Environmental and Experimental Botany 54 (2005) 131–141

Anatomical changes due to uptake and accumulation of

Zn and Cd in Indian mustard (Brassica juncea)
B.B. Maruthi Sridhar a,b , S.V. Diehl a , F.X. Han c , D.L. Monts b,c , Y. Su b,∗
a Department of Forest Products, Mississippi State University, Mississippi State, MS 39762, USA
b Diagnostic Instrumentation and Analysis Laboratory (DIAL), Mississippi State University, Mississippi State, MS 39762, USA
c Department of Physics and Astronomy, Mississippi State University, Mississippi State, MS 39762, USA

Accepted 21 June 2004

Abstract

Anatomical and physiological changes in Indian mustard (Brassica juncea) plants due to uptake and accumulation of Zn and
Cd were investigated. Potted plants were exposed to metal treatments of Zn and Cd for 15 and 16 days, respectively. Leaves,
stems and roots were harvested for studying anatomy and analyzing metal accumulation. Anatomical changes were documented
using light microscopy, scanning, and transmission electron microscopy. Accumulation of Zn and Cd in all parts of the plant
increased significantly with an increase in applied metal concentration. Microscopic studies revealed clotted depositions in roots
and stems, break down of parenchyma cells, and a decrease in starch content in leaves of plants treated with high concentrations
of Zn. Physiological and morphological changes of Zn-treated plants included a significant decrease in relative water content,
dry weight and plant height. Cd at higher concentrations resulted in structural changes only in stems and roots. Mustard plants
accumulated significant amounts of Zn and Cd without exhibiting symptoms of phytotoxicity. However, higher Zn (ZnT3 and
ZnT4) and Cd (CdT4) concentrations resulted in structural changes in roots, stems and leaves and altered physiological and
morphological characteristics. Our results systematically illustrate the physiological implications of structural alterations caused
by Zn and Cd at higher concentrations.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Anatomy; Brassica juncea; Cadmium; Microscopy; Phytoremediation; Zinc

1. Introduction as mining and smelting of metalliferous ores, electro-

Over many years, Zn and Cd contamination has built
up in the environment from industrial activities, such
∗ Corresponding author. Tel.: +1 662 325 3286;
fax: +1 662 325 8465.
E-mail address: su@dial.msstate.edu (Y. Su).
plating, fertilizers and pesticides (Raskin and Ensley,
2000). Zn is widely used in the paint, rubber, dye and
wood preservative industries (ATSDR, 1994) while Cd
is mainly used in batteries, metal coatings and plastic
industries (Raskin and Ensley, 2000; ATSDR, 1999).
Both Zn and Cd often combine with other elements at
hazardous waste sites to form compounds of chlorides,

