Compartmentalization and ultrastructural alterationsinduced by chromium in aquatic macrophytes

Pedro A. Mangabeira • Aluane S. Ferreira • Alex-Alan F. de Almeida •

Valeria F. Fernandes • Emerson Lucena • Vania L. Souza •

Alberto J. dos Santos Junior • Arno H. Oliveira • Marie F. Grenier-Loustalot •

Frederique Barbier • Delmira C. Silva

Received: 17 February 2011 / Accepted: 3 May 2011 / Published online: 12 May 2011

� Springer Science+Business Media, LLC. 2011

Abstract The aim of the present study was to identify

the sites of accumulation of Cr in the species of

macrophytes that are abundant in the Cachoeira river,

namely, Alternanthera philoxeroides, Borreria scabi-

osoides, Polygonum ferrugineum and Eichhornia

crassipes. Plants were grown in nutritive solution

supplemented with 0.25 and 50 mg l-1 of CrCl3�6H2O.

Samples of plant tissues were digested with HNO3/HCl

in a closed-vessel microwave system and the concen-

trations of Cr determined using inductively-coupled

plasma mass spectrometry (ICP-MS). The ultrastructure

of root, stem and leaf tissue was examined using

transmission electron microscopy (TEM) and secondary

ion mass spectrometry (SIMS) in order to determine the

sites of accumulation of Cr and to detect possible

alterations in cell organelles induced by the presence of

the metal. Chromium accumulated principally in the

roots of the four macrophytes (8.6–30 mg kg-1 dw),

with much lower concentrations present in the stems and

leaves (3.8–8.6 and 0.01–9.0 mg kg-1 dw, respec-

tively). Within root tissue, Cr was present mainly in

the vacuoles of parenchyma cells and cell walls of xylem

and parenchyma. Alterations in the shape of the

chloroplasts and nuclei were detected in A. philoxero-

ides and B. scabiosoides, suggesting a possible appli-

cation of these aquatic plants as biomarkers from Cr

contamination.

Keywords Aquatic plants � Heavy metals �Chromium � Cell ultrastructure � Transmission

electron microscopy � Secondary ion mass

spectrometry

Introduction

Chromium is not essential for plants and is frequently

toxic, particularly in the Cr?6 form (Shanker et al.

2005). According to Cervantes et al. (2001), plant

roots are able to reduce Cr?6 to Cr?3 via a detoxifi-

cation reaction mediated by chromium reductases, and

this could form the basis of a phytoremediation process

for toxic Cr?6. In this context, phytoremediation

comprises the removal by detoxification or stabiliza-

tion of contaminating metals in soils and water

systems by plants. Thus, high levels of metals that

Electronic supplementary material The online version ofthis article (doi:10.1007/s10534-011-9459-9) containssupplementary material, which is available to authorized users.

P. A. Mangabeira (&) � A. S. Ferreira �A.-A. F. de Almeida � V. F. Fernandes �E. Lucena � V. L. Souza � A. J. dos Santos Junior �A. H. Oliveira � D. C. Silva

Departamento de Ciencias Biologicas,

Centro de Microscopia Eletronica, Universidade Estadual

de Santa Cruz, Rodovia Ilheus-Itabuna km 16,

Ilheus, Bahia 45662-900, Brazil

e-mail: pamangabeira@uesc.br

M. F. Grenier-Loustalot � F. Barbier

Service Central d’Analyse, CNRS, Echangeur de Solaize,

Chemin du Canal, 69360 Solaize, France

123

Biometals (2011) 24:1017–1026

DOI 10.1007/s10534-011-9459-9

are normally toxic to other organisms may be tolerated

by plants that can absorb the pollutants through the

roots and either detoxify them or translocate them

directly to the aerial parts where they are stored at very

high concentrations (so-called hyperaccumulation)

(Mishra and Tripathi 2009). In this manner, phyto-

remediation allows a full or partial decontamination of

soils whilst preserving their biological activities, and

provides an attractive alternative to other remediation

processes involving the immobilisation or extraction

of metal contaminants, which are both expensive to

apply and limited to small areas (Baker et al. 1991).

