ORIGINAL PAPER

Accumulation and translocation of Cd metal and the Cd-inducedproduction of glutathione and phytochelatins in Vicia faba L.

Radomır Cabala • L’udmila Slovakova •

Manal El Zohri • Hartmut Frank

Received: 3 February 2010 / Revised: 21 October 2010 / Accepted: 23 November 2010 / Published online: 8 December 2010

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Translocation of cadmium (Cd) in the tissues of

Vicia faba, the water content in biomass, the biomass

production, and the glutathione and phytochelatin tissue

concentrations were studied and correlated with the plant

sensitivity and/or tolerance to Cd. The total concentrations

of Cd were determined by inductively coupled plasma/

mass spectrometry (ICP-MS), the concentrations of gluta-

thione (GSH) and phytochelatins 2 and 3 (PC2 and PC3)

were determined by on-line high performance liquid

chromatography/electrospray-ionization tandem mass

spectrometry (HPLC–ESI–MS–MS) in the roots and leaves

of the sensitive and the tolerant cultivars of V. faba grown

in Cd containing nutrient solutions (NS, 0–100 lmol l-1

Cd2?). Both the cultivars of V. faba accumulate a major

portion of Cd in the roots and only a minor part of ca. 4%

in the leaves. The differences between the cultivars con-

cerning Cd accumulation in leaves were apparent from

higher Cd concentrations in NS and the Cd amount in the

sensitive cultivar was approximately twice as high. In the

roots, the differences between the cultivars in the Cd

accumulation were only statistically significant with the

highest Cd concentrations in NS, with the tolerant cultivar

accumulating about 16% more of Cd compared to the

sensitive one. The biomass production of the sensitive

cultivar decreased approximately twice as fast with

increasing Cd concentration in NS. The biomass water

content decreased with increasing Cd concentration in NS

in both the cultivars. In general, the GSH concentration did

not linearly correlate with Cd accumulation, except for the

roots of the sensitive cultivar where it was independent,

and was higher in the sensitive cultivar than in the tolerant

one in both the leaves and roots. The GSH concentration in

leaves was approximately one order of magnitude higher

than that in the roots for both the cultivars. The relation-

ships between the PC and Cd concentrations in tissues were

found nonlinear. At lower Cd accumulation levels, the PC

concentrations followed an increase in the Cd accumula-

tion in both the roots and leaves, whereas at higher Cd

accumulations the relations differed between roots and

leaves. In the roots, the PC concentrations decreased with

increasing Cd accumulation, whereas the PC concentration

in the leaves followed the decrease in the Cd accumulation.

Keywords Vicia faba L. � Cd accumulation �Glutathione � Phytochelatins

Abbreviations

Cd Cadmium

CCd-leaves Cd concentration in the leaves

CCd-NS Cd concentration in the nutrient

solution

CCd-roots Cd concentration in the roots

Communicated by J. Ueda.

R. Cabala (&)

Department of Analytical Chemistry, Faculty of Science,

Charles University in Prague, Albertov 6,

128 43 Prague, Czech Republic

e-mail: cabala@natur.cuni.cz

L. Slovakova

Department of Plant Physiology, Faculty of Natural Sciences,

Comenius University in Bratislava, Bratislava, Slovak Republic

M. El Zohri

Botany Department, Faculty of Science,

Assuit University, Assuit, Egypt

H. Frank

Environmental Chemistry and Ecotoxicology,

University of Bayreuth, Bayreuth, Germany

123

Acta Physiol Plant (2011) 33:1239–1248

DOI 10.1007/s11738-010-0653-0

DW Dry weight

FW Fresh weight

GSH Glutathione, c-Glu-Cys-Gly

HPLC–ESI–MS–MS High performance liquid

chromatography electrospray-

ionization tandem mass

spectrometry

ICP-MS Inductively coupled plasma/mass

spectrometry

MTs Metallothioneins

NS Nutrient solution

PC, PCs Phytochelatin, phytochelatins

PC2 Phytochelatin 2 (c-Glu-Cys)2-Gly

PC3 Phytochelatin 3 (c-Glu-Cys)3-Gly

R Correlation coefficient

Introduction

Vicia faba beans are considered to be the main legume of

Middle Eastern countries, hence they have a potential

economic importance in many countries, especially in

Egypt. On the other hand, V. faba has proved to be very

sensitive biomonitor of water contaminants both in labo-

ratory and in situ treatments (Villalobos-Pietrini et al.

1994). Cadmium (Cd) is of special interest, as it is con-

sidered to be the fifth most toxic metal for vertebrates, and

the fourth most toxic metal for vascular plants (Jones

1939). It has been introduced into the environment mainly

through agricultural applications of Cd-contaminated

phosphate fertilizers, sewage sludge and pesticides and by

some industrial activities, such as metal mining, smelting

and founding, the use and disposal of batteries, and dis-

posal of metal-contaminated waste (Van Bruwaene et al.

