75, 100, 150,

200 lM) maintained in 800 ml of Hoagland’s nutrient

solution. For soluble and ionically bound CWP assay three

concentrations (5, 50 and 100 lM) of metal were selected.

At appropriate time intervals, whole plant samples were

removed and washed thoroughly with distilled water to

determine the root length, fresh weight, and other param-

eters. Each treatment and control was replicated thrice.

Plant growth parameters

Growth was measured in terms of fresh weight, root length,

tolerance index and root growth inhibition. Cadmium

toxicity was determined by measuring plant biomass pro-

duction and root elongation. Root length was measured and

marked before the metal was added. After 96 h of metal

stress, root length was again measured and growth rate was

calculated. Tolerance index of plant was calculated from

fresh weight by using the formula, (FW treated/FW con-

trol) 9 100 and represented in percent tolerance.

Measurements were performed on 5 plants per concentra-

tion per replicate and the experiment was repeated thrice.

Fresh weight of the whole plant was taken after 96 h of

cadmium treatment immediately after the harvesting. Cal-

culated values are the mean ± SD of five values (n = 5).

1262 Plant Cell Rep (2008) 27:1261–1269

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Lipid peroxidation

Lipid peroxidation in seedling roots was determined by

estimation of malonaldehyde (MDA) content following the

method of De Vos et al. (1989). Samples from control and

metal treated plants were homogenized in 10 ml of 0.25%

(w/v) 2-thiobarbituric acid in 10% (w/v) trichloroacetic

acid. The resulting mixture was heated at 95�C for 30 min

and then cooled quickly on ice bath. Mixture was centri-

fuged at 10,000g for 15 min and the absorbance of the

supernatant was taken at 532 and 600 nm. The non-specific

absorbance at 600 nm was subtracted from the absorbance

at 532 nm. The concentration of MDA content was cal-

culated by using extinction coefficient of 155 mM-1 cm-1

(Kwon et al. 1965).

Protein estimation

Protein was estimated by using method of Lowry et al.

(1951). A measure of 0.5 ml of 1 N NaOH (sodium

hydroxide) was followed by 0.1 ml of extracted sample.

After 10 min digestion on boiling water bath, 2.5 ml of

reagent B [48 ml of 5% Na2CO3 (sodium carbonate) and

2 ml of 0.5% CuSO4�5H2O (copper sulfate) in 1% sodium

potassium tartarate] was added. A measure of 0.5 ml of

folin-phenol reagent was added after 10 min. A 30-min

incubation developed a blue color complex in the mixture.

Absorbance was taken at 700 nm against a blank without

sample. Protein content was calculated by a standard curve

made by bovine serum albumin (BSA).

Cell wall preparation

Cell walls were prepared by homogenizing 30 mg tissue in

ice cold sodium acetate buffer (10 mM, pH 6.0), using

100 ll of buffer per mg of original fresh weight of tissue

(OFW). Homogenate was mixed and centrifuged at 3,000g

for 15 min at 3�C. The supernatant was collected and used

for assay of soluble peroxidase. Pellet was resuspended in

the same buffer and again centrifuged. This washing pro-

cedure was repeated up to six times to ensure that all the

soluble peroxidase had been washed out. Supernatant from

final wash was assayed for peroxidase activity to confirm

their reduction to a negligible level. Finally, washed pellet

was used as cell wall fraction to extract ionically bound

cell wall peroxidases.

Ionically bound CWP extraction

From cell walls, ionically bound CWP was extracted with

1 M NaCl (sodium chloride). The washed pellet was

resuspended in 100 ll mg-1 OFW of 100 mM sodium

acetate buffer, pH 6.0, containing 1 M NaCl. The

suspension was mixed thoroughly and incubated on ice

bath for 60 min with periodic shaking. After incubation

this sample was centrifuged at 3�C for 15 min at 3,000g.

The supernatant contains the salt extractable cell wall

peroxidase; this fraction was considered to represent that

fraction of peroxidase that is ionically bound to the cell

wall in vivo (Goldberg et al. 1987; Bacon et al. 1997).