Furthermore, phytoremediation is an environmentally

friendly technology that offers the possibility of in situ

recovery of metals for further use (Pilon-Smits 2005).

By virtue of its low-cost, phytoremediation has been

targeted by governmental agencies and private enter-

prises with limited budgets.

It has been demonstrated that aquatic macrophytes

have the potential to remove pollutants and can be

used as biomarkers for the occurrence of heavy metals

in aquatic environments (Maine et al. 2001). Metal

accumulation in macrophytes is frequently accompa-

nied by cell modifications that may contribute to their

metal tolerance (Prasad and Freitas 2003). Investiga-

tions involving high resolution ion microscopy are

scarce in biology (Jauneau et al. 1992; Grignon et al.

1996; Mangabeira et al. 2004), although the technique

can be used together with transmission electron

microscopy (TEM) for the precise detection of heavy

metals in plant tissues and cells. Thus, the location of

the metal in the sample can be determined by

comparison of ion secondary image (ISI) generated

by secondary ion mass spectrometry (SIMS) with the

image of the element under investigation (in the

present case Cr).

The aim of the present study was to employ TEM

and SIMS in order to identify the sites of accumu-

lation of Cr in the species of macrophytes that are

abundant in the Cachoeira river, namely, Alternan-

thera philoxeroides (Mart.) Griseb., Borreria scabi-

osoides Cham. and Schltdl., Polygonum ferrugineum

Wedd. and Eichhornia crassipes (Mart.) Solms. The

capacity of each macrophyte for phytoremediation

was assessed on the basis of accumulation of Cr in

tissues and cells, and the ultrastructure of root, stem

and leaf tissue was examined in order to evaluate the

effect of Cr and to detect possible alterations in the

cell organelles.

Materials and methods

Plant material and cultivation conditions

The macrophytes A. philoxeroides, B. scabiosoides,

P. ferrugineum and E. crassipes were collected from

Cachoeira river at locations in uncontaminated area

near to Ilheus (14�470–14�480S; 39�060–39�080W), and

transported in plastic buckets containing water to the

Centro de Pesquisa do Cacau (CEPEC) in Ilheus,

Bahia, Brazil. The experiment was conduced in

greenhouse (14�470S 39�160W 55 m a.s.l.) at the

follow conditions 30% of the total incidence radiation,

temperature of 23 ± 1�C, relative humidity of

84 ± 3%. Plants were then transferred to 30 l trays

containing 1/4 strength Hoagland and Arnon (1950)

nutrient solution and allowed to acclimate for a period

of 30 days. After this time, some plants were trans-

ferred to nutrient solution supplemented with CrCl3�6H2O (25 or 50 mg l-1) and others were maintained in

nutrient solution without Cr. Incubation was carried

out under constant aeration for 90 days, during which

time the level of nutrient solution within each tray was

maintained by adding deionised water, and the pH was

monitored and adjusted to 5.8 with NaOH or HCl on a

daily basis. The nutrient solution was replaced every

15 days.

Chemical analysis

The plants were separated into roots, stems and leaves,

except for E. crassipes which was separated into roots

and shoots. Samples (50 mg dry weight) of plant

material were digested with analytical grade HNO3

(4.0 ml) and HCl (1.0 ml) in closed Teflon digestion

bombs heated to 190�C for 12 min in a 1,000 W Ethos

Plus Microwave Labstation (Milestone Inc., Monroe,

CT, USA). After cooling, the concentrations of Cr in

the samples were determined using an inductively-

coupled plasma mass spectrometer (ICP-MS) model

PQ3 Excel (Thermo Scientific, Waltham, MA, USA).

