Turkish Journal of Biology
Turk J Biol
(2016) 40: 652-660
© TÜBİTAK
doi:10.3906/biy-1411-13
http://journals.tubitak.gov.tr/biology/
Research Article
Anatomical and ultrastructural responses of Brassica napus after long-term
exposure to excess zinc
1,
1
1
2
Seyed Mousa MOUSAVI KOUHI *, Mehrdad LAHOUTI , Ali GANJEALI , Mohammad H. ENTEZARI
1
Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
2
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
Received: 06.11.2014
Accepted/Published Online: 26.08.2015
Final Version: 18.05.2016
Abstract: Heavy metal contamination resulting from anthropogenic activities is one of the major environmental problems in the
modern world. Progress toward creating modified plants that are more efficient in phytoremediation requires an understanding of the
anatomical and ultrastructural changes involved in heavy metal accumulation. A large supply of zinc (Zn) is toxic for plants and can
lead to functional and structural disorders. The present study investigates the anatomical and ultrastructural responses of Brassica napus
(rapeseed) to excess Zn after long-term exposure. Results showed that Zn bioaccumulation in the roots and leaves of plants treated
with 350 µM Zn2+ was severely increased, leading to a variety of anatomical and ultrastructural alterations in several cell types. Overall,
although B. napus has been reported as a metal-accumulator species, our data revealed that B. napus cultivar Hayola 401 could not
tolerate a high concentration of Zn and thus is not a good candidate for Zn phytoremediation.
Key words: Heavy metals, phytoremediation, soil contamination, zinc
1. Introduction
The modern world is facing a major environmental
problem, that of heavy metal contamination (Li et al.,
2006). The progressive release of heavy metals into the
environment resulting from anthropogenic activities,
such as mining, smelting of metalliferous ores, and
electroplating, can detrimentally affect living components
of ecosystems (Maruthi Sridhar et al., 2005; Wang et al.,
2009). Heavy metal accumulation in agricultural soils has
raised concerns about food safety and subsequent adverse
effects on human health (Brunetti et al., 2011). Zinc (Zn)
is the second most ample transition metal after iron (Fe)
(Wang et al., 2009). Besides industrial activities, the use
of Zn-containing fertilizers and pesticides in agriculture
and the wide use of Zn in paint, rubber, dye, and wood
preservative industries have contributed to environmental
contamination with Zn (Maruthi Sridhar et al., 2005).
Zn is an essential element for plants, required for
the activity of many plant enzymes and for chlorophyll
biosynthesis (Taiz and Zeiger, 2010). Zn is also involved in
protein synthesis, carbohydrate metabolism, tryptophan
and indole acetic acid synthesis, and membrane integrity.
However, a large supply of Zn can be toxic for plants
and may lead to functional and structural disorders
(Marschner, 1995).
*Correspondence: [email protected]
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Use of plants to clean up or extract pollutants from
the environment is called phytoremediation (Li et al.,
2006; Katayama et al., 2013). In recent years, many plants
have been screened to find hyperaccumulator species that
accumulate high metal concentrations, have fast growth,
produce high biomass, and are fully harvestable (Marchiol
et al., 2004). Most hyperaccumulator species produce
little biomass and have slow growth rates (Li et al., 2006).
Moreover, metal uptake by plants and its bioaccumulation in
different parts of the plant are intrinsically limited (Maruthi
Sridhar et al., 2005). Progress toward creating modified
plants that are more efficient in phytoremediation requires
an understanding of the ultrastructural, biochemical,
physiological, and molecular mechanisms involved in
heavy metal accumulation in different parts of the plant (Li
et al., 2006). Since anatomical and ultrastructural changes
can reflect and/or lead to biochemical, physiological, and
molecular alterations in the plant (Todeschini et al., 2011),
investigating these changes can help to understand the
overall process of phytoremediation (Maruthi Sridhar et
al., 2005).
Fast-growing and high biomass-accumulating plants,
such as the Brassicaceae species, are good candidates for
phytoremediation (Brunetti et al., 2011). Brassica napus
(rapeseed), the third most important source of edible
MOUSAVI KOUHI et al. / Turk J Biol
vegetable oil after soybean and palm (Burbulis et al.,
2008), is mainly used in food applications (Grispen et
al., 2006), but it is also used as a medicinal food plant in
Central Asia, North Africa, and West Europe (Saeidnia
and Gohari, 2012). In recent years, it has also been used
in the production of biofuel. Thus, the cultivation of
rapeseed in heavy metal-contaminated soils is beneficial
both for phytoremediation and for biofuel production, in
addition to its importance in producing edible and healthy
oil (Grispen et al., 2006). Since, to the authors’ knowledge,
simultaneous studies on the long-term anatomical and
ultrastructural responses of plants to excess heavy metals
are scarce, the present study investigates the responses of
B. napus to Zn2+.
2. Materials and methods
2.1. Plant culture and experimental design
Seeds of rapeseed cultivar Hayola 401 were prepared by
the Agricultural and Natural Resources Research Center
of Khorasan-Razavi, Mashhad, Iran. Seeds were sterilized
in 10% sodium hypochlorite solution for 10 min, rinsed
thoroughly three times with deionized water, soaked
in deionized water for about 2 h, and then cultured
hydroponically with perlite as a supporting material.