0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2004.06.011
132 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141
oxides and sulfides (ATSDR, 1994, 1999). These con-
taminants move into the soil and remain persistent for
long periods of time by binding strongly to soil parti-
cles. Excess Zn and Cd in soil cause adverse human
health effects, animal fatalities, and the disruption of
the natural ecosystems (Brown et al., 1994).
Phytoremediation is a cleanup alternative where
plants are used to degrade, extract, contain or immobi-
lize contaminants from soils and water (Raskin and En-
sley, 2000). For effective phytoremediation of metals
they must be translocated and accumulated in the aerial
parts of the plant. Progress of metal phytoextraction is
limited by metal uptake and translocation efficiency
within different plant parts. In non-hyperaccumulator
plants, Zn accumulation is localized as globules in
roots of Deschampsia caespitosa (Van Steveninck et
al., 1987), electron dense granules in roots of Betula
pendula (Denny and Wilkins, 1987) and as globules in
leaves of Lemna minor (Van Steveninck et al., 1990).
Among hyperaccumulators Zn is localized as globular
crystals in the roots and leaves of Thlaspi caerulescens
(Vazquez et al., 1994, 1992a; Kupper et al., 1999; Keller
et al., 2000). Accumulation and localization of Cd oc-
curred as electron dense granules in roots of Agrostis
gigantean and Zea mays (Rauser and Ackerley, 1987),
as cell wall deposition in roots of Z. mays (Khan et al.,
1984) and in roots of bush bean plants (Vazquez et al.,
1992b).
Uptake and accumulation of metals at higher con-
centrations can be cytotoxic in some plant species,
causing structural and ultrastructural changes affecting
the growth and physiological well being of the plants
(Barcelo et al., 1988; Zhao et al., 2000; Vazquez et al.,
1992a; Han et al., 2004). Zn hyperaccumulation results
in a decrease in mesophyll cell size in Arabidopsis hal-
leri (Zhao et al., 2000) while Cd accumulation causes a
break down of chloroplasts in bush bean plants (Barcelo
et al., 1988) and decreases plant growth in Brassica
juncea (Haag-Kerwer et al., 1999).
Indian mustard has been identified as a high biomass
producing plant with the capacity to accumulate Zn and
Cd at higher concentrations in plant cells (Kumar et
al., 1995; Salt et al., 1995). With in this context, the
physiology, morphology and anatomical characteris-
tics of mustard plants are important. It is also important
to study the structural changes in different plant parts
to understand the overall process of phytoremediation.
Hence in this study the physiological, morphological
and anatomical characters of mustard plants were eval-
uated with reference to their Zn and Cd phytoextraction
potential. The main objective of our study was to iden-
tify the structural and ultrastructural changes caused
by Zn and Cd accumulation in leaves, stems and roots
of mustard plants; and to correlate the metal accumula-
tion with anatomical, physiological and morphological
changes.
2. Materials and methods
2.1. Plant culture and phytoremediation
experimental design
The mustard seeds were a commercial variety ob-
tained locally. The soil used for the pot study was
Miracle-Gro Potting Mix from Miracle-Gro Lawn
Products Inc. (Marysville, OH). The seeds were sown
in plastic pots, each containing approximately 2.0 kg
of potting mix. The plants were kept outdoors in an
enclosed area except during extreme weather condi-
tions. The seedlings were thinned to two plants per
pot at the 2–3-leaf stage. Modified Hoaglands Nutri-
ent solution (Hoagland and Arnon, 1950) was sup-
plied to the plants daily, after the plants attained
3–4 leaves. The composition of the nutrient solution
was 0.5 mM Ca(NO3 )2 , 3.1 mM NH4 NO3 , 0.01 mM
KH2 PO4 , 50.0 ␮M KCl, 0.2 ␮M CuSO4 , 12.0 ␮M
H3 BO4 , 0.1 ␮M NiSO4 ·6H2 O, 2.0 ␮M MnSO4 ·H2 O,
0.5 ␮M ZnSO4 ·7H2 O, and 0.2 mM MgSO4 . Metal
treatments with six replicates in each group were
supplied with 2 mM (ZnT1), 10 mM (ZnT2), 50 mM
(ZnT3), and 100 mM (ZnT4) of Zn solution, supplied
as ZnSO4 ·7H2 O; 50 ␮M (CdT1), 500 ␮M (CdT2),
2.5 mM (CdT3), and 10 mM (CdT4) of Cd solution,
supplied as CdCl2 .
All the treatment groups along with control (T0)
were arranged in a completely randomized design. The
metal treatment at the rate of 100 ml pot−1 day−1 was
started at 5 weeks after plants emerged. The metal
treatments were supplemented with nutrient solution
to avoid any water and nutrient deficiencies. Fifteen
days after the start of metal treatment, all the Zn treated
groups were harvested, except the ZnT4 group is har-
vested 4 days earlier because of severe phytotoxicity
symptoms. Since large number of samples were col-
lected for microscopic study, the Cd treated groups
 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141
were harvested 16 days after the start of metal treat-
ment (1 day later than most of the Zn treated groups).
2.2. Procedure for microscopic study
Stem and root samples of 5 mm length were ex-
cised from 2 cm above and 2 cm below the stem–root
intersection, respectively. Leaf samples of 5 mm length
were excised from the middle portion of the third leaf
(lower leaf) from the base of the plant. The leaf, stem
and root samples were prepared for light microscopy
(LM), scanning electron microscopy (SEM), and trans-
mission electron microscopy (TEM). All the samples
were excised and quickly immersed in H2 S saturated
water as pretreatment for 30 min at room temperature
to precipitate Cd and Zn (Khan et al., 1984). The sam-
ples were immediately fixed in formaldehyde acetic
acid (FAA) saturated with H2 S water for LM and in
glutaraldehyde for SEM and TEM studies.
2.2.1. Light microscopy (LM)
The plant samples were alcohol dehydrated, paraffin
embedded, ultramicrotomed and subjected to different
stains including sulfide-silver to localize the Cd and Zn
in stem samples and with potassium iodide (KI) to vi-
sualize the starch content (Sass, 1958) in leaf samples.
For the sulfide–silver method, the LM stem sections
embedded in paraffin were microtomed to obtain four
microsections, placed on glass slides, cleaned in citri-
solve, and dehydrated in an ethanol series. The sections
were then treated in developer solution in the dark at
22 ◦ C for 1 h. Finally they were washed in an ethanol
series, counter stained with 2% safranin and then de-
hydrated and mounted (Pearse, 1972).
2.2.2. Scanning electron microscopy (SEM)
Leaf, stem and root samples were also prepared
for scanning electron microscopy (SEM). Stem sam-
ples of approximately 5 mm length were collected from
2 cm above the stem–root intersection. The procedures
for collection of leaf and root samples remain the
same as described for light microscopy. All the sam-
ples were immediately fixed in 2.5% glutaraldehyde
in 0.05 M potassium phosphate buffer (pH 7.1) for 8 h
(Sass, 1958; Johansen, 1940), and then dehydrated in
an ethanol series. The samples were sealed in parafilm,
frozen in liquid nitrogen and fractured transversely
using a pre-cooled knife. The cryofractured speci-
133
mens were critical point dried through carbon dioxide,
mounted on stubs, and coated with gold–palladium. All
materials were observed with a LEO SEM.
2.2.3. Transmission electron microscopy (TEM)
Stem and root segments of approximately 3 mm
length were collected for transmission electron mi-
croscopy (TEM). The samples were fixed in 2.5% glu-
taraldehyde in 0.05 M potassium phosphate buffer (pH
7.1) for 8 h, and post fixed with OsO4 . The samples
were dehydrated in an ethanol series and embedded in
Spurrs epoxy resin. Ultrathin sections were obtained
using an ultramicrotome and stained with uranyl ac-
etate and basic lead citrate for observation under JEOL
TEM (Johansen, 1940).
2.3. Chemical analysis
The plants were cut about 2 cm above the soil level,
upper leaves, lower leaves, stems and roots were har-
vested, and the roots were washed with de-ionized wa-
ter and the samples were dried at 80 ◦ C in an oven for
48 h for chemical analysis. Among the total 6–7 leaves
of the plant, the first three leaves counting from the base
of the plant were considered as lower leaves and the rest
as upper leaves when collecting leaves for chemical
analysis. Dry leaves, stems and roots were then ground
and weighed. Plant samples (approximately 0.5 g) were
digested with concentrated HNO3 and H2 O2 (Jackson,
1958). The digested solution was filtered and then an-
alyzed for Zn and Cd concentration using inductively
coupled plasma-atomic emission spectrometry (ICP-
AES) (Han and Banin, 1997).
2.4. Measurements and statistical analysis
The plant heights from the root–stem intersection to
the growing tip of the stem were measured at the end
of the experiment. The fresh weights and dry weights
of the upper leaves, lower leaves and stems were ob-
tained using an electronic balance. The relative wa-
ter content (RWC) of the plants were obtained using
the formula (Fresh weight–Dry weight)/Fresh weight.
Statistical analysis was conducted with SAS statistical
software (SAS Institute Inc. NC). The GLM procedure
was used for the analysis of different metal treatments,
with means separated by Duncans multiple range test at
p < 0.05. The CORR procedure was used for correlation
134 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141