1984; Cabrera et al. 1998). In addition to these anthropo-

genic emissions, Cd is also released by natural sources,

such as volcanoes and continental dusts or by weathering

of the underlying bedrock (Cabrera et al. 1998).

Plant species and even varieties differ significantly in

their resistance to Cd and in Cd accumulation capacity

(Hart et al. 1998; Zhao et al. 2002), but this is not neces-

sarily correlated with the tolerance to the metal (Greger

et al. 1991). For example, resistant and tolerant cultivars of

Silene cucubalus (Mathys 1975) and Agrostis (Mathys

1973) accumulate Cd to the same level. Cd accumulation

also differs among organs or tissues in the same plant.

Adriano (1986) reported that Cd concentrations in plant

tissues decrease in the order: old roots [ new roots [stems [ leaves [ reproductive organs.

Complexation, compartmentation and sequestration

belong to the main protective mechanisms of plants against

negative influence of heavy metals (e.g. Cd). Together with

metallothioneins (MTs), glutathione (GSH) and phyto-

chelatins (PCs), which plays an important role in detoxi-

fication of heavy metals, are members of a group of

biogenic ligands produced by plants (Meister 1995). GSH,

usually the most abundant intracellular nonprotein thiol

(tripeptide c-Glu-Cys-Gly), is enzymatically converted into

PCs, peptides of general structure (c-Glu-Cys)n-Gly,

n = 2–11, by glutamyl-cysteine dipeptidyl transpeptidase

(Beck et al. 2003). Cd2? catalyzed PC biosynthesis is

influenced, in dependence on their concentrations, by free

heavy metals and enzymes, and inhibited by Cd–PC and

Cd–GSH complexes. All the reactions form together a self-

regulating loop. The amount of PCs induced by Cd2?

seems to be roughly related to the Cd2? concentration and

the time. It is generally supposed that, within species-

specific and organ-specific limits of toxicity, the more

Cd2? is supplied, the greater induction of PCs is obtained

(Sanita di Toppi and Gabbrielli 1999). In addition,

Cd-stressed cells have to restore their physiological GSH

concentration by activating the enzyme catalyzing GSH

biosynthesis, which requires an energy supply (Ruegsegger

and Brunold 1992). Using purified recombinant PC-syn-

thase from Arabidopsis thaliana, it has been shown that the

Cd–(GSH)2 complex is a necessary substrate along with

free GSH, which explains the metal dependence of the PC

synthesis reaction mechanism (Vatamaniuk et al. 2000).

PCs belong to the group of sulfhydryl-rich peptides,

which exhibit strong metal-binding properties. Originally,

the basic physiological function of PCs has been suggested

to keep the homeostasis of heavy-metal nutrients, such as

Cu and Zn, in the cytoplasm (Steffens 1990). However,

there is currently no direct evidence that PCs have func-

tions other than in metal detoxification (Cobbett 2000). In

heavy-metal tolerance, PCs have shown various effects.

They seem to be involved in the main mechanism for high

tolerance to Cd in tomato and Arabidopsis (Howden et al.

1995). In other cases, such as S. vulgaris, the PCs seem to

have a negative influence on the metal tolerance, as the PC

production depletes the GSH in the cell, needed for pro-

tection against free radicals generated by the metal toxicity

(Ric De Voss et al. 1992). Studies on Cd-tolerant and

Cd-sensitive lines of the plant Datura innoxia, also known

as Jimsonweed, show that both lines accumulate similar

levels of PC–Cd complexes and synthesize similar

amounts of PCs at similar rates, but the onset of synthesis

of the PC–Cd complexes is more rapid in the metal-

tolerant plant line than in the metal-sensitive line (Yen

et al. 1999).

An important objective for scientific research is to select

crop species and cultivars, which are tolerant to Cd and

retain it in the roots, i.e. translocate less Cd from a con-

taminated medium to the biomass. This idea has

1240 Acta Physiol Plant (2011) 33:1239–1248

123

successfully been applied with durum wheat and sunflower

(Penner et al. 1995; Li et al. 1995). Accordingly, the aims

of this study were to investigate (a) the differences in the

Cd accumulation, the GSH and PC concentrations in bio-

mass between Cd-sensitive and Cd-tolerant cultivars of

V. faba; (b) the differences in Cd translocation patterns

within the plant between the cultivars; (c) the correlations

among the Cd, GSH and PC concentrations in the plant

tissues of both the cultivars.

