Zinc alleviates mercury-induced oxidative stress in Pfaffiaglomerata (Spreng.) Pedersen

Niceia Spanholi Calgaroto • Denise Cargnelutti • Liana Veronica Rossato •

Julia Gomes Farias • Sibila Trojahn Nunes • Luciane Almeri Tabaldi •

Fabiane Goldschmidt Antes • Erico Marlon Moraes Flores •

Maria Rosa Chitolina Schetinger • Fernando Teixeira Nicoloso

Received: 7 January 2011 / Accepted: 19 April 2011 / Published online: 8 May 2011

� Springer Science+Business Media, LLC. 2011

Abstract The possible role of zinc (Zn) to reverse

the oxidative stress caused by mercury (Hg) was

investigated in Pfaffia glomerata plantlets. Thirty-

day-old acclimatized plantlets of P. glomerata were

exposed to four treatments: control, 50 lM Zn,

50 lM Hg and 50 lM Zn ? 50 lM Hg for 9 days.

In Zn ? Hg treatment, shoot and root Hg concentra-

tions were 59 and 24% smaller than that plants

exposed to 50 lM Hg added alone. An increase in the

Zn concentration in the shoot of plants exposed to

Zn ? Hg occurred, although in the roots Zn concen-

tration was not altered, when compared to the control.

Fresh and dry weights, as well as the activity of

d-aminolevulinic acid dehydratase (d-ALA-D) in

Hg-treated plants were significantly reduced. Per-

centage survival, fresh and dry weights and d-ALA-D

activity of plants treated by 50 lM Zn ? 50 lM Hg

were greater than of that treated by Hg alone.

Moreover, Zn treatment reduced the lipid peroxida-

tion caused by Hg, being this effect related to

increased root superoxide dismutase activity, and

shoot catalase and ascorbate peroxidase activities. In

conclusion, the presence of Zn in the substrate caused

a significant reduction in the oxidative stress induced

by Hg.

Keywords Antioxidant system � Brazilian ginseng �Mercury � Oxidative stress � Pfaffia glomerata � Zinc

Introduction

Over the past 200 years emissions of toxic heavy

metals have risen tremendously and significantly

exceed those from natural sources for practically all

metals (Clemens 2006). Mercury (Hg) pollution is a

ubiquitous problem resulting both from natural events

and anthropogenic activities and it has caused deep

concern because of its toxicity, mobility, bioaccumu-

lation, methylation and dispersion in the biosphere

(Rodrıguez et al. 2003). Mercury is extremely toxic

to plants and animals (Patra and Sharma 2000). To

combat Hg toxicity, plant cells activates the antiox-

idant system that include non-enzymatic scavengers

N.S. Calgaroto and D. Cargnelutti contributed equally to this

work.

N. S. Calgaroto � J. G. Farias � S. T. Nunes �F. T. Nicoloso (&)

Departamento de Biologia, Centro de Ciencias Naturais

e Exatas, Universidade Federal de Santa Maria, Santa

Maria, RS 97105-900, Brazil

e-mail: ftnicoloso@yahoo.com

D. Cargnelutti � L. V. Rossato � L. A. Tabaldi �F. G. Antes � E. M. M. Flores �M. R. C. Schetinger (&)

Departamento de Quımica, Centro de Ciencias Naturais

e Exatas, Universidade Federal de Santa Maria, Santa

Maria, RS 97105-900, Brazil

e-mail: mariaschetinger@gmail.com

123

Biometals (2011) 24:959–971

DOI 10.1007/s10534-011-9457-y

(glutathione, ascorbate, carotenoids, proline, and

a-tocopherol) and enzymatic scavengers (superoxide

dismutase, ascorbate peroxidase, catalase, and thio-

redoxin peroxidase) that participate in scavenging

reactive oxygen species (ROS) comprising both free

radical (O2_�, OH�, HO2

� ) and non-radical (molecular)

forms (H2O2) (Edreva 2005; Cargnelutti et al. 2006;

Zhou et al. 2008; Calgaroto et al. 2010).

Zinc (Zn) is an essential component of thousands

of proteins in plants (Broadley et al. 2007) and is

involved in cell membrane stabilization, metallothi-

onein synthesis, protein synthesis, carbohydrate

metabolism, and tryptophan and indoleacetic acid syn-

thesis (Marschner 2002). Studies with animals showed

that Zn is an important antioxidant, decreasing ROS

production (Zago and Oteiza 2001; Fernandez et al.

2003). Some studies have reported the ability of Zn to

interact with essential elements such as Cu and Fe,

decreasing their content in tissues and hence retard-

ing the oxidative processes when these two micro-

nutrients are present in excess (Zago and Oteiza

2001; Santon et al. 2003; Broadley et al. 2007).

Generally, Zn applications decrease Cd uptake and

accumulation in plants (McLaughlin et al. 1994;

Oliver et al. 1997). Hart et al. (2002) attributed

the competitive interaction between Cd and Zn to the

existence of a common transport system in the

plasma membrane. Wu and Zhang (2002) found that

increasing Zn application could alleviate Cd toxicity

stress in barley plants by improving growth and

reducing membrane damage.