Seeds were sown in 1.5-L plastic pots filled with perlite and
arranged in two groups. In the control group, pots were
fed with 10%-strength Hoagland nutrient solution (Taiz
and Zeiger, 2010). In the other group, pots were fed with
same strength of Hoagland nutrient solution containing
350 µM Zn2+, prepared by dissolving appropriate amounts
of ZnSO4.7H2O in deionized water. The treatment
concentration (350 µM) was selected after a primary
study to find a first estimate of toxicity thresholds. For
this purpose, the effect of different concentrations of Zn2+
on the growth of rapeseed was investigated, and then the
maximum Zn2+ concentration tolerated by rapeseed was
selected according to the growth parameters. Moreover,
our previous study, investigating the effect of a wide range
of concentrations of Zn2+ on some growth parameters
of rapeseed seedlings, provided a good estimation of Zn
toxicity (Mousavi Kouhi et al., 2014).
Plants were grown at 25 ± 1 °C under a 16/8-h
photoperiod. The nutrient solution of each group was
renewed every 10 days. For this purpose, the pots were
fully rinsed with distilled water and then fed with a
relevant nutrient solution, where the rinsing water was
removed by leaching. Nutrient solution strength was
increased to 25% and 50% in the second and subsequent
renewals, respectively. Pots were weighed daily and weight
loss was reversed by adding nutrient solution. Two months
after planting, the plants were harvested for subsequent
analyses.
2.2. Microscopic analyses
Root segments of about 2 mm in length were cut 1 cm above
the root tip and about 1 cm above the junction between
branch and taproot. Leaf rectangular segments (1 × 2 mm)
were sectioned from the sixth leaf, between the third and
the fourth principal lateral veins (Stefanowska et al., 1999),
and immediately transferred to 4% glutaraldehyde in 0.1
M cacodylate buffer (pH 7.4) at 4 °C for overnight fixation.
After rinsing three times with the same buffer for 15 min
each, the samples were postfixed in 1% OsO4 in the same
buffer for 2 h, followed by washing three times in the same
buffer. Segments were dehydrated in a graded series of
ethanol (30%, 50%, 70%, 90%, and 100%) and were then
infiltrated with resin overnight. Semithin (1 µm) and
ultrathin (80 nm) sections were cut on an ultramicrotome
(Ultracut UCT, Leica, Austria). Semithin sections were
observed with a light microscope (Olympus BX 51, Japan)
connected to a digital camera (Olympus DP 71) after
staining with 1% toluidine blue in 1% borax. Ultrathin
sections were mounted on copper grids and then observed
with transmission electron microscopy (TEM; LEO 912
AB, Zeiss, UK) (Mousavi Kouhi et al., 2015).
Different sections were prepared in the present study.
However, the best section, being a good representative,
was selected to study anatomical and ultrastructural
modifications. The modifications that were completely
distinct from those of the control plant were descriptively
investigated without quantitative analysis. For instance,
although the size of starch grains is a qualitative parameter,
their modifications relative to the control were so obvious
that they did not need to be measured quantitatively.
However, parameters such as cell size, number of
chloroplasts, and size of mitochondria were investigated
quantitatively. In order to measure the cell size, the
diameter of ten cells was determined and then the mean
size was calculated. To measure the size of cell organelles,
the diameter of ten organelles from a given cell was
determined and the mean size was calculated. The number
of chloroplasts was counted in more than 20 mesophyll
cells and the mean number was recorded for each cell.
2.3. Determination of Zn content of roots and leaves
Plant roots and leaves were dried at 70 °C for 48 h. The
dried samples were ground to a fine powder and 0.5 g
of each powder was digested with 5 mL of HNO3 and 1
mL of 30% H2O2, followed by heating at 90 °C. The two
latter stages were repeated until the digest was clear. After
clearing the sample digest, heating was continued until
near dryness. Finally, digest residue was dissolved with
diluted HNO3 and brought to a final volume of 25 mL
with deionized water (Kalra, 1998). The Zn content of root
and leaf extracts was determined with atomic absorption
spectrometry (AA-670, Shimadzu, Japan).