Peroxidase assay

Soluble and cell wall peroxidase was assayed using sub-

strate 3, 30, 5, 50-tetramethyl benzidine (TMB) (Bos et al.

1981). TMB was made up at 20 mg ml-1 in dimethyl-

sulphoxide (DMSO) and stored in aliquots at -20�C. A

measure of 5 ll of sample was added to each assay tube

followed by 100 ll of 100 mM sodium acetate buffer, pH-

6.0 containing 0.1 mg ml-1 TMB and 0.5 ll ml-1 of 6%

(v/v) H2O2 (Hydrogen peroxide). Mixture was incubated

for 60 min at 4�C and reaction was stopped by 100 ll of

0.6 M sulphuric acid (H2SO4). Absorbance was recorded at

450 nm. There was no reaction in the absence of hydrogen

peroxide. Enzyme activity was calculated by using

extinction coefficient of the terminal oxidation product

formed, a yellow diimine that absorb light at 450 nm

(extinction coefficient = 5.9 9 104 M-1 cm-1).

Statistical analysis

All values in this work are mean of at least three inde-

pendent experiments. The mean ± SD and exact number

of experiments are given in figures and tables. The sig-

nificance of differences between control and each treatment

was analyzed using Student’s t test.

Results

Growth parameters of Brassica juncea seedlings were

analysed by measuring fresh weight and root length. Fig-

ure 1 show the effect of cadmium toxicity on fresh weight

(Fig. 1a), root growth (Fig. 1b) and lipid peroxidation

(Fig. 1C) after 96 h of metal treatment. Increasing con-

centrations of cadmium from 5 to 200 lM progressively

decreased fresh weight up to 91.66% and root growth to

96.92% (Fig. 1a, b). A decrease in fresh biomass was found

to be significant on increasing cadmium concentration from

25 to 200 lM (P B 0.05).The reduction in fresh biomass

and relative growth rate of roots is correlated with an

increased lipid peroxidation level (Fig. 1c). Increase in

MDA content was found to be significant after 50 lmol

cadmium concentration (P B 0.05).

Root growth was affected more severely as compared to

overall growth. Figure 2 shows correlation between

Plant Cell Rep (2008) 27:1261–1269 1263

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decreased tolerances (Fig. 2a) to cadmium with increasing

root inhibition (Fig. 2b). A continuous significant decrease

(P B 0.05) in tolerance and a significant increase

(P B 0.05) in root inhibition was found in seedlings treated

with increasing cadmium concentrations for 96 h. Seed-

lings treated with 200 lM of cadmium for 96 h show

reduced tolerance of 6.24% (Fig. 2a) with a total increase

in root inhibition of 97.52% (Fig. 2b).

Both ionically bound CWP activity and soluble per-

oxidase activity in roots of seedling plants (15-day old)

and 3–4 leaf stage plants (30-day old) of Brassica juncea

was compared and correlated with lipid peroxidation level

after treating them with increasing concentrations (5, 50

and 100 lM) of cadmium for 72 h (Fig. 3). Ionically

bound CWP activity showed stimulation at lower cad-

mium concentration followed by a slight decrease at

higher concentrations (Fig. 3a). A significant increase in

ionically bound CWP activity can be seen in seedling

roots at 5 lM cadmium concentration (P B 0.05) which

further show a slight decrease on increasing the level of

metal toxicity up to 100 lM. But the increase in activity

was significant (P B 0.05). Whereas, no significant

change was seen in ionically bound CWP activity in 3–4

leaf stage plants with increased cadmium toxicity

(P B 0.05) (Fig. 3a). An overall enzyme activity was

found to be low in 3–4 leaf stage plants as compared to

Fig. 1 Effects of cadmium on fresh weight (a), root growth (b) and

MDA content (c) in Brassica juncea seedlings. Root length, fresh

weight and MDA content were analyzed after 96 h of treatment.