The genus of Pfaffia belongs to the Amaranthaceae

family and has about 90 species distributed through-

out Central and South America. In Brazil, 27 species

have been described (Taniguchi et al. 1997). Carneiro

et al. (2002) showed that an undetermined species of

the genus Pfaffia exhibited high tolerance to soil

contamination, growing quite abundantly in soil

mixtures with 90 and 1,450 mg kg-1 of Cd and Zn,

respectively. Skrebsky et al. (2008) showed that

Pfaffia glomerata (Spreng.) Pedersen plantlets grown

hydroponically seemed to have reasonable degree of

Cd tolerance. In a recent study, we also observed

that the growth and the antioxidant mechanisms of

P. glomerata plants were significantly affected by

high levels of Hg (25 and 50 lM) in the substrate

(Calgaroto et al. 2010). However, no information has

been found in plants on whether Zn is capable of

ameliorating Hg toxicity. Under this context, the

present work was designed to analyze the effect of Zn

on the biochemical and physiological alterations

caused by Hg in P. glomerata plants.

Materials and methods

Plant material and growth conditions

Pfaffia glomerata (Spreng.) Pedersen plantlets for

tissue culture were obtained from the Brazilian

Ginseng Germplasm Program, Universidade Federal

de Santa Maria, RS, Brazil. Nodal segments (1.0 cm

long) without leaves were micropropagated in MS

medium (Murashige and Skoog 1962), supplemented

with 30 g l-1 of sucrose, 0.1 g l-1 of myo-inositol

and 6 g l-1 of agar according to Nicoloso et al.

(2001). One thirty-day-old in vitro-grown plantlet

was transferred per pot (1.2 l) which contains 1.0 l of

acid-washed sand. Each experimental unit consisted

of 10 plantlets, totalizing five replicates per treat-

ment. Throughout cultivation, sand was maintained at

80% of field capacity, determined with a sample

altered on a tension table. Irrigation was performed

daily by replacement of transpired water, calculated

by weighing the pots. The plantlets were supple-

mented daily with nutrient solution containing the

following composition: 65.1 mg l-1 NH4Cl, 76.2

mg l-1 MgSO4�7H2O, 135.2 mg l-1 MgCl2�6H2O,

33.1 mg l-1 KH2PO4, 181.5 mg l-1 KCl, 575.3

mg l-1 Ca(NO3)2�4H2O, 0.11 mg l-1 CuSO4�5H2O,

0.39 mg l-1 MnCl2�4H2O, 0.57 mg l-1 ZnSO4�7H2O, 0.04 mg l-1 NiSO4, 1.54 mg l-1 H3BO3,

0.09 mg l-1 H2MoO4�H2O and 13.34 mg l-1 FeSO4�7H2O. After 1 month of plantlet acclimatization, four

treatments (control; 50 lM Zn; 50 lM Hg; and

50 lM Zn ? 50 lM Hg) were added to the nutrient

solution. Hg and Zn were added as HgCl2 and ZnCl2,

respectively. Both in vitro and ex vitro cultured

plantlets were grown in a growth chamber at

25 ± 1�C on a 16/8 h light/dark cycle with

35 lmol m-2 s-1 of irradiance by cold fluorescent

lamps. After 9 days of Hg exposure, three plantlets

per replicate were harvested randomly either for

growth or biochemical analyses. Three independent

and representative tissue samples were used for Hg

and Zn determination. Fresh samples were used for

measurements of H2O2, MDA, chlorophyll and

carotenoids concentrations, antioxidant enzyme

960 Biometals (2011) 24:959–971

123

activities and non-enzymatic antioxidant concentra-

tions. All chemicals used were of analytical grade

purchased from Sigma Chemical Company (USA).

Plant growth parameters

At harvest, the plants were divided into root and

shoots. Roots were rinsed twice with distilled water.

Subsequently, growth and biochemical parameters

were determined. Plant biomass was measured on

fresh and dry weight basis. For fresh weight, excess

water from the root was dried with tissue paper and

weighed on an electronic balance. To obtain dry

weight, roots and shoots were dried at 65�C until

reaching a constant weight. The percentage survival

was calculated based on the difference of the total

number of plants present before the addition of

treatments and the number of surviving plants after

9 days of treatment.

Tissue Hg and Zn concentration

Between 70 and 100 mg of shoots and roots were

digested with 5 ml HNO3 and 0.2 ml H2O in closed

Teflon vessels, which were heated at 100�C for 3 h in

a digester block (Tecnal TE 007D). The samples were

then diluted to 50 ml with high-purity water. Hg

concentrations were determined using a Varian

Atomic Absorption Spectrophotometer (Spectr AA

600, Australia) equipped with a cold-vapor genera-

tion device (Varian VGA-76).

Zinc concentration was determined by inductively

coupled plasma optical emission spectrometry (ICP-

OES) using a PerkinElmer Optima 4300DV (Shelton,

USA) equipped with a cyclonic spray chamber and a

concentric nebulizer. The emission lines used were

213.857 nm. Instrumental parameters were adjusted

according manufacturer recommendations. Nebu-

lizer, intermediate and principal gas flow rates were

set to 0.65, 0.20 and 14 l min-1, respectively. Sample

decomposition was carried out in an open digestion

system, using a heating block from Velp Scientifica

(Milano, Italy) equipped with glass vessels.

Chlorophyll and carotenoid determination

Fresh biomass (leaves plus stem) was homogenized

in 80% ice-cold acetone in the dark and then

centrifuged at 10,000 rpm for 10 min at 4�C and

the supernatant was used for the immediate determi-

nation of pigments. Absorbance of the solution was

measured at 663, 645, 510 and 480 nm in a spectro-

photometer (Celm E-205D) in order to determine the

concentration of carotenoids, chlorophyll-a and chlo-

rophyll-b, respectively. Pigment concentration was

calculated following the equation used by Lichtent-

haler (1987).