analysis with means separated at p < 0.05. Least signif- (Table 1). Cd also accumulated significantly in leaves,
icant difference (LSD) was used for comparisons be- stems and roots with higher applied metal concentra-
tween the treatment means. tions (CdT3 and CdT4) (Table 2). The Cd accumulation
in leaves and stems are close to the accumulation in
roots in the CdT1- and CdT2-treated groups (Table 2)
3. Results whereas the CdT3- and CdT4-treated groups showed a
higher concentration in roots followed by lower leaves,
3.1. Effect of Zn and Cd accumulation on plant upper leaves and stems. The dry weight and plant height
growth decreased for CdT4-treated plants compared to control
plants – T0 (Table 2). The metal accumulations listed
Mustard plants grew steadily in all of the Zn- and in Tables 1 and 2 was based on the dry weight basis of
Cd-treated groups and chlorosis was not visually ob- harvested plant material.
served during the treatment process except in higher Pearson’s correlation coefficients were calculated to
Zn-treatment groups (ZnT3 and ZnT4) towards the end identify the effect of metal accumulation on the phys-
of the experiment. General side effects of metal treat- iological and morphological characters of the plant.
ment included stunted growth with an increase in metal Zn accumulation showed significant (p < 0.01) nega-
concentration. The Zn accumulation in leaves, stems tive correlation with plant dry weight and highly sig-
and roots increased significantly with an increase in nificant (p < 0.001) negative correlation with RWC and
applied metal solution concentration (Table 1). Zn ac- plant height (Table 3). Among the different plant parts
cumulation increased almost linearly in all plant parts, analyzed, Zn accumulation in stem was significantly
resulting in high Zn concentration in roots followed by negatively correlated to the RWC (r = −0.94*** ) and
stems, lower leaves and upper leaves (Table 1). The plant height (r = −0.74*** ). Cd-treated plants showed
dry weight, relative water content (RWC), and plant (p < 0.01) negative correlations with only plant dry
height significantly decreased for ZnT4-treated plants weight (Table 4).
Table 1
Averaged Zn accumulation in upper leaves, lower leaves, stems and roots (in mg kg−1 dry weight), shoot dry weight, relative water content
(RWC), and height of plants treated with Zn (n = 6) at the end of the experiment
Treatment Upper leaf Lower leaf Stem con- Root con- Dry RWC (%) Height (cm)
concentration concentration centration centration weight (g)
T0 82 c 76 c 91 c 72 c 11.7 a 91 a 12.8 cb
ZnT1 326 c 348 c 437 c 318 c 12.8 a 89.1 a 18.2 a
ZnT2 1073 cb 1311 c 1790 c 1541 c 11.9 a 90.2 a 15 b
ZnT3 3022 b 4388 b 10523 b 10953 b 12.1 a 83.6 b 11 c
ZnT4a 7280 a 8190 a 15807 a 16482 a 5.9 b 81.3 b 6.7 d
Means followed by different letters are significantly different at the 0.05 probability level, grouped into classes a, b, c and d.
a ZnT4 group was harvested 4 days earlier than other Zn-treated groups.