Vertical bars represent mean ± standard errors (n = 5). Data points

marked with the letter a show significant increase, b significant

decrease and c insignificant differences (P B 0.05)

Fig. 2 Tolerance index (a) and root growth inhibition (b) in Brassicajuncea seedlings. Tolerance index and growth inhibition were

calculated from fresh biomass {(FW treated/FW control) 9 100}

and root length, respectively, after 96 h of cadmium treatment. Barsare mean ± SD of 5 values (n = 5). Data points marked with the

letter a, show significant increase, b significant decrease and cinsignificant differences (P B 0.05)

1264 Plant Cell Rep (2008) 27:1261–1269

123

seedlings (Fig. 3a). On the same experimental set lipid

peroxidation level in roots of seedlings was analyzed and

shown in Fig. 3b and which further support these results.

However, no significant change in MDA content was

found at 50 and 100 lmol concentration of cadmium

(P B 0.05). The increase in H2O2 content in cadmium

treated Brassica juncea seedling roots was found to be

significant in comparison to control (P B 0.05) on

increasing cadmium concentrations from 5 to 100 lM

(Fig. 6a), which is directly correlated with increased

ionically bound CWP activity, whereas in roots of 3–4

leaf stage plants the accumulation of H2O2 was found

very less (Fig. 6a). Plants of 3–4 leaf stage when treated

with 5 and 100 lM cadmium shows a significant increase

in H2O2 level but this increase was found to be insignif-

icant in case of 50 lM treated plants. Activity of soluble

peroxidase decreased significantly at 50 and 100 lmol

cadmium concentrations (P B 0.05, in roots of Brassica

seedlings. However, there is no significant change in

soluble peroxidase activity was found in 3–4 leaf stage

plants (P B 0.05) (Fig. 3c). Activity induction of soluble

peroxidase is again high in seedlings in comparison to its

activity in 3–4 leaf stage plants (Fig. 3c).

Induction of ionically bound CWP activity by cadmium

toxicity in roots of Brassica seedlings (15-day old) was

further proved by analyzing its activity induction by

another heavy metal, i.e. copper. A progressive significant

increase (P B 0.05) in ionically bound CWP activity in

comparison to cadmium treated seedlings was found with

increasing copper concentrations to 5, 50 and 100 lM

(Fig. 4a). A 6-fold increase in comparison to control was

found in ionically bound CWP activity in roots of seedlings

treated with 100 lM copper for 72 h. These results were

further supported by MDA content in roots of seedlings of

the same experiment. A significant increase 78.56%

(P B 0.05) in MDA content was found in roots of seedlings

treated with 100 lM copper as shown in Fig. 4b. A pro-

gressive significant increase (P B 0.05) of H2O2 level in

roots of copper treated seedlings was found on increasing

copper concentrations from 5 to 100 lM (Fig. 6b). How-

ever, we found that cadmium is more effective in H2O2

generation in roots of Brassica juncea seedlings in com-

parison to copper. This increased level of H2O2 in 5, 50 and

100 lM copper treated seedlings was further correlated

with increased activity of ionically bound CWP in roots of

5, 50 and 100 lM copper treated seedlings. As a whole the

induction of ionically bound CWP activity and H2O2 level

was found to be higher in case cadmium treated roots in

comparison to copper. Soluble peroxidase activity in roots

of copper treated seedlings after 72 h showed a signifi-

cant increase at 5 and 100 lM copper concentrations

(P B 0.05) (Fig. 4c), whereas in case of 50 lM copper

treated seedlings it show a significant decrease in its

activity (P B 0.05). However, in case of cadmium treated

seedlings, soluble peroxiadse activity shows a significant

decrease at 50 and 100 lM (P B 0.05). An overall activity

of soluble peroxidase in roots was found to be low in

cadmium in comparison to copper treated seedlings

(Fig. 4c).

Fig. 3 Changes in ionically bound CWP (a), soluble peroxidase (c)

activities and MDA content (b) in roots of seedlings (15 days) and

3–4 leaf stage (30 days) plants of Brassica juncea. Plants were treated

with cadmium for 72 h. Vertical bars represent standard errors

(n = 3). Data points marked with the letter a show significant

increase, b significant decrease and c insignificant differences

(P B 0.05)

Plant Cell Rep (2008) 27:1261–1269 1265

123

Ascorbic acid pretreatment results shown in Fig. 5

reveal that 50 lM cadmium induces ionically bound CWP

activity significantly (P B 0.05) in seedling roots. A sig-

nificant decrease in its activity (P B 0.05) in roots of

50 mM ascorbate pretreated seedlings was found when

exposed to 50 lM cadmium (Fig. 5).