Materials and methods

Reagents, chemicals and standards

Cadmium chloride (p.a.) was used as supplied (Sigma-

Aldrich, Steinheim, Germany). Bidistilled water (Buchi

F-285, Flawil, Switzerland) and all other reagents and sol-

vents were of analytical grade quality and used as received.

PC standards (lyophilized) were kindly provided by prof.

M. H. Zenk (University of Wittenberg-Halle, Germany).

Plant material and cultivation

Seeds of Cd-tolerant (Giza 40) and Cd-sensitive (Giza 2)

cultivars of broad bean (V. faba L.), obtained from the

Cairo Agricultural Research Center (Cairo, Egypt), were

germinated on moist filter paper in Petri dishes for 5 days.

The seedlings were further grown in 1/10 strength modified

Hoagland’s solution in a greenhouse with the 16/8 light

cycle (light intensity 35,000 lux) and the day/night tem-

perature of 23/18�C. When the plants were 19 days old,

they were transferred to a fresh nutrient solution containing

CdCl2 at concentrations of 0, 1, 10, 40 and 100 lmol l-1,

five replicates of each. After another 15 days, the samples

were collected: roots and leaves separated, washed with

deionized water, weighed, frozen in liquid nitrogen, and

stored at –80�C until the sample preparation.

Sample preparation, separation and quantification

A detailed description of the sample preparation, separation

and quantification method with the Cd detection limit,

0.5 lg l-1 (ICP-MS) and the limits of quantification

(LOQ), 0.2 lmol kg-1 (HPLC–ESI–MS–MS), for GSH,

PC2 and PC3 has been published in El Zohri et al. (2005).

Statistical testing

To judge rightly and exactly the statistical significance of

differences between the data compared t test at a confi-

dence level (1 - a) equal to 0.95 was applied.

Results

During the investigation, a huge multidimensional dataset

was obtained, composed of total Cd-, GSH- and PCs-con-

centrations in plant tissues and the biomass parameters

corresponding to respective Cd concentrations in the

nutrient solution. This large set of data was difficult to treat

and present in full and thus simple two-dimensional

parameter correlations are presented and discussed in the

text below.

Correlation between external and internal

Cd concentration

The general response of the broad been to the increasing

Cd concentration in the nutrient solution (CCd-NS) differs

for roots and leaves as well as for the two cultivars. The Cd

uptake into the roots and translocation into the leaves was

found time-dependent and influenced by the total Cd con-

centration in the medium.

No significant difference in the concentration of Cd in

the roots (CCd-roots) was observed between the two culti-

vars. The CCd-roots increased almost linearly with CCd-NS

(R [ 0.9980) and reached a level of 100 mmol Cd kg-1

(DW) at CCd-NS of 100 lmol l-1 (Fig. 1).

A different behavior is observed for leaves, where the Cd

concentration (CCd-leaves) is almost identical for a CCd-NS of

up to 10 lmol l-1 and starts to differ significantly above this

value for both the cultivars (Fig. 2). Above this CCd-NS value,

the CCd-leaves of tolerant cultivar decreases only slightly,

whereas for the sensitive cultivar the maximum CCd-leaves,

3.3 mmol kg-1 (DW), is reached at CCd-NS of 40 lmol l-1.

0 1 10 40 1000

20

40

60

80

100

120

Cd

in r

oo

ts (

mm

ol.k

g-1 D

W)

Cd in nutrient solution (µmol.l-1)

Tolerant

Sensitive

Fig. 1 Dependence of the Cd concentration (mmol kg-1 DW) in the

roots of Vicia faba L. cultivars (Cd-tolerant Giza 40 and Cd-sensitive

Giza 2) on the Cd concentration (lmol l-1) in nutrient solution. Data

points represent mean ± standard error (n = 5)

Acta Physiol Plant (2011) 33:1239–1248 1241

123

Correlation between the Cd concentration

and biomass production

Total (roots and leaves) fresh (Fig. 3) and dry biomass

weights (the data are not shown) of broad bean decreased

with increased CCd-NS for both the tolerant and the sensitive

cultivars. This decrease was more evident in the sensitive

cultivar where the CCd-NS of only 1 lmol l-1 decreased the

total FW and total DW to about 64 and 60% of the control

weight, respectively. In the tolerant cultivar, the effect was

not statistically significant at this CCd-NS value.

The roots of the sensitive cultivar were more sensitive to

Cd than tolerant ones. The lowest CCd-NS used (1 lmol l-1)

decreased their total biomass to 58%, whereas this

parameter was not influenced in the tolerant cultivar.

FW of roots of the sensitive and tolerant plant dimin-

ished with the Cd concentration to 10 and 50% of the

control at CCd-NS of 100 lmol l-1, respectively.