Table 2
Averaged Cd accumulation in upper leaves, lower leaves, stems and roots (in mg kg−1 dry weight), shoot dry weight, relative water content
(RWC), and height of plants treated with Cd (n = 6) at the end of the experiment
Treatment Upper leaf Lower leaf Stem con- Root con- Dry weighta RWCa (%) Heighta (cm)
concentration concentration centration centration (g)
T0 3c 7b 4.8 c 1.3 b 11.7 91.0 12.8
CdT1 24 c 55 b 32 c 32 b 12.3 90.0 13.2
CdT2 90 bc 163 b 145 cb 134 b 13.4 91.1 14.2
CdT3 271 b 512 b 303 b 520 b 10.7 91.5 13.3
CdT4 1037 a 1850 a 1125 a 4530 a 8.1 90.6 10.8
Means followed by different letters are significantly different at the 0.05 probability level, grouped into classes a, b and c.
a Not significantly different at the 0.05 probability level.
 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141 135

Table 3
Correlation coefficients between Zn concentrations in root, stem, lower leaf and upper leaf (in mg kg−1 dry weight) and plant characters: dry
weight, relative water content (RWC) and plant height (n = 6)
Plant characters Root concentration Stem concentration Lower leaf concentration Upper leaf concentration
RWC −0.91*** −0.94*** −0.87*** −0.85***
Dry weight −0.42** −0.50** −0.45** −0.39**
Plant height −0.64*** −0.74*** −0.66*** −0.64***
** Significant at <0.01 probability level.
*** Significant at <0.001 probability level.