Signal drift due to matrix effects was monitored by

adding internal standard (10 lg l-1 of Cr) to both

sample and standard solutions according to the estab-

lished protocol of the manufacturer. All estimations

were conduced in triplicate and the concentrations

were expressed in mg per kg dry weight. The overall

recovery associated with the digestion process was

found to be in the range 87–95%.

1018 Biometals (2011) 24:1017–1026

123

TEM analysis

TEM analyses were carried out in the Electron

Microscopy Center of the Universidade Estadual de

Santa Cruz, Ilheus, BA, Brazil. In order to avoid

confusion between stained cell structures and sites of

Cr accumulation, TEM slides were not stained with

uranyl acetate and lead citrate. Root, stem and leaf

tissues were immersed in 3% glutaraldehyde in 0.1 M

sodium cacodylate buffer, pH 6.9, cut into small

fragments (ca. 1 mm3), submitted to a weak vacuum

for 30 min and subsequently maintained under nor-

mal pressure for a further 1 h. Samples were then

submitted to six washes (10 min each) with sodium

cacodylate buffer, fixed with 1% osmium tetroxide in

0.1 M sodium cacodylate buffer for 4 h at 4�C,

washed six times (10 min each) with sodium caco-

dylate buffer, and dehydrated in an ethanol gradient

(30, 50, 75, 85 and 95% ethanol, followed by three

washes in 100% ethanol). Finally, samples were

covered sequentially with solutions of ethanol:epoxy

resin (Spurr 1969) in proportions of 3:1, 1:1 and 1:3,

and then three times with pure Spurr resin, the final

treatment being continued overnight at room temper-

ature. Next day, samples were placed in silicon

moulds, covered with pure Spurr resin and polymer-

ised overnight. The polymerised resin blocks were

trimmed with a razor blade, thinly sectioned (2 lm)

with a glass blade, and ultra-thinly sectioned

(60–70 nm) with a diamond blade using a ultrami-

crotome (model EM FC6 LEICA Microsystems).

Thinly-cut sections were placed between glass slides

and cover slips for structural examination. Ultra-thin

sections were cut and placed onto copper mesh grids

and examined using a MorgagniTM 268D TEM (FEI

Company, Hillsboro, OR, USA) equipped with a

CCD camera and controlled by software running

under Windows OS.

High-resolution SIMS analysis

Samples were rapidly frozen by plunging into liquid

propane cooled to -196�C with liquid nitrogen,

transferred to a cryogenic vessel (model EM-AFS,

Leica Microsystems, Wetzlar, Germany) containing

20 ml of acetone pre-cooled to -92�C, and left for

1 week. Frozen specimens were embedded in Lowc-

ryl K4M resin, polymerised at -20�C for 2 days, cut

into 2 lm sections using a Reichert-Jung Ultracut E

ultramicrotome and placed either on a square gold

plate for analysis by SIMS or between glass slides

and cover slips for correlation with light microscopy.

High resolution SIMS analyses were performed at

the Division of Biological Sciences, Department of

Medicine, Enrico Fermi Institute, University Chi-

cago, IL, USA. A Finnigan MAT 90 magnetic sector

mass spectrometer (Thermo Scientific, Waltham,

MA, USA) was combined with a scanning ion

microprobe (Strick et al. 2001), by which the samples

were illuminated with a gallium focused 40 nm ion

beam in order to obtain high lateral resolution mass-

resolved images of their surfaces. Secondary ions

were detected using an ETP AF820 active film

electron multiplier (Scientific Instruments Services,

Ringoes, NJ, USA) operating in the pulse counting

mode at count rates up to 50 MHz. In order to locate

Cr, samples were scanned in the positive mode with

the 52Cr? SIMS signal displayed on a CRT at fields of

view 80 and 160 lm wide. SIMS images (512 9 512

pixels) formed by single square raster scans were

stored and analysed using Kontron IMCO imaging

system (Kontron, Fremont, CA, USA).