Delta-aminolevulinic acid dehydratase activity

Shoots were homogenized in 10 mM Tris–HCl

buffer, pH 9.0, at a proportion of 1:1 (w/v). The

homogenate was centrifuged at 12,0009g at 4�C for

10 min to yield a supernatant (S1) that was used for

the enzyme assay. The supernatant was pre-treated

with 0.1% Triton X-100 and 0.5 mM dithiotreithol

(DTT). Delta-aminolevulinic acid dehydratase

(d-ALA-D; E.C. 4.2.1.24) activity was assayed as

described by Barbosa et al. (1998) by measuring the

rate of porphobilinogen (PBG) formation. The incu-

bation medium for the assays contained 100 mM

Tris–HCl buffer, pH 9.0 and 3.6 mM ALA. Incuba-

tion was started by adding 100 ll of the tissue

preparation to a final volume of 400 ll and stopped

by adding 350 ll of the mixture containing 10%

trichloroacetic acid (TCA) and 10 mM HgCl2. The

product of the reaction was determined with the

Ehrlich reagent at 555 nm using a molar absorption

coefficient of 6.1 9 104 l-1 mol-1 cm-1 (Sassa

1982) for the Ehrlich-porphobilinogen salt. The

d-ALA-D activity was expressed as nmol PBG

mg-1 protein h-1.

Determination of hydrogen peroxide

The H2O2 concentration was determined according to

Loreto and Velikova (2001). Approximately 0.1 g of

both roots and shoot was homogenized at 4�C in 2 ml

of 0.1% trichloroacetic acid (TCA) (w/v). The

homogenate was centrifuged at 12,0009g for

15 min. Then, 0.5 ml of the supernatant was added

to 0.5 ml of 10 mM potassium phosphate buffer

(pH 7.0) and 1 ml of 1 M KI. The H2O2 concentra-

tion of the supernatant was evaluated by comparing

its absorbance at 390 nm with a standard calibration

curve. The hydrogen peroxide concentration was

expressed as lmol g-1 fresh weight.

Biometals (2011) 24:959–971 961

123

Estimation of lipid peroxidation

The level of lipid peroxidation products was esti-

mated following the method of El-Moshaty et al.

(1993) by measuring the concentration of malondi-

aldehyde (MDA) as an end product of lipid perox-

idation by reaction with thiobarbituric acid (TBA).

Fresh tissue samples (0.1 g fresh weight) were

ground in 2 ml of 0.2 M citrate–phosphate buffer

(pH 6.5) containing 0.5% Triton X-100, using mortar

and pestle. The homogenate was filtered through two

layers of paper and centrifuged for 15 min at

20,0009g. One milliliter of the supernatant fraction

was added to an equal volume of 20% (w/v) TCA

containing 0.5% (w/v) TBA. The mixture was heated

at 95�C for 40 min and then quickly cooled in an ice

bath for 15 min. After centrifugation at 10,000 g for

15 min, the absorbance of the supernatant was

measured at 532 nm. A correction for non-specific

turbidity was made by subtracting the absorbance

value taken at 600 nm. The lipid peroxides were

expressed as nmol MDA (mg protein)-1, by using an

extinction coefficient of 155 l mol-1 cm-1.

Catalase assay

Catalase (CAT, E.C. 1.11.1.6) activity was assayed

according to the method of Aebi (1984) with some

modifications. Fresh samples (1 g) were homogenized

in 5 ml of 50 mM KH2PO4/K2HPO4 (pH 7.0), 10 g l-1

PVP, 0.2 mM EDTA and 10 ml l-1 Triton X-100. The

homogenate was centrifuged at 12,0009g at 4�C for

20 min and the supernatant was used for the enzyme

assay. CAT activity was determined by monitoring

the disappearance of H2O2 by measuring the decrease

in absorbance at 240 nm from a reaction mixture

containing 2 ml of 15 mM H2O2 in KPO4 buffer (pH

7.0) and 30 ll extract. Activity was expressed as

DE min-1 mg-1 protein.

Ascorbate peroxidase assay

Ascorbate peroxidase (APX, E.C. 1.11.1.11) activity

was measured according to Zhu et al. (2004). The

reaction mixture, at a total volume of 2 ml, contained

25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM

EDTA, 0.25 mM ascorbate, 1.0 mM H2O2 and 100 ll

enzyme extract. H2O2-dependent oxidation of ascor-

bate was monitored by a decrease in absorbance at

290 nm (E = 2.8 l mol-1 cm-1) and activity was

expressed as lmol ascorbate oxidated min-1 mg-1

protein.

Superoxide dismutase assay

Superoxide dismutase (SOD, E.C. 1.15.1.1) activity

was assayed according to Misra and Fridovich

(1972). About 200 mg of roots and shoots were

homogenized in 5 ml of 100 mmol l-1 K-phosphate

buffer (pH 7.8) containing 0.1 mmol l-1 EDTA,

0.1% (v/v) Triton X-100 and 2% PVP (w/v). The

homogenate was centrifuged at 22,0009g at 4�C for

10 min. The assay mixture consisted of a total

volume of 1 ml, containing glycine buffer (pH

10.5), 1 mmol l-1 epinephrine and enzyme material.