Discussion

Plant metabolism must be highly regulated in order to

allow effective integration of a diverse spectrum of bio-

synthetic pathways that are reductive in nature. This

regulation does not completely avoid photodynamic or

reductive activation of molecular oxygen to produce

reactive oxygen species (ROS) particularly superoxide,

H2O2 and singlet oxygen (Halliwell 1981; Foyer and

Noctor 2005). Plant cells produce ROS, particularly

superoxide and H2O2, as second messengers in many pro-

cesses associated with plant growth and development

(Schroeder et al. 2001; Foreman et al. 2003).

Metal ions may stimulate the generation of ROS either

by direct transfer of electrons in single electron reactions

involving metal cations or as a consequence to metal

inactivated metabolic reactions (Dietz et al. 1999). Cad-

mium toxicity was evident from reduced growth of roots

(Fig. 1b) and fresh biomass (Fig. 1a). Root growth appears

to be strongly regulated by ROS (Foreman et al. 2003). It is

so because roots are the first site of exposure and toxicity to

the metal and thus root growth was more severely affected.

Both redox active (Cu) and non-reodox active (Cd) are

reported to increase lipid peroxidation via ROS generation

in plants (Gallego et al. 1996; Chaoui et al. 1997).

Excessive concentrations of both Cu and Cd are known to

cause cellular oxidative damage, lipid peroxidation (Dixit

et al. 2001) and H2O2 accumulation (Schutzendubel et al.

2001). We observed similar results that excess Cd and Cu

cause an increase in lipid peroxidation (Figs. 1c, 3b, 4b).

De Vos et al. (1989) and Rama Devi and Prasad (1998)

Fig. 4 Changes in ionically bound CWP (a), soluble peroxidase

activities (c) and MDA content (b) in roots of Brassica junceaseedlings. Seedlings were treated with cadmium and copper sepa-

rately in two independent experiments. Vertical bars represent

standard errors (n = 3). Data points marked with the letter a, show

significant increase, b significant decrease and c insignificant

differences (P B 0.05)

Fig. 5 Changes in ionically bound CWP in roots of Brassica junceaseedlings. Seedlings were pretreated with 50 mM ascorbic acid and

then exposed to 50 lM cadmium. Vertical bars represent mean ± SD

(n = 3). Data points marked with the letter a show significant

increase and b significant decrease (P B 0.05)

1266 Plant Cell Rep (2008) 27:1261–1269

123

reported a similar increase in lipid peroxidation when

plants were treated with Cu. Cadmium almost always

adopts only one oxidation state, which is a bivalent cation.

Unlike redox active metals like copper, cadmium is not

able to induce production of ROS through a Fenton type

reaction or Haber–Weiss reaction (Bartosz 1997). Cad-

mium causes oxidative stress probably through indirect

mechanisms such as interaction with the antioxidative

defense, disruption of the electron transport chain or

induction of lipid peroxidation (Chen et al. 2003; Leon

et al. 2002). It is possible that the observed changes in the

metabolic system occurred as a result of unspecific cellular

degradation processes. However, another possibility is that

cadmium triggers common defense pathways in plant cells

like other biotic or abiotic environmental stresses. A joint

initial event of these pathways is an accumulation of H2O2,

which acts as a substrate in cross-linking reactions cata-

lyzed by cell wall peroxidases.

Cadmium and copper both cause increased accumula-

tion of H2O2 in roots of Brassica juncea seedlings (Fig. 6).

Similar to our results, Raeymaekers et al. (2003) reported a

specific, rapid and concentration dependent accumulation

of H2O2 in copper treated tobacco BY-2 cell cultures.

Increase in H2O2 content has been also reported in copper

treated A. thaliana (Drazkiewicz et al. 2004). Thus, ele-

vated levels of ionically bound CWP activity, H2O2 level

and MDA content is a common response to oxidative stress

caused by cadmium and copper in roots of seedlings by

different mechanisms involved in various metabolic

pathways.