The influence of Cd on the growth parameters expressed

in the DW percentage of FW is illustrated in Fig. 4. The

root DW of the tolerant cultivar increased monotonously

from 5.5 to 7.2% following the increase in CCd-NS. In other

words, the water content linearly decreases with increasing

CCd-NS. This almost perfectly linear decrease is character-

ized by high values of DW - CCd-NS correlation coeffi-

cients of 0.9755. A more complicated and less distinct

relationship between DW and CCd-NS was found for the

sensitive cultivar where mean values for DW for roots

increased from 6.5% at control to its maximum of 8% at

CCd-NS of 10 lmol l-1 and decreased surprisingly to 7% at

the maximum Cd concentration (Fig. 4). It is probable that

in our case the roots of the sensitive broad bean cultivar

were somewhat degraded (decomposed) which could

enhance the water content.

The FW of leaves of the sensitive and tolerant plant

diminished with the Cd concentration to 15 and 42% of the

control at CCd-NS of 100 lmol l-1, respectively.

Similarly as for the roots, the leaves DW of the tolerant

cultivar increased monotonously from 10.5 to 15.6% fol-

lowing the increase in CCd-NS. The water content linearly

decreases with increasing CCd-NS and this decrease is

characterized by DW - CCd-NS correlation coefficients of

0.9839.

Leaves DW of the sensitive cultivar decreased slightly at

CCd-NS of 1 lmol l-1, followed by a continuous DW

0 1 10 40 1000

1

2

3

4

Cd

in le

aves

(m

mo

l.kg

-1 D

W)

Cd in nutrient solution (µmol.l-1)

Tolerant

Sensitive

Fig. 2 Dependence of the Cd concentration (mmol kg-1 DW) in the

leaves of Vicia faba L. cultivars (Cd-tolerant Giza 40 and Cd-

sensitive Giza 2) on the Cd concentration in nutrient solution. Data

points represent mean ± standard error (n = 5)

0

10

20

30

40

50

0 1 10 40 100

Cd Concentration in Nutrient Solution, µmol.L-1

To

tal F

resh

Bio

mas

s, g

TolerantSensitive

100%

97.4

%

64.4

%

85,5

%

39,3

% 68.4

%

26,0

% 46,8

%

12,2

%

Fig. 3 Dependence of total plant biomass production of Vicia faba L.

cultivars (Cd-tolerant Giza 40 and Cd-sensitive Giza 2) on the Cd

concentration in nutrient solution (lmol l-1). Numbers represent

biomass production expressed in terms of % of the control

(0 lmol l-1 Cd) (n = 5)

0

5

10

15

Leaves -Tolerant

Leaves -Sensitive

Roots -Tolerant

Roots -Sensitive

% D

ry W

eig

ht

of

FW

0 1 10 40 100

Cd concentration in nutrient solution, µmol.l-1

Fig. 4 Dependence of dry weight biomass production (leaves and

roots) of Vicia faba cultivars (Cd-tolerant Giza 40 and Cd-sensitive

Giza 2) on the Cd concentration in nutrient solution (lmol l-1).

Biomass production is expressed in terms of % of the fresh weight.

Data points represent mean ± standard error (n = 5)

1242 Acta Physiol Plant (2011) 33:1239–1248

123

increase up to 13.6% at the highest Cd concentration which

is the opposite tendency to the roots.

This irregular correlation between the water content in

FW and CCd-NS is reflected in the correlation coefficient

values below 0.42 for both roots and leaves of the sensitive

cultivar.

Correlation between tissue concentrations

of Cd, GSH and PCs

The response of the tissue GSH and PC concentrations

toward Cd accumulation was generally stronger in roots of

the tolerant cultivar and in leaves of the sensitive one.

The GSH concentration in roots of the sensitive cul-

tivar was independent on the CCd-NS (Fig. 5a) and

remained approximately at the same level as in control

broad bean plants. The GSH concentration was approxi-

mately ten times higher in roots of the sensitive cultivar

than in the tolerant one at higher Cd root concentrations.

On the contrary, a dramatic decrease in the GSH root

concentration occurred in the tolerant cultivar by appli-

cation of CCd-NS as low as 1 lmol l-1 and continued up

to CCd-NS of 10 lmol l-1. At higher Cd accumulation in

roots, the GSH level remained low and almost unchanged

(Fig. 5a).