Epinephrine was the last component added. Adeno-

chrome formation over the next 4 min was spectro-

photometrically recorded at 480 nm. One unit of

SOD activity is expressed as the amount of enzyme

required to cause 50% inhibition of epinephrine

oxidation under the experimental conditions used.

This method is based on the ability of SOD to inhibit

the autoxidation of epinephrine at an alkaline pH.

Since the oxidation of epinephrine leads to the

production of a pink adrenochrome, the rate of

increase of absorbance at 480 nm, which represents

the rate of autoxidation of epinephrine, can be

conveniently followed. The enzyme has been found

to inhibit this radical-mediated process.

Ascorbic acid concentration

Ascorbic acid (AsA) determination was performed as

described by Jacques-Silva et al. (2001). Both roots

and shoots were homogenized in a solution contain-

ing 50 mmol l-1 Tris–HCl and 10 ml l-1 Triton

X-100 (pH 7.5), centrifuged at 6,8009g for 10 min.

To the supernatant, 10% TCA was added at a

proportion of 1:1 (v/v) followed by centrifugation

(6,800 g for 10 min) to remove protein. An aliquot of

the sample (300 ll) was incubated at 37�C in a

medium containing 100 ll 13.3% TCA, 100 ll

deionized water and 75 ll 2,4-dinitrophenylhydra-

zine (DNPH). The DNPH solution contained 2%

DNPH, 0.23% thiourea, 0.27% CuSO4 diluted in 49%

H2SO4. After 3 h, 500 ll of 65% H2SO4 was added

and samples were read at 520 nm. A standard curve

was constructed using L(?) ascorbic acid.

962 Biometals (2011) 24:959–971

123

Non-protein thiols group concentration

Non-protein thiol (NPSH) concentration was mea-

sured spectrophotometrically with Ellman’s reagent

(Ellman 1959). Root and shoot samples were homog-

enized in a solution containing 50 mmol l-1 Tris–

HCl and 10 ml l-1 Triton X-100 (pH 7.5), centri-

fuged at 6,8009g for 10 min, and NPSH was

determined in a fraction obtained after mixing 1

volume of supernatant with 1 volume of 10% TCA

followed by centrifugation (6,800 g for 10 min) and

neutralization (to pH 7.4) with 1 M Tris–HCl as

described by Jacques-Silva et al. (2001). The reaction

was read at 412 nm after the addition of 0.05 ml of

10 mM 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB).

A standard curve using cysteine was used to calculate

the concentration of NPSH in the samples and was

expressed as lmol SH g-1 fresh weight.

Protein extraction

In all the enzyme preparations, protein concentration

was determined by the method of Bradford (1976)

using bovine serum albumin as standard and was

expressed in mg l-1.

Statistical analysis

The analyses of variance were computed on statisti-

cally significant differences determined based on the

appropriate F-tests. The results are the means ± SD

of at least three independent replicates. The mean

differences were compared utilizing Tukey test with

P \ 0.05.

Results

Tissue metals concentration

Mercury (Hg) concentration was greater in roots than

in shoots upon addition of Hg in the substrate

(Table 1). Conversely, Hg concentrations were,

approximately, 15 and 6.8% greater in shoots than

in roots, respectively, in control and Zn treatments. In

Zn ? Hg treatment, shoot and root Hg levels were 59

and 24% smaller than that plants exposed to 50 lM

Hg added alone. In addition, an increase in the Zn

levels in the shoot of plants exposed to Zn ? Hg

occurred, although in the roots, Zn concentration was

not altered, when compared to the control.

Percentage survival and fresh and dry weight

of plantlets

In comparison to the control, fresh and dry weight, as

well as percentage survival in the Hg-exposed

P. glomerata plants decreased significantly, while

the addition of Zn together with Hg reversed the

negative effect caused by Hg (Fig. 1). The percentage

survival was about 47 and 20% lower that of control in

plants exposed to Hg alone and Zn ? Hg treatments,

respectively. In 50 lM Hg treatment, shoot fresh

weight decreased about 71.5% when compared to the

control, whereas root fresh weight decreased by 59%.

On the other hand, in plant treated with Zn ? Hg,

shoot and roots fresh weight decreased about 51.4 and

24.4% in relation to the control, respectively.

Shoot dry weight in Hg-exposed plants decreased

69% in relation to the control. In plants exposed to

Table 1 Hg and Zn concentrations in shoot and roots of Pfaffia glomerata plantlets exposed to the treatments for 9 days

Treatments Hg concentration (lg g-1 dry weight) Zn concentration (lg g-1 dry weight)

Shoot Root Shoot Root

Control 16.9 ± 1.5 cA* 2.53 ± 0.25 bB 79.7 ± 4.0 bB 161.7 ± 10.0 aA

50 lM Zn 15.5 ± 0.7 cA 1.06 ± 0.52 bB 116 ± 4.6 bB 176 ± 37.9 aA

50 lM Hg 351.5 ± 39.5 aB 797 ± 149 aA 73.7 ± 4.7 bB 143 ± 34.0 aA

50 lM Zn ? 50 lM Hg 208 ± 94.0 bB 605.3 ± 159.9 aA 216.7 ± 65.6 aA 150.7 ± 23.4 aB

Data represent mean values ± SD based on independent determination

* Mean values followed by the same small letters in the column, and capital letters in the line did not differ significantly by Tukey test

of P \ 0.05

Biometals (2011) 24:959–971 963

123

Zn ? Hg treatment, the dry weight also decreased,

but this value was only about 26% to the control.