Experimental design and statistical analysis

The experiment followed a randomised design and

involved the treatment of plants with two different

concentrations of Cr (25 and 50 mg l-1) with five

repetitions each. The mean concentrations of Cr

accumulated in root, stem and leaf tissues were

compared by analysis of variance and the Tukey test

(q\ 0.05).

Results

Accumulation of Cr in plant tissues

ICP-MS analyses of plant tissues demonstrated that Cr

accumulated mainly in the roots of the macrophytes,

with significantly (q\ 0.05) smaller levels in the

stems and, with the exception of E. crassipes, almost

negligible amounts in the leaves (Table 1). Moreover,

the levels of accumulation of the metal in the roots of

A. philoxeroides, P. ferrugineum and E. crassipes

differed significantly (q\ 0.05) according to the

treatment applied (i.e. 25 or 50 mg l-1 of Cr). The

largest accumulations of Cr were observed in the roots

Biometals (2011) 24:1017–1026 1019

123

of A. philoxeroides that had been grown in 50 mg l-1

of Cr. A maximum accumulation (29.16 mg Cr kg-1

dw) of Cr was observed in the roots of A. philoxero-

ides that had been grown in the presence of 50 mg l-1

of CrCl3, although high levels (ca. 29.16 mg Cr kg-1

dw) were also found in the roots of P. ferrugineum and

B. scabiosoides grown under similar conditions. The

total uptake of Cr into the roots appeared to reflect the

concentration of metal in which the plant was grown.

However, the total Cr accumulation in leaf tissue,

which was generally insignificant, was not propor-

tional to the treatment concentrations indicating that

translocation of the metal from roots to leaves/shoots

was a rate-limiting step.

Effects of Cr treatment on the ultrastructure

of plant tissues as determined by TEM and SIMS

Alterations in the ultrastructure of leaves, stems and

roots of A. philoxeroides that had been grown in the

presence of Cr could be readily detected by TEM

(Fig. 1) and SIMS (Fig. 2) analysis. The presence of

Cr at 50 mg l-1 induced the most severe modifica-

tions, which included changes in nuclear shape and

envelop integrity together with modifications in the

shape of leaf chloroplasts, resulting in the structural

disarrangement of thylakoids and stroma in compar-

ison with control plants. Modifications in cell ultra-

structure detected by TEM analyses of leaves, stems

and roots of B. scabiosoides, P. ferrugineum and

E. crassipes that had been incubated in 50 mg l-1 of

Cr are shown in Fig. 3 and Supplementary Figs. 5

and 7, respectively, while those revealed by SIMS

analysis are presented in Fig. 4 and Supplementary

Fig. 6, respectively. Of particular note were the

modifications in the mitochondrial cristae induced

by Cr in root cells of B. scabiosoides (Fig. 3i).

Discussion

Of all of the species and plant parts analyzed, the roots

of A. philoxeroides exhibited the highest concentration

of Cr. Naqvi and Rizvi (2000) reported significant

accumulations of Cr in the roots of A. philoxeroides. In

the case of E. crassipes, while the roots were the

preferential site of metal accumulation, with the cell

walls and vacuoles accumulating high levels of Cr,

there was a modest translocation to the aerial partsTa

ble

1C

on

cen

trat

ion

so

fC

rin

tiss

ues

of

mac

rop

hy

tes

cult

ivat

edfo

r9

0d

ays

inn

utr

ien

tso

luti

on

sco

nta

inin

gd

iffe

ren

tam

ou

nts

of

CrC

l 3�6

H2O

CrC

l 3�6

H2O

(mg

l-1)

A.

phil

oxe

roid

es(m

gC

rkg

-1

dw

)

B.

scabio

soid

es(m

gC

rkg

-1

dw

)

P.

ferr

ugin

eum

(mg

Cr

kg

-1

dw

)

E.

crass

ipes

(mg

Cr

kg

-1

dw

)