Quite different results were observed for the leaves. The

GSH concentration in both the cultivars was generally

higher than in roots and differed between the two cultivars

even in the control plants (in all other cases, the concen-

trations of GSH and PCs were identical for the control

plants within the experimental precision). In the sensitive

cultivar, following an increase in the Cd accumulation (see

Fig. 2), the GSH concentration decreased to a minimum,

corresponding to the Cd accumulation at a CCd-NS of

10 lmol l-1 (Fig. 5b), whereas with a higher Cd accu-

mulation the GSH content was elevated but this increase

did not correspond to the amount of Cd determined in the

leaf tissues. In the tolerant cultivar, the amount of GSH in

leaves was three times lower than in the sensitive one. The

curve describing the correlation between the GSH con-

centration and the Cd tissue accumulation forms a loop

where the GSH concentration decreases continuously to its

minimum at CCd-NS of 10 lmol l-1 with increasing Cd

accumulation and remains almost unchanged at Cd accu-

mulation values corresponding to increased CCd-NS (com-

pare Figs. 2, 5b).

In both the cultivars, the production of PCs (PC2 and

PC3) was measured as the response to an increased CCd-NS

and correlated with Cd accumulation in broad bean tissues.

Application of Cd at very low concentrations induced

biosynthesis of PCs, as confirmed by the increase in the PC

concentrations from values below the limit of quantifica-

tion in control plants of both the cultivars to values above

them for the exposed plants. Careful inspection of Fig. 5

yields the following findings.

The PC concentrations in roots were higher for the tol-

erant cultivar than for the sensitive one (one exception for

CCd-NS of 100 lmol l-1 can be seen in Fig. 5c), whereas in

the leaves the relation was reciprocal. The highest PC2

concentration in leaves (Fig. 5d) is approximately one-

third lower compared to roots (Fig. 5c) for the tolerant

cultivar, whereas the highest PC2 in leaves is approxi-

mately twice as high as that in roots for the sensitive cul-

tivar. The highest PC3 concentration in leaves (Fig. 5f) is

approximately three times lower than in roots (Fig. 5e) of

the tolerant cultivar, whereas in the sensitive one it is 1.7

times lower than that in roots (opposite to PC2). This effect

corresponds to a higher Cd amount sequestered in the root

tissues as a consequence of an increased Cd concentration

in the cultivation medium.

The PC3 concentration is approximately one order of

magnitude higher than that of PC2 for leaves and roots of

both the cultivars. The courses of the dependence of PC3

concentration on the Cd accumulation follow the shape of

the courses for PC2 for roots and leaves of both the cul-

tivars, but the shapes are fundamentally different between

roots and leaves.

Discussion

Correlation between external and internal

Cd concentration

In accordance with the published results, the Cd uptake

into the roots and translocation into the leaves was found

time-dependent (Dixit et al. 2001) and influenced by the

total Cd concentration in the medium (Szalai et al. 2002).

In agreement with our results, a continuous increase in the

Cd amount in roots was also observed in maize (Szalai

et al. 2002) and wheat (Sun et al. 2005) where its con-

centration was much higher in the roots than in the leaves.

An approximately linear correlation between CCd-roots and

CCd-NS is described in this paper. Results for V. faba could

be an evidence for simple (passive) partitioning of Cd

between NS and the root tissue in V. faba. Similarly, in

Silene dioica, the young parts of roots significantly par-

ticipate in substantial uptake of Cd, as most Cd is present in

the rhizodermal cell walls (Martinka and Lux 2006).

Unfortunately, our experimental technique applied did not

allow to differentiate the Cd binding locations within the

tissues and cells. We have found that the amount of Cd in

leaves was approximately 30 times lower than in roots for

both the cultivars. This translocation pattern is observed in

some bush bean (Phaseolus vulgaris) which accumulates

Cd in the root more than in the shoot (Rodecap et al. 1981),

Acta Physiol Plant (2011) 33:1239–1248 1243

123

in soybeans, 84–92% of Cd are retained within the root

(Cataldo et al. 1981), and the Lupinus albus roots retain up

to 88% (Zhao et al. 2002). The same pattern is observed for

other plants, such as durum wheat (Cieslinski et al. 1996),

maize (Lozano-Rodriguez et al. 1997), and rice (Liu et al.

2004). On the contrary, higher natural Cd concentrations

have been observed in leaves than in roots for spinach and

lettuce (Kabata-Pendias and Pendias 1992). Relatively low

Cd accumulation in leaves found at our experiments can be

explained by the presence of transport barriers in roots;

moreover, the oxidative damage imposed by Cd is probably

inhibited by an overall increase in the activity of antioxi-

dative enzymes (Dixit et al. 2001).