Interestingly, root dry weight in Hg-treated plants

was slightly but not significantly, decreased upon

presence of Hg, when compared to the control

(Fig. 1C), whereas, Zn-treated plants showed greater

root dry weight than that of Hg-treated.

Concentration of chlorophyll and carotenoids

and delta-aminolevulinic acid dehydratase

activity

In comparison to control, the concentrations of

chlorophyll-a and chlorophyll-b were not altered

upon addition of Hg, Zn and Zn ? Hg treatments

(Fig. 2A) On the other hand, Hg-treated plants

showed higher chlorophyll-a concentration than those

treated with Zn alone. Carotenoids concentration

increased in plants grown in the presence of

Zn ? Hg, when compared with control and Zn

treatments. d-ALA-D activity decreased in plant

treated with Zn or Hg alone compared with the

control, whereas there were no changes in activity

when both metal were added together (Fig. 2B).

Fig. 1 Percentage survival (A), fresh weight (B) and dry

weight (C) of P. glomerata plantlets exposed to different

treatments. Control: without Zn and Hg; Zn: 50 lM Zn as

ZnCl2; Hg: 50 lM Hg as HgCl2; Zn ? Hg: 50 lM

Zn ? 50 lM Hg. Data represent the mean ± SD of three

replicates. Identical letters indicate no significant differences

among the treatments according to Tukey0s multiple range test

(P \ 0.05)

Control Zn Hg Zn + Hg

Treatments

0

1

2

3

4

5P

igm

ents

con

cent

ratio

n (m

g g

-1 F

W)

chlorophyll a

aba a

a

abb

chlorophyll b carotenoids

bc c

ab

a

a

a

Control Zn Hg Zn + Hg

Treatments

0

2

4

6

8

10

ALA

-D a

ctiv

ity (

nmol

PB

G m

in-1 m

g pr

otei

n-1

)

Shoot

a

b

a

b

A

B

Fig. 2 Pigments concentration (A) and d-aminolevulinic acid

(d-ALA-D) activity (B) of shoots of P. glomerata plantlets

exposed to different treatments. Control: without Zn and Hg;

Zn: 50 lM Zn as ZnCl2; Hg: 50 lM Hg as HgCl2; Zn ? Hg:

50 lM Zn ? 50 lM Hg. Data represent the mean ± SD of

three replicates. Identical letters indicate no significant

differences among the treatments according to Tukey0smultiple range test (P \ 0.05)

964 Biometals (2011) 24:959–971

123

Hydrogen peroxide and lipid peroxidation

The concentration of H2O2 augmented in shoots of

plants treated with Hg and Zn ? Hg (Fig. 3). Con-

versely, root H2O2 concentration was only changed

upon addition of Zn ? Hg treatment, where it

decreased when compared to other treatments. Shoot

lipid peroxidation was significantly increased upon

addition of Hg, while it decreased significantly in the

treatment with Zn ? Hg added together, when com-

pared to the control (Fig. 3B). On the other hand, lipid

peroxidation in roots did not alter upon all treatments.

Antioxidant enzymes activity

SOD activity was higher in shoots than in roots

(Fig. 4A). Mercury-treated plants showed 2.3-fold

higher shoot SOD activity than the control. Treat-

ments either with Zn applied alone or together with

Hg showed greater shoot SOD activity that the

control, but these increase was lower than that

observed in plants treated exclusively with Hg. In

the roots, however, the highest SOD activity occurred

Control Zn Hg Zn + Hg

Treatments

0.00

0.30

0.60

0.90

1.20

1.50

H2O

2 (µ

mol

g-1

fres

h w

eigh

t)

Shoot

a

b

a

b

Root

aa b

a

Control Zn Hg Zn + Hg

Treatments

0.00

0.06

0.12

0.18

0.24

0.30

TB

AR

S (

nmol

MD

A m

g-1

pro

tein

)

Shoot

a

b

abc

Root

aa a

c

B

A

Fig. 3 Hydrogen peroxide concentration (A) and lipid

peroxidation (B) of P. glomerata plantlets exposed to different

treatments. Control: without Zn and Hg; Zn: 50 lM Zn as

ZnCl2; Hg: 50 lM Hg as HgCl2; Zn ? Hg: 50 lM

Zn ? 50 lM Hg. Data represent the mean ± SD of three

replicates. Identical letters indicate no significant differences

among the treatments according to Tukey0s multiple range test

(P \ 0.05)

Control Zn Hg Zn + Hg

Treatments

0

160

320

480

640

800

SO

D a

ctiv

ity (

units

SO

D m

g-1

pro

tein

)

Shoot

a

d

b

b

Root

d

a

c

c

Control Zn Hg Zn + Hg

Treatments

0

14

28

42

56

70

CA

T a

ctiv

ity (

109

E m

in-1 g

-1) Shoot

a

abb

b

Root

ba

bab

Control Zn Hg Zn + Hg

Treatments

0.00

0.16

0.32

0.48

0.64

0.80

AP

X a

ctiv

ity (

units

mg

-1 p

rote

in)

Shoot

cb

a

b

Root

abb

ca

A

B

C

Fig. 4 Superoxide dismutase (SOD) activity (A), catalase

(CAT) activity (B) and ascorbate peroxidase (APX) activity

(C) of P. glomerata plantlets exposed to different treatments.