Table 4
Correlation coefficients between Cd concentrations in root, stem, lower leaf and upper leaf (in mg kg−1 dry weight) and plant characters: dry
weight, relative water content (RWC) and plant height (n = 6)
Plant characters Root concentration Stem concentration Lower leaf concentration Upper leaf concentration
RWC −0.06 0.04 0.11 0.07
Dry weight −0.45** −0.46** −0.52*** −0.41
Plant height −0.44 −0.38 −0.54*** −0.28
** Significant at <0.01 probability level.
*** Significant at <0.001 probability level.

Fig. 1. SEM micrographs showing the transverse section of lower leaves of control (A and C), ZnT4-(B) and CdT4-(D) treated plants. The
leaves of ZnT4-treated plants show the breakdown of epidermal, palisade and spongy parenchyma cells resulting in a decrease in intercellular
spaces compared to the control (T0) plants (A) by 11th day of metal treatment. The changes in CdT4-treated (D) leaves were less significant
compared to control (T0) leaves by 16th day of metal treatment. Note: ZnT4-treated plants were harvested 5 days earlier than Cd-treated plants,
hence different controls (T0) were used here.
136 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141

Fig. 2. Light micrographs showing the transverse section of control (A and C) and ZnT4-treated (B and D) leaves stained with potassium iodide.
The leaves were sampled on the 7th (A and B) and 12th day (C and D) after starting the ZnT4-treatment. The starch appears as black to dark
brown spots in the leaf. Note: the decrease in starch content in the ZnT4-treated leaves from 7th day (B) to 12th day (D) after starting the metal
treatment compared to control (T0) leaves.

3.2. Effect of Zn and Cd accumulation on plant
structural changes
The Zn-treated plants showed gradual changes in
leaf structure with an increase in metal concentration.
The ZnT3 and ZnT4 groups showed significant fo-
liar structural changes compared to the control group
(T0). The SEM micrographs of leaf samples showed
a reduction in the palisade and epidermal cells of the
ZnT4 group (Fig. 1B) compared to the control group
(Fig. 1A) by 11th day of metal treatment. The leaves of
ZnT4-treated plants also show a breakdown of spongy
and palisade parenchyma cells (Fig. 1B) followed by
further loss of cell shape and decrease in intercellu-
lar spaces compared to the control group (Fig. 1A).
For leaves of Cd-treated plants (Fig. 1D), the changes
in epidermal and mesophyll cells were not significant
compared to the control (Fig. 3C) by 16th day of metal
treatment. The ZnT4-treated plants also show a de-
crease in starch content by seventh day (Fig. 2B) of
metal treatment compare to the control plants (Fig. 2A).
The starch content appears as black to dark brown spots
in the transverse section of leaves stained with potas-
sium iodide. The starch content was significantly re-
duced by the 12th day of the treatment (Fig. 2D) com-
pared to the control group (Fig. 2C), indicating an inhi-
bition in the formation and accumulation of starch with
uptake of Zn at higher concentrations. The starch con-
tent was compared qualitatively based on microscopic
observations only.
 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141 137

Fig. 3. Light micrographs showing the transverse section of stems of control (A and C), ZnT4 (B) and CdT4 (D) treated plants sampled on the
last day of metal treatment and stained with sulfide–silver for localization of Zn and Cd. The Zn (B) and Cd (D) precipitates were seen as black
deposits along the walls of xylem and phloem vessels compared to clean control-T0 (A and B) stems. Also note the increased black depositions
in ZnT4 (B) treated stems compared to CdT4 (D) treated stems. Note: ZnT4-treated plants were harvested 5 days earlier than Cd-treated plants,
hence different controls (T0) were used here.