Roots

Ste

ms

Lea

ves

Roots

Ste

ms

Lea

ves

Roots

Ste

ms

Lea

ves

Roots

Shoots

00.0

2±

0.0

01

Aa

0.0

1±

0.0

01

Aa

0.0

1±

0.0

01

Aa

0.0

2±

0.0

01

Aa

0.0

1±

0.0

02

Aa

0.0

1±

0.0

01

Aa

0.0

2±

0.0

01

Aa

0.0

1±

0.0

01

Aa

0.0

1±

0.0

01

Aa

0.0

1±

0.0

01

Aa

0.0

1±

0.0

01

Aa

25

21.1

8±

0.1

92

Ba

4.5

4±

0.0

39

Bb

0.1

0±

0.0

03

Bc

13.7

0±

0.2

04

Ba

3.7

8±

0.0

33

Bb

0.1

0±

0.0

01

Bc

8.6

1±

0.1

14

Ba

5.2

5±

0.1

66

Bb

0.0

2±

0.0

04

Bc

10.9

1±

0.4

29

Ba

4.6

8±

0.0

53

Bb

50

29.1

6±

0.4

80

Ca

5.7

5±

0.0

81

Bb

0.1

5±

0.0

04

Bc

19.0

7±

0.0

81

Ba

4.9

4±

0.3

34

Bb

0.1

3±

0.0

02

Bc

19.4

8±

0.0

8C

a8.6

6±

0.1

63

Bb

0.0

4±

0.0

01

Bc

17.7

5±

0.6

68

Ca

9.0

2±

0.0

33

Bb

Val

ues

repre

sent

the

mea

nof

thre

ere

pet

itio

ns

±st

andar

der

ror

Val

ues

bea

ring

dis

sim

ilar

upper

case

(colu

mns)

or

low

erca

se(r

ow

s)le

tter

sar

esi

gnifi

cantl

ydif

fere

nt

acco

rdin

gto

Tukey

test

(q\

0.0

5)

1020 Biometals (2011) 24:1017–1026

123

particularly when the treatment involved a high

concentration of Cr (Table 1). Other authors (Maine

et al. 2001; Ingole and Bhole 2003; Paiva et al. 2009)

have also described the greater accumulation of Cr in

roots of E. crassipes in comparison with the aerial

parts, whilst Mishra and Tripathi (2008) demonstrated

that E. crassipes was more efficient in the removal of

heavy metals than Pistia stratiotes or Spirodela

polyrrhiza. On the other hand, Mang et al. (2008)

recently reported that Cr absorption by A. philoxeroides

roots was greater than by roots of E. crassipes, a finding

that is agreement with the results of the present study.

These researchers employed Fourier transform infrared

(FTIR) spectrometry to show that the root cell walls of

Cr-treated plants exhibited a significant shift in –OH

absorption peaks in comparison with control plants,

and suggested that –OH and COO– groups were

associated with Cr binding in aqueous solutions and

that this might constitute a mechanism of Cr

accumulation.

Fig. 1 TEM electron micrographs of transversal sections of

leaf, stem and root tissue of A. philoxeroides following

treatment of plants with 50 mg l-1 Cr: a electron-dense

material (arrowed) in cell wall (cw) of leaf, bar 2 lm;

b deformed chloroplast (cl) and nucleus (n) in leaf cell, bar2 lm; c Cr deposits (arrowed) in the cell wall (cw) of stem

xylem, bar 5 lm; d Cr deposits in the cell wall (cw) of stem

parenchyma, bar 5 lm; e deformed chloroplast (cl) and

nucleus (n) in stem cells, chromium deposits (arrows), bars2 lm; f disintegration of nucleus (n) in root cell (arrow), bars1 lm