Control: without Zn and Hg; Zn: 50 lM Zn as ZnCl2; Hg:

50 lM Hg as HgCl2; Zn ? Hg: 50 lM Zn ? 50 lM Hg. Data

represent the mean ± SD of three replicates. Identical letters

indicate no significant differences among the treatments

according to Tukey0s multiple range test (P \ 0.05)

Biometals (2011) 24:959–971 965

123

at the control, whereas, the minimum SOD activity

occurred in the Hg alone treatment, being 60% lower

than to control. Plants exposed to Zn alone and

Zn ? Hg concomitantly, showed approximately 37

and 73% of decrease in the SOD activity compared to

control plants.

Shoot CAT activity decreased upon addition of

Hg, whereas there was no significant differences in

plants supplied with Zn or Zn ? Hg. Root CAT

activity had a different response, it significantly

increased (60%) upon addition of Hg, when com-

pared to the other treatments.

The treatment with Zn applied alone showed

greater root APX activity than control, whereas

treatments containing Hg, either added alone or

together with Zn, showed a significant decrease,

when compared to the control. On the other hand,

shoot APX activity only increased upon addition of

Zn ? Hg.

Antioxidant non-enzymatic concentration

The NPSH concentration was much higher in the

shoot than in roots (Fig. 5A). Shoot NPSH concen-

tration increased only upon addition of Zn ? Hg,

when compared to the other treatments. Treatments

with Hg added alone or together with Zn showed

greater root NPSH concentration than the control.

Conversely, root NPSH concentration did not change

upon addition of Zn treatment, when compared to the

control.

In general, AsA concentration in shoots and roots

followed a similar pattern as that found for NPSH

concentration, with the exception of Hg-treated

plants, which shoot AsA increased above control

levels. Moreover, plants exposed to Zn ? Hg showed

the highest shoot AsA concentration (Fig. 5B).

Discussion

This study was performed to analyze the mechanism

by which Zn antagonizes Hg toxicity in P. glomerata,

an important medicinal plant. This interaction

between a nutrient and a toxic element may be

important for understanding, analyzing and improv-

ing the defense strategies through various parameters.

In the present study, plants of P. glomerata exposed

to Hg added alone in the nutrient solution showed

significant oxidative stress symptoms, observed as an

increase in shoot H2O2 concentration (Fig. 3A) and

lipid peroxidation (Fig. 3B). Similar results were

reported in our previous work (Calgaroto et al. 2010).

These alterations were followed by significant

changes in the antioxidant system (Fig. 4, 5). How-

ever, the antioxidant system was not sufficient to

avoid damage, resulting in 52.8% of mortality in Hg-

treated plants (Fig. 1A). Percentage survival was

significantly higher in plants exposed to Zn ? Hg,

when compared to Hg added alone, indicating that Zn

treatment alleviated the oxidative damage induced

by Hg.

In the present study, shoot Hg concentration

decreased concomitantly with a significant increase

of Zn concentration in plants exposed to Zn ? Hg

(Table 1), indicating a possible negative interaction

between these metals. Zinc, Cd and Hg are divalent

metals that constitute group II B of the periodic table

Control Zn Hg Zn + Hg

Treatments

0.00

0.30

0.60

0.90

1.20

1.50

NP

SH

(µ

mol

SH

g-1 fr

esh

wei

ght) Shoot

bb

b

b

Root

aba

a

Control Zn Hg Zn + Hg

Treatments

0

30

60

90

120

150

Asc

orbi

c ac

id (

µg A

sA g

-1 fr

esh

wei

ght)

Shoot

b

c

b

c

Root

ab

a

a

B

A

Fig. 5 Non-protein thiols (NPSH) concentration of

P. glomerata plantlets exposed to different treatments. Control:

without Zn and Hg; Zn: 50 lM Zn as ZnCl2; Hg: 50 lM Hg as

HgCl2 and; Zn ? Hg: 50 lM Zn ? 50 lM Hg. Data represent

the mean ± SD of three replicates. Identical letters indicate no

significant differences among the treatments according to

Tukey0s multiple range test (P \ 0.05)

966 Biometals (2011) 24:959–971

123

of elements. Although Zn is an essential trace metal

while the remaining two are toxic metals, they share

biological responses such as affinity towards thiol

groups, antagonism to biological cations, participa-

tion in redox reaction and modulation of nutrients

homeostasis (Peixoto et al. 2003). Hart et al. (2002)

observed that the concentration-dependent uptake of

both Cd2? and Zn2? in both Triticum aestivum L. and

Triticum turgidum L. var. durum consisted of a

combination of linear binding by cell walls and

saturable, Michaelis–Menten influx across the plasma

membrane. Several other data suggest the existence

of antagonist effect of Zn on other essential transition

metals like Cu and Fe (Zago and Oteiza 2001;

Marschner 2002). In the present study, the concen-

tration of Zn in shoots of plants exposed to Zn ? Hg

might be had some positive influence on the antiox-

idant system, which resulted in significant decrease of

shoot lipid peroxidation (Fig. 3B) associated with a

slight increase of CAT and APX activities (Fig. 4B

and C). Therefore, Zn might interact negatively with

Hg and it also suggests that Zn is a pivotal component

of the antioxidant defense network that protects

membranes from oxidation.