The calculation of the CCd-roots/CCd-leaves ratio from our

experiments yields values in the range from 10 to 100 and

from 10 to 40 for the tolerant and the sensitive cultivars,

respectively. The highest ratio was found at CCd-NS of

100 lmol l-1 and the lowest one at CCd-NS of 10 lmol l-1

(the results are not shown). This also indicates that above

the CCd-NS of 10 lmol l-1, the leaves of the sensitive

cultivar contain an approximately twice as high Cd con-

centration compared to the tolerant one. These results are

in agreement with publications where the root/shoot con-

centration ratio of Cd was found to be usually higher in the

resistant ecotypes of S. dioica than in the sensitive one

(Martinka and Lux 2006). The different distribution of Cd

0,000

0,001

0,002

0 50 100

Cd in roots (mmol.kg -1 DW)

GS

H in

ro

ots

(m

mo

l.kg

-1 F

W)

TolerantSensitive

100

100

40

40

10

10

1

1

0,000

0,001

0,002

0,003

0 50 100

Cd in roots (mmol.kg-1 DW)

PC

2 in

ro

ots

(m

mo

l.kg

-1 F

W) Tolerant

Sensitive

0

100

10040

40

10

10

1

1

0,00

0,02

0,04

0,06

0 50 100

Cd in roots (mmol.kg-1 DW)

PC

3 in

ro

ots

(m

mo

l.kg

-1 F

W) Tolerant

Sensitive

100

100

40

40

10

101

1

0

0,000

0,005

0,010

0,015

0 1 2 3 4

Cd in leaves (mmol.kg -1 DW)

GS

H in

leav

es (

mm

ol.k

g-1

FW

)

TolerantSensitive

100

100

40

40

10

10

1

1

0

0

0,000

0,002

0,004

0 1 2 3 4

Cd in leaves (mmol.kg-1 DW)P

C2

in le

aves

(m

mo

l.kg

-1 F

W) Tolerant

Sensitive

100100

40

40

10

101

1

0

0,00

0,01

0,02

0 1 2 3 4

Cd in leaves (mmol.kg-1 DW)

PC

3 in

leav

es (

mm

ol.k

g-1

FW

) TolerantSensitive

100100

40

40

10

10

1

1

A B

C D

F E

Fig. 5 Correlation of the GSH,

PC2 and PC3 concentrations

(mmol kg-1 FW) with the Cd

concentration (mmol kg-1 DW)

in roots and leaves of Vicia fabaL. cultivars (Cd-tolerant Giza

40 and Cd-sensitive Giza 2).

The numbers at data points

represent the corresponding Cd

concentration in nutrient

solution (lmol l-1). Data points

represent mean ± standard

error (n = 5)

1244 Acta Physiol Plant (2011) 33:1239–1248

123

among the tissues could explain the differences in the

sensitivity to this metal (Lozano-Rodriguez et al. 1997).

Correlation between the Cd concentration

and biomass production

It was published that the Cd-treated plants showed lower

solute potential values, which resulted in higher turgor

potential values. This observation may suggest that Cd

decreases cell wall extensibility, which may cause a

reduced cell expansion (Poschenrieder et al. 1989). The

irregular correlation between the water content in FW and

CCd-NS found at our plants is inconsistent with most pub-

lished results. Indeed, in agreement with our results,

a longer cultivation of roots of sensitive Mung bean in

Cd-medium caused degradation processes and desiccation

(Slovakova et al. 2007). Similarly, it was published that

higher Cd concentrations caused growth retardation and the

roots appeared to be darker than the control lettuce plants,

perhaps due to root death (Maier et al. 2003). Roots of Cd-

tolerant pea mutant developed and growth by Cd concen-

trations by which root growth of wild-type plants was

completely inhibited (Tsyganov et al. 2007). To increased

Cd concentration in S. vulgaris, the twice as high Cd

concentration was necessary to inhibit the growth of tol-

erant plant roots compared to the roots of the sensitive one

(Knecht et al. 1994). Exposure to Cd reduced transpiration

in Brassica juncea and this correlated with reduced leaf

expansion growth whereas the photosynthesis was not

affected (Haag-Kerwer et al. 1999). Disturbances in

physiological functions may also be caused by malfunc-

tioning of plasma membrane. A longer exposure of a pea

plant to Cd was associated with increased degree of un-

saturation of the fatty acids, causing changes in the root

plasma membrane phospholipid composition and fluidity

(Hernandez and Cooke 1997).

Correlation between tissue concentrations

of Cd, GSH and PCs

In many works about molecular response of plant to Cd

exposure is presented that legumes have the unique

capacity to synthesize in higher ratio homoglutathione

(hGSH) and homophytochelatins (hPCs) than GSH and

PCs. This was reported for Vigna angularis (Oven et al.

2001) and for soybean (Vazqez et al. 2009). But in other

kind of legumes such as pea and white lupin (Vazqez et al.

2009) the prevalent type were PCs synthesized form GSH.