Biometals (2011) 24:1017–1026 1021

123

Fig. 2 SIMS images of

stem and root tissue of

A. philoxeroides following

treatment of plants with

50 mg l-1 Cr: a ISI of stem

parenchyma (p); b Cr

deposits in cell wall and

vacuole of stem

parenchyma (p) at a depth

of 20 nm; c ISI of the vessel

element (ve) and

parenchyma (p) of stem

xylem; d Cr deposits in cell

wall of the vessel element

of stem at a depth of 20 nm;

e ISI of vessel element (ve)

of root xylem; f Cr deposits

(arrowed) in the vessel

element (ve) of root xylem

at a depth of 20 nm

(arrow); g ISI of root

parenchyma; h Cr deposits

in the cell wall (cw) and

vacuole (v) of root

parenchyma at a depth of

20 nm

1022 Biometals (2011) 24:1017–1026

123

The results of the present study clearly indicate that

Cr is strongly adsorbed to the cell walls of the roots

and that translocation to the aerial parts is negligible,

as described in an earlier report (Pulford and Watson

2003). It is possible, however, that part of the metal

taken up by the roots may cross the plasma membrane

and become bound to macromolecules, organic acids,

or sulphur-rich polypeptides, such as phytochelatins,

thereby accumulating in the cytoplasm or the vacuole

and becoming detoxified (Harmens et al. 1994).

Indeed, it has been suggested that the formation of

complexes between Cr and organic acids may play an

important role in the inhibitor/stimulator effect of Cr

on the translocation of different minerals (Panda and

Choudhury 2005).

Mangabeira et al. (2004) employed ion micros-

copy to detect large amounts of Cr in the vascular

cylinder of E. crassipes roots and stems, particularly

around the secondary xylem. In addition to the

presence of Cr in the root parenchyma, these authors

observed Cr in the transport parenchyma, indicating

that such cells were responsible for conveying the

metal to the leaves. It was concluded that compart-

mentalisation of Cr in the vacuoles was important for

the detoxification and tolerance of macrophytes

towards the metal. Srivastava et al. (1999) proposed

Fig. 3 TEM electron micrographs of transversal sections of

leaf, stem and root tissue of B. scabiosoides following treatment

of plants with 50 mg l-1 Cr: a Deformed chloroplast (arrows)

in leaf cell, bar 7 lm; b normal chloroplast with thylakoid and

starch granule (sg) in control leaf, bar 1 lm; c deformed

chloroplast (cl; arrowed) in leaf cell, bar 5 lm; d normal

nucleus (n) in control leaf, cl (chloroplast), bar 5 lm;

e deformed nucleus (n) in leaf cell, bar 5 lm; f Cr deposits

(arrowed) in stem cell wall (cw), bar 2 lm; g Cr deposits

(arrowed) in the cell wall and cell wall of stem xylem, bar5 lm; h Cr deposit (arrowed) in cell wall (cw) and vacuole of

root cells, bar 1 lm; i electron dense material (arrowed) in

mitochondria (m), alterations in mitochondrial cristae (asterisk)

and Cr deposit in vacuole (v) in root cells, bar 1 lm

Biometals (2011) 24:1017–1026 1023

123

that Cr forms metal chelates with organic acids

within the vacuoles or small cell vesicles, which are

responsible for the detoxification and tolerance to

metal stress. On the basis of such evidence, it is

possible to state that the macrophyte species studied

are, to some extent, tolerant to Cr, since they are

capable of accumulating the metal in vacuoles.

Other floating macrophytes including A. sessilis,

Salvinia herzogii and P. stratiotes also accumulate

large amounts of Cr in the roots, and it has been shown

that in such plants the metal tends to be immobilised

in the radicular system in order to limit aerial toxicity

(Sinha et al. 2002; Maine et al. 2004). In this context,

Mishra and Tripathi (2009) explained the accumula-

tion of heavy metals in the roots of macrophytes in

terms of an active uptake of heavy metals by

plasmolysed cells and the impregnation of cell walls

via passive diffusion. However, a different explana-

tion has been given by Barbosa et al. (2007) who

stressed the tendency of metal ions to either bind to

other ions or precipitate within the cell walls, thus

preventing the translocation of the metal to the aerial

parts. These authors have emphasised that Cr is not

only toxic but is also unnecessary for the development

of plants, thus accounting for the absence of specific

mechanisms for Cr transport from roots to leaves.