Light micrographs of ZnT4 – (Fig. 3B) and CdT4
– (Fig. 3D) treated stems showed Zn and Cd precip-
itates as black deposits along the walls of xylem and
phloem vessels compared to their respective controls
(Fig. 3A,C) by 11th and 16th day, respectively. The
transverse section of the stems were pretreated with
H2 S to precipitate metal contents and stained with
sulfide–silver for localization of Zn and Cd. The metal
precipitates were seen as black deposits on the safranin
background. The ZnT4-treated stems showed thick
black depositions along the walls of xylem and phloem
vessels (Fig. 3B). In CdT4-treated stems (Fig. 3D),
the black depositions were not uniform, but were seen
along the walls of vascular bundles.
The SEM micrographs of sulfide fixed root samples
from ZnT3 – (Fig. 4B), ZnT4 – (Fig. 4C) and CdT4 –
(Fig. 4D) treated groups show precipitates binding all
along the cell walls of vascular bundles compared to the
control (T0) group (Fig. 4A) by the end of metal treat-
ment period. These clotted depositions increase from
ZnT3 – (Fig. 4B) to ZnT4 – (Fig. 4C) treated plants.
The precipitates were more intense in Zn-treated plants
(ZnT3 and ZnT4) compared to CdT4-treated plants
(Fig. 4D). In control group (Fig. 4A), the clotted de-
positions were not observed along the cell walls of the
vascular bundles. TEM observations of the sulfide fixed
root sections showed black electron dense depositions
along the cell walls of ZnT4-treated plants (Fig. 5B).
The black depositions were not observed in the cell
walls (Fig. 5A) of the roots of control group (T0). The
roots of CdT4-treated plants show an increase in the
number of vacuoles (Fig. 5C) and precipitation along
the cell walls, when compared to the cross sections
of control roots (Fig. 5A). The increase in number of
138 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141

Fig. 4. SEM micrographs of transverse sections of roots of control-T0 (A), ZnT3 (B), ZnT4 (C) and CdT4 (D) treated plants sampled on the
last day of metal treatment. The SEM micrographs showed clotted depositions along the cell walls of vascular bundles in ZnT3 (B), ZnT4 (C)
and CdT4 (D) treated plants compared to control (A).