Works with Lotus japonicus (Loscos et al. 2006; Ramos

et al. 2007) indicate that synthesis of both kind of hGSH

and hPCs as well as GSH and PCs is also tissue specific

and Lotus is able to synthesize both of them. The HPLC–

ESI–MS–MS analytical method used for determination of

GSH and PCs (El Zohri et al. 2005) proved absence of

hGSH and hPCs in V. faba. The possible oxidation of GSH

and PCs in the sample was studied and discussed in the

same article. Briefly, in plant extracts prepared with addi-

tion of DTT at the very beginning of the extraction, GSH

and PCs were completely reduced, whereas in its absence

they were present predominantly in the oxidized form. This

is especially important for V. faba homogenates which are

known to contain high concentrations of redox-active

polyphenols, oxidative enzymes, and transition metals, all

of which promote oxidation during sample preparation.

Our results show that the response of GSH concentration

in roots to Cd is in agreement with the generally accepted

opinion that the formation of a Cd–PC complex reduces the

free Cd concentration in cytosol and could lead to a

decrease in the GSH content (Lozano-Rodriguez et al.

1997). Similarly, it has been published that the GSH con-

tent of CdCl2-treated cells of Mesorhisobium huakui,

which express gene for PC-synthase of A. thaliana,

decreased, whereas the GSH content in untreated cells was

higher (Sriprang et al. 2003). On the other hand, in the

sensitive cultivar the increased amount of GSH at higher

Cd content could result either from diminished PC syn-

thesis or from the enhanced synthesis of GSH as published

for poplar transformants with an augmented PC synthesis

(Noctor et al. 1996, 1998). Nonlinear relationships among

CCd-NS, GSH and Cd tissue concentrations are described in

this paper. Results point to relatively complicated feed-

back-controlled processes of production, regulation and

accumulation of the species of interest.

The differences in the PC content, found in our work,

clearly demonstrate that the tolerant cultivar could block

the Cd influx at the first barrier more effectively, in the

roots, whereas PCs probably bind Cd ions predominantly in

the leaves of the sensitive cultivar. As reported by Liu et al.

(2007), Cd resistance in both Brassica chinensis L. and

Brassica pekinensis (Lour.) Ropr. is based on an exclusion

mechanism in which the roots accumulate the metal ions

and then prevent the Cd translocation to the shoot. A

similar metal accumulation pattern has been published for

wheat. The PC concentrations in the stem and roots of

wheat were increased whereas no distinct difference was

found in the leaves and the PC synthesis was closely linked

to metal uptake and accumulation (Sun et al. 2005). Also,

tobacco plants overexpressing of Arabidopsis PC-synthase

gene showed higher content of PCs than controls and it was

higher in roots than in shoots (Pomponi et al. 2006). We

have found that in the roots the PC concentrations increase

up to their maximum at the Cd accumulation level corre-

sponding to CCd-NS of 10 lmol l-1 and decrease even at

increasing Cd accumulation. This behavior could be

explained by the limited PCs production capacity and/or

GSH depletion (compare Fig. 5a, c, e) which is more

Acta Physiol Plant (2011) 33:1239–1248 1245

123

apparent for the sensitive cultivar. The concentrations of

PCs in the leaves do not increase so steeply with increasing

Cd accumulation and beyond its maximum, at the Cd

accumulation level corresponding to CCd-NS equal to

10 lmol l-1, decreases sharply following only moderate

reduction in the Cd accumulation. This effect could be an

evidence of a Cd removal mechanism or a Cd input barrier

which is more effective in the tolerant cultivar than in the

sensitive one. Another evidence for a more effective Cd

removal mechanism in leaves follows from the fact that the

Cd accumulation level is substantially lower in the tolerant

cultivar above CCd-NS of 10 lmol l-1.

The fact that the highest PC concentrations are found at

the same levels of the Cd accumulation in respective tis-

sues corresponding to CCd-NS of 10 lmol l-1 could result

from the activity of a similar Cd-controlled feedback

mechanism of PC production and GSH level regulation in

both the cultivars.

Our measurements have shown that the PC3 concen-

tration was approximately one order of magnitude higher

than that of PC2 for leaves and roots of both the cultivars.

Synthesis of higher concentration of higher molecular

weight PC3 than PC2 was recorded also by Beraud et al.

(2007) as reaction of V. faba plants to the increased Cd

concentration exposure. Oven et al. (2001) has reported

about Cd-induced synthesis of the PCs in Azuki bean

where the PC2 concentration increased 15-fold upon

exposure to the Cd ions. PC(n ? 1)/PC(n) ratio is species

specific and depends on the Cd accumulation as can be

found in the literature (for Phragmites australis see Ederli

et al. 2004, for Salix viminalis Landberg and Greger 2004,

for Cuscuta reflexa Srivastava et al. 2004, for S. vulgaris

Knecht et al. 1994, for barley genotypes with different

tolerance to Cd Persson et al. 2006, for B. juncea Haag-

Kerwer et al. 1999).

