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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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