vacuoles is seen in both epidermal and cortex cells of
CdT4-treated plants.
The stems of ZnT4-treated plants (Fig. 6B) show
thickened cell walls in both xylem and phloem vessels,
which were not seen in control group (Fig. 6A) by the
end of metal treatment period. The TEM micrographs
of the sulfide fixed stems of ZnT4-treated plants show
electron dense depositions along the cell walls of xylem
and phloem vessels (Fig. 6C). These depositions, inten-
sively stained with uranyl acetate and lead citrate, were
seen deposited along the phloem vessels (Fig. 6D). All
the plant sections were treated with H2 S to precipitate
their respective metal contents.
4. Discussion
Our study shows that Zn-treatments at higher con-
centrations results in a linear increase in metal accu-
mulation in roots followed by stems, lower leaves and
upper leaves. Since Zn acts as a growth promoting
micronutrient at lower concentrations, the ZnT1- and
ZnT2-treated groups show no significant changes in dry
weight, RWC, and increase in plant height compared
to the control (T0) group, suggesting that mustard has
a high tolerance and uptake ability for Zn. Kumar et al.
(1995) suggested that 100 mg l−1 of Zn concentra-
tion was not phytotoxic to B. juncea when added
to a soil mixture. However, at higher concentrations
(ZnT3 and ZnT4), the plants show significant changes
in physiological and morphological characters, such
as reduction in RWC, dry weight and plant height
(Table 1).
Apart from these physiological and morphologi-
cal changes, Zn accumulation also results in structural
changes in leaves, stems and roots. Structural changes
in leaves include shrinkage of epidermal, palisade and
spongy parenchyma cells and a decrease in starch con-
tent and RWC. The Zn accumulation in stems of ZnT4-
treated plants were seen as depositions in light mi-
crographs and as electron dense depositions along the
walls of vascular bundles in TEM micrographs. Sim-
ilar electron dense depositions were also observed all
along the cell walls in TEM micrographs of their roots.
 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141
Fig. 5. TEM micrographs of transverse section of roots of ZnT4
(B), CdT4 (C) treated plants along with control-T0 (A) sampled
on the last day of metal treatment. Electron dense depositions
are seen along the walls of root cells in ZnT4 (B) treated plants
compared to control (A). The roots of CdT4 (C) treated plants
and depositions showed vacuoles and depositions along the cell
walls.
Several studies verified these precipitations and depo-
sitions as compounds of Zn through X-ray probe mi-
croanalysis (Van Steveninck et al., 1987, 1990; Denny
and Wilkins, 1987; Vazquez et al., 1994, 1992a; Kup-
per et al., 1999). These depositions were found to be
139
either simple salts of Zn or large organic molecules
such as proteins and carbohydrates complexes
with Zn.
Most of these studies did not correlate the metal ac-
cumulation or structural changes to physiological and
morphological changes. In our study, we found that
at higher Zn concentrations (ZnT3 and ZnT4) an ex-
cess deposition of Zn resulted in decreased translo-
cation or uptake of metals and water from roots and
stems to leaves. This is confirmed by the significant
decrease in RWC (Table 1) and by the high nega-
tive correlation of RWC with Zn accumulation in the
stems (r = −0.94*** ) and roots of Zn-treated plants
(r = −0.91*** ).
In the case of Cd-treated plants at higher concen-
trations, metal accumulation was higher in roots fol-
lowed by lower leaves, stems and upper leaves. No
significant changes in RWC, dry weight, and plant
height at lower concentrations for Cd-treatment groups
(CdT1, CdT2 and CdT3) suggest the applied levels
of Cd were within the tolerance limit. This is consis-
tent with mustard being a Cd accumulator (Kumar et
al., 1995; Salt et al., 1995). However, due to signifi-
cant metal accumulation in the CdT4-treated plants, the
cross sections of roots showed precipitation and an in-
crease in the number of vacuoles in TEM micrographs
of roots (Fig. 5) and as black depositions in light mi-
crographs of stems (Fig. 3). The Cd accumulation also
results in a decrease in dry weight and plant height in
CdT4-treated plants, confirming again that Cd affects
the growth of plants at higher concentrations. This is
in accordance with the studies of Haag-Kerwer et al.
(1999) that Cd accumulation resulted in a decrease in
growth rate and reduction in transpiration of mustard
plants.
Mustard plants can accumulate significant amounts
of Zn and Cd without showing phytotoxicity or reduc-
tion in plant growth when the environmental metal
concentrations are relatively low. However, higher
Zn and Cd concentrations will result in structural
changes in roots, stems and leaves and altered phys-
iological and morphological characters. Our study
presented here documented some of the physiolog-
ical implications of structural alterations caused by
Zn and Cd due to metal uptake, translocation and
accumulation. We believe that our results on the
localization patterns and their effect on plant struc-
tural, morphological and physiological characteris-
140 B.B.M. Sridhar et al. / Environmental and Experimental Botany 54 (2005) 131–141

Fig. 6. SEM micrographs of transverse section of stems of ZnT4-treated plants (B) showed thickened cell walls compared to control-T0 (A).
TEM micrographs of ZnT4-treated plants showed electron dense depositions along the walls of vascular bundles (C) in transverse section of
stem and along phloem vessels (D) in longitudinal section of the stem. The plants were sampled on the last day of metal treatment.

tics will contribute to understand and optimize the References

processes of phytoremediation of Zn and Cd by mus-
tard plants.
Acknowledgements
We acknowledge Mr. Dharmendra K. Singh, Mr.
Cheng Wang, Mr. Shyam S. Balasubramaniam, and
Mr. Thomas W. Hallmark, for their contributions to
data collection and plant culture activities. The au-
thors thank Ms. Yunju Xia, Mr. Dean Patterson, and
Dr. Thomas Meaker of DIAL for their help in chem-
ical analysis. We also thank Mr. Richard F. Kuk-
linski, Mr. William A. Monroe, Ms. Kay N. Milam
for expert assistance in microscopy; Ms. Amanda M.
Lawrence for help in sample processing, providing
Microtome and other accessories; and Dr. Gary W.
Lawrence for providing the knife holder. This work
was supported by funding from U.S. Department of En-
ergy through Cooperative Agreement DE-FC26-98FT-
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