In our experiments a comparatively good correlation

was found between PC2 and PC3 concentrations in leaves

of the sensitive cultivar (R2 = 0.9455, compare Fig. 5d

and f) and in roots of the tolerant cultivar (R2 = 0.9082,

compare Fig. 5c and e), whereas correlations were mod-

erate in leaves of the tolerant cultivar (R2 = 0.8907) and

roots of the sensitive cultivar (R2 = 0.7863). It is inter-

esting that the correlation is better in roots of the tolerant

cultivar where the PC concentrations are higher compared

to those in the sensitive cultivar and these relations are

opposite in the leaves. The correlations between GSH and

PC concentrations were only moderate and the corre-

sponding R2 are in the range from 0.4285 (GSH-PC2 in the

roots of the tolerant plants) to 0.8182 (GSH-PC3 in the

leaves of sensitive cultivar). The absence of a straightfor-

ward correlation between the GSH and PC concentrations

is a sign of a more complicated reaction mechanism of the

PC production from the GSH precursor.

As can be concluded from these findings, the tolerant

broad bean cultivar Giza 40 has a better ability to sequester

higher Cd amounts in roots which is confirmed by a higher

production of both PC2 and PC3 compared to the sensitive

cultivar Giza 2. The higher PCs production in leaves of the

Giza 2 cultivar indicate that much more of free Cd can

penetrate into the leaves and can negatively influence the

metabolic processes, e.g., photosynthesis (Gorinova et al.

2007), which consequently affects the whole plant metab-

olism and growth. As the next step of the investigation, it

would be necessary to study Cd accumulation and trans-

location, together with the GSH and PC tissue concentra-

tions, during the whole life cycle of V. faba, from the

germination period until the bean-fruit production.

In conclusion, V. faba accumulates more than 95% of Cd

in the roots and only less than 4% in the leaves. The differ-

ences between the cultivars were statistically apparent only

at higher levels of Cd accumulation. The CCd-leaves was found

to be nonlinearly dependent on CCd-NS, ranging from 0.01 up

to 1.71 and from 0.01 to 3.27 mmol kg-1 DW for tolerant

and sensitive cultivars, respectively. The differences

between the cultivars in Cd accumulation were apparent at

higher CCd-NS. On the contrary, in the roots, the CCd-roots

depended linearly on CCd-NS and was found in the range from

0 to 93.9 and 0 to 108.5 mmol kg-1 DW for tolerant and

sensitive cultivar, respectively. The difference between the

cultivars in the Cd accumulation in roots was only statisti-

cally significant with the highest CCd-NS. The production of

biomass and its water content decreased nonlinearly with

increasing Cd accumulation and this decrease was several

times steeper for the sensitive cultivar. The biomass weight

reached only 12.2 and 46.8% of the control for sensitive

and tolerant cultivar, respectively, at CCd-NS equal to

100 lmol l-1 Cd2?. The GSH concentration did not linearly

correlate with Cd accumulation, except for the roots of the

sensitive cultivar where the GSH concentration was almost

constant and equal to 0.0018 mmol kg-1 FW. The GSH

concentration was higher in the sensitive cultivar than that in

the tolerant one in both the leaves and roots. The GSH con-

centrations were in the range of 0.0002–0.0018 mmol kg-1

FW for roots, of 0.0001–0.005 mmol kg-1 FW and of

0.0001–0.0135 mmol kg-1 FW for leaves of tolerant and

sensitive cultivars, respectively. The relationships between

the PC and Cd concentrations in tissues were found nonlin-

ear. The concentrations of PC2 and PC3 were found in ranges

of 0–0.003 mmol kg-1 FW and 0–0.035 mmol kg-1 FW,

respectively. For roots, the PC concentrations were higher in

tolerant cultivars compared to the sensitive one, whereas the

relation was opposite in leaves (except at the highest Cd

accumulation where the difference in the PC concentrations

between the cultivars was negligible). The highest PC con-

centrations were found at CCd-NS of 10 lmol l-1. The rela-

tions among the Cd accumulation and GSH and PC tissue

1246 Acta Physiol Plant (2011) 33:1239–1248

123

contents were found nonlinear and relatively complex,

indicating the presence of some feedback control system in

the two cultivars.

Acknowledgments R. Cabala would like to acknowledge the

research project VZ MSM 0021620857 from the Ministry of Educa-

tion, Youth and Sports of the Czech Republic. L’. Slovakova grate-

fully acknowledges a partial financial support from the grants of the

Slovak grant agencies VEGA No. 1/4354/07, COST 0004-06 APVV.

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