In common with many previous workers (Goldberg et al.

1987; Thompson et al. 1998; Andrews et al. 2000) our

results also strongly supports that the salt extractable wall

bound peroxidase is located within the cell wall in vivo.

Several authors have shown that changes in growth are

usually associated with the apoplast rather than with the

cytoplasm of assayed tissue (Cordoba-Pedregrosa et al.

1996). Changes in cytoplasmic activity are often associated

with induction of plant antioxidant system.

An appreciable increase in ionically bound CWP

activities in roots of seedlings (Fig. 3a) could reflect the

modification of mechanical properties of the cell wall,

which in turn, can be correlated with metal stress. Similar

to our results, activities of lignifying peroxidase were

stimulated in response to copper-induced oxidative stress

(Chen et al. 2000; Jouili and El Ferjani 2003). In the same

way, the growth reduction caused by copper (Chen et al.

2002) and by cadmium (Chaoui and El Ferjani 2005) was

closely associated with the increased activity of cell wall

bound peroxidase. Like NAD(P)H oxidase and Xanthine

oxidase, cell wall bound peroxidases could also trigger the

oxidative burst in plants (Bolwell et al 1998). The pro-

duction of ROS in the apoplast can derive the oxidative

cross-linking of cell wall components, such as hydroxyl

proline-rich glycoproteins. The cross-linking of structural

proteins in the wall has been proposed as a metabolism to

restrict cell growth (Iiyama et al. 1994) and to limit cell

elongation (De Cnodder et al. 2005).

Concerning the decrease in soluble peroxidase activi-

ties (Fig. 3c) in cadmium treated seedlings it has been

reported that a loss in antioxidant capacities (e.g. a

decrease in ascorbate peroxidase activity) results in an

intrinsic accumulation of signaling molecules like H2O2

(Lee et al. 2007) which is involved in triggering sec-

ondary reactions: mechanical strengthening of cell walls

including lignifications. Barcelo et al. (1987) employed a

similar extraction procedure with lupin hypocotyls and

showed that cell wall material contained salt extractable

peroxidases but no significant quantity of cytoplasmic

marker enzymes.

As far as the relationship between plant growth stage

and ionically bound CWP activity is concerned, an inverse

Fig. 6 Changes in H2O2 level in roots of seedling stage and 3–4 leaf

stage plants of Brassica juncea treated with cadmium (a) and in

seedling roots treated with equimolar concentrations of cadmium and

copper (b). Treatment of cadmium and copper to seedlings and 3–4

leaf stage plants were given for 72 h. Vertical bars represent standard

errors (n = 3). Data points marked with the letter a, show significant

increase and c insignificant differences (P B 0.05)

Plant Cell Rep (2008) 27:1261–1269 1267

123

relationship between growth rate and cell wall bound per-

oxidase activity has been reported in many plant systems

(Carpita and Gilbeaut 1993; Chen and Kao 1995; Fry 1986;

Lee and Lin 1995). Thus, ionically bound CWP is gener-

ally believed to be a putative wall rigidification enzyme

(Cosgrove 1997). It is similar to our results that no sig-

nificant change has been observed in ionically bound CWP

and soluble peroxidase activities in roots of 3–4 leaf stage

plants (Fig. 3a, c) against cadmium toxicity.

Decrease in ionically bound CWP activity in roots of

ascorbate pretreated seedlings (Fig. 5) shows an antioxi-

dant behavior of ascorbate that suppresses cadmium

toxicity induced via ROS. From here we can suggest a

defensive role of ionically bound CWP activity in response

to metal stress. Antioxidants such as ascorbic acid which

are found at high concentrations in chloroplasts and other

cellular compartments (5–20 mM ascorbic acid) are crucial

for plant defense against oxidative stress (Noctor and Foyer

1998; Mittler 2002).

Acknowledgments G. S. Shekhawat is thankful to Professor Aditya

Shastri, Vice chancellor, Banasthali University, Rajasthan, India for

kind cooperation.

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