Shoot and root fresh weight from plants exposed to

Hg added alone showed reduction of about 70% and

60%, respectively, when compared to the control

(Fig 1B). Such result might be related to the effect of

Hg on aquaporins. Mercury induces conformational

changes in aquaporins because it is thought to bind to

–SH groups of the proteins, then blocking the

channels and reducing their hydraulic conductivity

(Tyerman et al. 1999). The addition of HgCl2(0.1 mM) reduced pressure-induced water flux and

root hydraulic conductivity in the roots of 1-year-old

aspen (Populus tremuloides Michx.) seedlings by

about 50% (Wan and Zwiazek 1999). In the present

work, the symptoms of wilting showed by plants

treated with Hg can explain the decrease in fresh

weight and this data corroborates with other reports

(Zang and Tyerman 1999; Cargnelutti et al. 2006).

Interestingly, in P. glomerata plants treated with

Zn ? Hg, shoot and roots fresh weight showed a

significant recover when compared to the Hg treat-

ment (Fig. 1B).

Zinc is an essential component of many vital

enzymes, a structural stabilizer for proteins, mem-

brane and DNA-binding proteins (Epstein and Bloom

2005). On the other hand, Zn excess in substrate

might causes nutritional disorders in plants (Marschner

2002; Reichman 2002). In the present study, signif-

icant decrease of shoot fresh and dry weight in plants

exposed to Zn alone might be due to Fe deficiency

once that shoot Fe concentration was about 53%

lower than that control plants (data not shown).

Tewari et al. (2008) also found that excess Zn led to a

decrease in Fe level in tissues of Morus alba L.

Moreover, both H2O2 and MDA accumulated in

leaves of Zn–stressed plants. Iron is an essential

element for plants and plays an important role in

several plant biological processes including the

catalytic groups for many redox enzymes (Epstein

and Bloom 2005). The antagonist interaction between

Zn and Fe can lead to nutrient deficiencies and crop

yield reduction (Marschner 2002; Reichman 2002).

The toxicity of heavy metals, such as non-essen-

cial ones, Hg and Cd, is generally accepted to occur

by inactivation of enzymes and/or functional proteins

by directly binding to them. However, several studies

also showed that the toxicity may be due, in part at

least, to oxidative damage by generation of ROS due

to the presence of heavy metals (Cargnelutti et al.

2006; Zhou et al. 2008; Sobrino-Plata et al. 2009).

Mercury ions can also substitute metal ions in

photosynthetic pigments, causing a decrease in pho-

tosynthesis rates (Xylander et al. 1996). Decreased

pigments content due to high levels of Hg in substrate

has been reported in Licopersicum esculentum L.

(Cho and Park 2000), Cucumis sativus L. (Cargnelutti

et al. 2006) and Medicago sativa cv Aragon (Sobrino-

Plata et al. 2009). In the present study, no effect was

observed in pigments concentration from P. glomer-

ata plants exposed to Hg (Fig. 2A). This result might

be related to the reduction in fresh weight (Fig. 1B)

and diminution of leaf expansion (data not shown),

which would lead to an increase in the concentration

of cellular components. Very similar results were

reported in our previous work (Calgaroto et al. 2010).

The enzyme d-aminolevulinic acid dehydratase

(d-ALA-D) is an important enzyme for the synthesis of

porphyrins, hemes and chlorophylls, essential factors

of an adequate aerobic metabolism and photosynthe-

sis (Pereira et al. 2006). In the present study, in

response to the imposed Hg toxicity, a significant

decrease in the d-ALA-D activity, by about 53%

when compared to the control, was observed

(Fig. 2B). Due to its sulphydryl nature, d-ALA-D is

highly sensitive to the presence of heavy metals such

Biometals (2011) 24:959–971 967

123

as Hg (Emanuelli et al. 1996). Therefore, the

significant decrease in d-ALA-D activity observed

in this study could be the result of Hg-binding with

–SH groups of d-ALA-D enzyme. Similar results

were reported in our previous work (Calgaroto et al.

2010). Plants exposed to Zn ? Hg, in contrast,

showed a sharp increase in d-ALA-D activity, so

that was significantly equal to the control. The

present study indicated that decrease in d-ALA-D

activity induced by Hg was prevented by increase of

Zn concentration in shoot (Table 1). Probably, Zn

avoided Hg-binding with –SH groups of this enzyme,

also avoiding its inactivation.

Oxidative stress occur either due to excess

production of free radicals, or inadequate availability

of antioxidants or a combination of both (Zhou et al.

2008; Wang et al. 2009). Lipid peroxidation is a basic

deteriorating change in unsaturated fatty acids of the

cell membranes induced by excess free radicals

(Haliwell and Gutteridge 1999). Estimation of mal-

onyldialdehyde (MDA) continues to be a reliable

method to assess the degree of peroxidative damage

to cell membrane, as it is the most abundant aldehyde

formed as a by-product during this process (Zhou

et al. 2008; Wang et al. 2009). In the present study,

roots MDA concentration was not altered in presence

of Zn or Hg (Fig. 3B), although other stress symp-

toms had occurred in Hg-stressed plants, such as

growth reduction (Fig. 1B and C) and root darkening

(data not shown). In shoot, however, the MDA

reached higher concentration in plants exposed to Hg

added alone, when compared to the control. This

result is in agreement with the report of Cho and Park

(2000), which showed that tomato plants exposed to

50 lM HgCl2 had a significant increase on MDA

concentration. Using alfalfa plants grown on an inert

solid substrate in a hydroponic culture, Sobrino-Plata

et al. (2009) also observed that Hg leaded to a higher

lipid peroxidation in shoots, with much more mod-

erate response in roots. These results are in agreement

with previous work by Vazquez and Carpena-Ruiz

(2005), which compared the toxic effects of Cd in

Lupinus albus grown in a liquid-hydroponic medium

or a semi-hydroponic system. They found that the

inert substrate perlite limited the rate of Cd absorp-

tion, and the toxic effects on roots were moderate.