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2.4. Statistical analyses
There were three replicates for each group, arranged in
a completely randomized design. A one-way analysis of
variance (ANOVA), followed by Duncan’s multiple range
test, was used to test significant differences and means
comparisons with MSTAT-C. Data were reported as mean
± standard deviation. Significant difference was defined as
P < 0.05.
2500
3. Results
3.1. Zn bioaccumulation in roots and leaves
Zn bioaccumulation in the roots and leaves of the plants
treated with 350 µM Zn2+ was severely increased compared
to the control (Figure 1). The results showed that the Zn
content of roots and leaves of Zn-treated plants was 5.7
and 6.42 times higher than that of the control plants,
respectively.
3.2. Root anatomy and ultrastructure
Cross-sections of the root tips of Zn-treated plants
showed a decreased number of cortical and stellar cells
and a decreased diameter of root tip compared to the
control (Figures 2a and 2b). Light micrographs revealed
that the most vulnerable tissue of the root tip cells under
Zn treatment was the epidermis. Root epidermal cells of
Zn-treated plants were deformed and shrunken, having
a smaller size than that of the control (Figures 2c and
2d). The stele diameter and size of the pericycle cells also
decreased under Zn stress (Figures 2e and 2f). Although
the number of vascular cells appeared lower compared to
the control, their size seemed to be larger (Figure 2f). Light
micrographs of about 1 cm above the junction between the
branch and the taproot revealed that under excess Zn, the
stele diameter and its cell numbers were decreased, and
2000
Leaf
1000
1500
b
c
c
0
500
mg / kg dry weight
a
Root
CONTROL
350 µM Zn
Figure 1. Zn content of roots and leaves of B. napus after 2
months of exposure to 350 µM Zn2+. Bars with different letters
indicate significant difference (ANOVA, F = 259.6; df = 3, 8; P
< 0.05).
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conducting cells of the xylem and phloem were undersized
compared to the control (Figure 3a–3d).
TEM micrographs of the cortical cells of the root tips of
Zn-treated plants revealed Zn deposition in the cell walls.
These cell walls had different thicknesses at different sites
and were deformed at certain sites (Figures 4a and 4b).
Other detectable and prominent ultrastructure alterations
in the cortical cells of Zn-treated plants were not observed.
The TEM image of the vascular tissue of the root tip of the
control plants showed a large starch grain in the plastids
of several vascular cells (Figure 5a). However, this feature
was not observed in Zn-treated plants. Moreover, several
plastids of later cells were deformed and misshaped in Zntreated plants. Zn deposition was observed in vacuoles and
intercellular spaces of vascular tissue (Figures 5b and 5c).
The stellar cells of the root tips revealed excess Zn-induced
disintegration of mitochondria (Figure 5c).
3.3. Leaf anatomy and ultrastructure
The leaves of the plants treated with high Zn showed
general chlorosis and had a small leaf area compared to
the control plants (data not shown). Light micrographs
of the leaf section of Zn-treated plants revealed that the
number and sizes of chloroplasts decreased and the size of
the epidermal cells was larger than in the control (Figure
6). Moreover, disorganization of spongy parenchyma cells
(mainly their chloroplasts) and decreased size of vascular
bundles were also observed in Zn-treated plants (Figure
6a–6d).
TEM micrographs of leaf mesophyll cells revealed
certain ultrastructural changes induced by excess Zn,
including breakdown of cell walls, structural disorder of
chloroplasts including decreased number of thylakoid and
grana, increase in size and number of plastoglobuli in the
chloroplasts, decrease in the size of the nucleus, and Zn
deposition in vacuoles (Figure 7).
4. Discussion
The Zn content of soils is between 60 and 89 mg/kg
(Kabata-Pendias, 2011). The critical toxicity level of Zn
in the leaves of crop plants is approximately 300 mg/kg
dry weight (Marschner, 1995). In the present study, the
Zn content of leaves was 1600 mg/kg dry weight. Many
studies have reported that high Zn accumulation can
result in functional and structural changes in different
parts of the plant (Maruthi Sridhar et al., 2005; Jiang et
al., 2007; Todeschini et al., 2011). Since the root is the
first target tissue exposed to high concentrations of heavy
metals, it seems that functional and structural disorders
appear more often in roots than in the aboveground parts
of the plant.
It has long been known that Zn toxicity can lead to
stunted growth and leaf chlorosis (Zeng et al., 2011). Leaf
chlorosis may result from induced deficiency of some
MOUSAVI KOUHI et al. / Turk J Biol
Figure 2. Light micrographs of cross semithin sections of the root tip of B. napus. a, c, and e: Control plants; b, d, and f: plants
treated with 350 µM Zn2+; pc: pericycle; en: endoderm.
ions with the same electrical charge or ion radius such as
magnesium and iron (Marschner, 1995). It has also been
known for many years that interference of Zn with Fe
metabolism can lead to chlorosis (Zeng et al., 2011) due to
competition between Zn and Fe, and/or interference with
the chelation processes of Fe during root uptake (KabataPendias, 2011).