Panda and Choudhury (2005) suggested that

Cr-induced oxidative stress results in the peroxidation

of membrane lipids, causing severe damage to the

cell membranes and degradation of photosynthetic

pigments. These authors also claimed that high

concentrations of Cr may cause damage to chloro-

plast ultrastructure and affect photosynthesis. Since

Cu, Fe and Cr present different redox potentials, their

Fig. 4 SIMS images of stem and root tissue of B. scabiosoidesfollowing treatment of plants with 50 mg l-1 Cr: a ISI of stem

parenchyma (p); b Cr deposits in stem parenchyma (p) at a

depth of 20 nm; c ISI of the root xylem (x); vessel element

(ve); d Cr deposits in vessel element (ve) of root xylem (x) and

parenchyma cell wall (p) at a depth of 20 nm

1024 Biometals (2011) 24:1017–1026

123

influence regarding induction of oxidative stress

exceeds that of other metals such as Co, Zn and Ni.

Moreover, Cr toxicity has been attributed to the

interference by the metal in photosynthetic electron

transport (Larcher 1995). On this basis, it is possible

that Cr may have exerted an effect on the photosyn-

thetic processes in the aquatic macrophytes studied

herein, possibly negatively influencing the growth

and development of these species. However, in the

present study no alterations were observed in the

chloroplasts of P. ferrugineum or E. crassipes

following treatment of the plants with 50 mg l-1

Cr. In contrast, Lage-Pinto et al. (2008) reported

structural changes, including thylakoid disorganisa-

tion, in the chloroplasts of samples of E. crassipes

originating from industrial areas. Indeed, according to

Panda and Choudhury (2005), alterations in chloro-

plast shape induced by biotic and abiotic stress result

from an increase in stroma volume and disorganisa-

tion of thylakoids.

Alternanthera philoxeroides and B. scabiosoides

that had been treated with 50 mg l-1 Cr presented

alterations in cell nuclei, including disintegration of

the nucleus, suggesting that high concentrations of Cr

may lead to cell death. Similar findings have been

reported by Rocchetta et al. (2007) following the

analysis of Cr-treated Euglena gracilis. Additionally,

roots of B. scabiosoides exhibited electron dense

deposits in the cell walls together with alterations in

mitochondrial cristae, in agreement with results

reported for Cr- and Ni-treated Allium cepa (Liu

and Kottke 2003).

The occurrence of Cr deposits in the cell walls of

root parenchyma and in the xylem vessel elements of

the macrophytes studied may be explained by the

slow diffusion of the metal together with cation

exchange at specific sites in the roots cells (Shanker

et al. 2005). In addition, the occurrence of Cr deposits

in the stem xylem of A. philoxeroides, B. scabioso-

ides and E. crassipes could be due to active uptake of

Cr by plasmolysed cells and impregnation of cell

walls via passive diffusion (Zayed and Terry 2002).

In this context, metallothioneins and organic acids are

important components of the cells walls of the

secondary xylem that contribute to the tolerance

and detoxification of Cr. For example, cell walls

contain large amounts of polygalacturonic acids, the

ionised groups of which are located on the external

surface of the cells and can bind to cations

(Grignon et al. 1996). According to the evidence

revealed by the profiles of cationic abundance in

plant tissues, such cations are linked to structural

polymers or may be precipitated with pyroantimoni-

ate (Jauneau et al. 1992; Ripoll et al. 1993).

Acknowledgments The authors wish to thanks Dr. Lionel

Dutruch (Service Central d’Analyse, CNRS), and Drs. Ricardo

Levi-Setti and Konstantin Gavrilov (Enrico Fermi Institute,

University of Chicago) for their kind assistance with ICP-MS

and ion microscopy imaging. This research was supported by

CNPq (Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico, Brazil).

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