Goncalves et al. (2009) also reported that the

influence of Cd on nutrient concentration in Solanum

tuberosum was related to many factors such as the

level of Cd in the substrate, potato cultivar, plant

organ, essential element, growth medium and expo-

sure time. In the present study, the higher MDA

concentration in the shoot indicates that above

ground tissues are more sensitive to the metals than

roots, and Zn addition had an important role in the

ROS detoxification induced by Hg.

H2O2 concentration is, in part, a result of SOD

activity, the first antioxidant enzyme involved with

ROS detoxification. Although H2O2 is a ROS not

reactive as the superoxide radical, accumulation of

H2O2 may lead to OH� due to the reaction with

transition metals like Fe and Cu, causing extensive

cellular damage (Zago and Oteiza 2001). To avoid a

potentially hazardous situation, APX and CAT anti-

oxidant enzymes act degrading these molecules to

H2O (Mittler 2002). H2O2 concentration (Fig. 3A)

and SOD activity (Fig. 4A) were higher in the shoot

of plants exposed to Hg alone, indicating that

occurred an over-production of superoxide radicals

resulting then, in increase by 2.3-fold in the SOD

activity when compared to the control. Perhaps Zn

addition in nutrient solution had a major role in

detoxification of superoxide radicals, because its

presence allowed the increase of SOD activity

(Fig. 4A). The higher SOD activity observed in shoot

of plants exposed to Zn and Zn ? Hg can be due

presence of Zn, which has a structural role in the

Cu-ZnSOD enzyme (Wang et al. 2009). As a result of

SOD action in plants exposed to Hg, shoot H2O2 was

produced, inducing slightly the scavenging-activity

of APX enzyme in this tissue (Fig. 4C). Conversely,

SOD activity was lower in roots exposed to Hg alone,

whereas CAT activity was greater. It seems that

excess of ROS production in presence of Hg in roots

would have inhibited antioxidant enzymes such as

SOD and APX. Similar results were reported in our

previous work (Calgaroto et al. 2010). However,

Zhou et al. (2008) found that high Hg level (40 lM)

increased the activities of SOD, CAT and APX in

Medicago sativa plants. Therefore, the varied

responses showed in the present paper and in others

might be due to differences related to many factors as

reported by Vazquez and Carpena-Ruiz (2005) and

Goncalves et al. (2009). In addition, the experiments

of the present study were carried out twice and gave

reproducible results. Besides that, the values mea-

sured of SOD and CAT activities are in the range

already reported in P. glomerata (Gupta et al. 2011)

968 Biometals (2011) 24:959–971

123

and in Zea mays (Gupta et al. 2009) under Pb stress

conditions.

ROS levels can be also controlled by non-

enzymatic antioxidants (Noctor and Foyer 1998).

Therefore, we measured the accumulation of AsA

and NPSH, the two major components of plant cells

that mediate H2O2 in some cellular compartments

(Mittler 2002). In the present study, Hg-treated

plants had significant increase in shoot NPSH con-

centration (Fig. 5A). Similar results were reported in

our previous work (Calgaroto et al. 2010). The

Hg-induced increase in NPSH levels might reflect the

defensive mechanism against oxidative stress trig-

gered by Hg (Zhou et al. 2008; Sobrino-Plata et al.

2009). The increase in NPSH concentration might

reflect enhanced GSH cellular concentration and,

subsequently, an increase in PCs production, metab-

olites that are known to detoxify heavy metals

(Ortega-Villasante et al. 2007; Zhou et al. 2008;

Sobrino-Plata et al. 2009). Recently Chen et al.

(2009), elucidated the important roles of PCs in Hg

tolerance of Brassica chinensis, not only because of

their sequestration of free Hg2? but because they

reduce oxidative stress in cells. As shown in Fig. 5B,

the shoot and root AsA concentration increased upon

addition of Hg, when compared to the control. In

addition, our results also indicate that AsA concen-

tration was much more affected in shoot than in roots,

which is consistent with the results of Calgaroto et al.

(2010) for Pfaffia glomerata and of Sobrino-Plata

et al. (2009) for Medicago sativa. The concentration

of AsA augmented also in the roots of Zea mays

plants exposed to 200 lM Pb (Gupta et al. 2009).

Interestingly, in the present study, shoot AsA con-

centration was significantly higher in plants exposed

to Zn ? Hg, when compared to Hg added alone,

indicating that Zn treatment might have alleviated the

oxidative damage induced by Hg through enhanced

AsA accumulation. Exposure to oxidative stress

increases tissue ascorbate accumulation and results

in an enhanced of ascorbate-dependent detoxification

processes (Noctor and Foyer 1998; Mittler 2002;

Sobrino-Plata et al. 2009).

In conclusion, the supplementation of Zn to

Hg-treated plants did alleviate the Hg-toxic effects

in P. glomerata plants by decreasing Hg uptake and

translocation, decreasing lipid peroxidation, increas-

ing the d-ALA-D activity, and increasing the protec-

tive effect of the antioxidant system.

Acknowledgments The authors wish to thank the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico

(CNPq), Coordenacao e Aperfeicoamento de Pessoal de

Nıvel Superior (CAPES) and Fundacao de Amparo a

Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for

the research fellowships.

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