The most vulnerable root tip tissue under Zn treatment
was the epidermis. For this reason, root epidermal cells are
the first cells exposed to excess Zn; thus, they are likely to
be the most negatively affected cells. Decreased diameter
of the root tip may result from a decreased number of
cortical and stellar cells, which may subsequently result
from the fact that metal-induced toxicity can lead to arrest
of cell division (Gangwar et al., 2014). Decreased number
of vascular cells of the root tip and their increased size
in Zn-treated plants may also be due to decreased cell
division. Decreased cell division induced by heavy metals
(Hossain et al. 2012) may allow preexisting vascular cells
to grow further as a result of potential competition.
A study by Sresty and Madhava Rao (1999) on the root
cells of pigeonpea under excess Zn and nickel revealed
many ultrastructural alterations, including major changes
in the nucleus, highly condensed chromatin, disruption
and dilation of nuclear membranes in several cortical
cells, withdrawal of the plasma membrane from cell walls,
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MOUSAVI KOUHI et al. / Turk J Biol
Figure 3. Light micrographs of cross semithin sections of about 1 cm above the junction between branch and taproot of B. napus.
a and b: Control plants; c and d: plants treated with 350 µM Zn2+. b and d are higher magnifications of the frames in a and c,
respectively. mt: metaxylem.
Figure 4. TEM micrograph of cross ultrathin sections of the root cortical cells of B. napus. a: Control plants; b and c: plants treated
with 350 µM Zn2+; cw: cell wall; dp: metal deposition; v: vacuole.
structureless cytoplasm, disintegration of cell organelles,
and development of vacuoles.
Deposition of metals in different cell compartments is
one of the processes used by plants to tolerate the excess
656
concentration of heavy metals. Sites of deposition depend
on the metal and plant species. In tolerant species, Zn
accumulates in the vacuole, whereas in nontolerant species
it accumulates in the cytosol (Marschner, 1995). However,
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Figure 5. TEM micrograph of cross ultrathin sections of the root vascular tissue cells of B. napus. a and b: Control plants; c, d, e,
and f: plants treated with 350 µM Zn2+; cw: cell wall; mc: mitochondrion; v: vacuole; dp: metal deposition; N: nucleus; pl: plastid;
s: starch grain; ER: endoplasmic reticulum.
Zn may also be sequestrated in chloroplasts (KabataPendias, 2011). Jiang et al. (2007), using TEM microscopy,
reported the deposition of Zn with P in the vacuoles
of roots. Zn deposition as electron-dense globules in
vacuoles may play a role in the tolerance of excess Zn by
decreasing its levels in the cytoplasm and nucleus (Sresty
and Madhava Rao, 1999).
Cell walls with certain electronegative ions, such as
HPO42–, H2PO4–, C1–, and SO42–, can sequestrate metals and
thus prevent their translocation from root to shoot (Jiang
et al., 2007). It has been reported that metal sequestration
in cell walls may lead to a decrease in their elasticity and
then to deformity or breakdown. On the other hand, under
heavy metals, decreased cell turgidity due to reduced
vacuole size can lead to deformity or breakdown of cell
walls, resulting in irregular cell alignments (Pokhrel and
Dubey, 2013).
Similarly to the present study, a breakdown of spongy
parenchyma cells was reported in Brassica juncea treated
with high concentrations of Zn. However, other symptoms
observed in B. juncea, including reduction in the palisade
and epidermal cells, breakdown in the palisade parenchyma
cells, loss of cell shape, decrease in intercellular spaces,
shrinkage of epidermal cells, and decrease in the starch
content of leaves (Maruthi Sridhar et al., 2005), were not
observed in B. napus exposed to toxic levels of Zn in the
present study. In another study, consistent with the present
study, increased size of leaf epidermal cells and decreased
numbers of chloroplasts of mesophyll cells were reported
in poplar treated with high Zn concentration (Todeschini
et al., 2011).
As with the present study, several studies on the Brassica
species have reported negative effects of excess Zn on the
disorganization of chloroplasts and a decreased number
of thylakoid and grana (Ebbs and Kochian, 1997; Jiang
et al., 2007; Ebbs and Uchil, 2008; Wang et al., 2009) that
can induce chlorosis (Wang et al., 2009). The decreased
number of chloroplasts in the present study was also
reported in Brassica oleracea treated with nickel (Molas,
2002). Furthermore, it was reported that the chloroplasts
of high Zn-treated plants are undersized. Disintegration
of double-membrane organelles, such as chloroplasts
and mitochondria, was also reported in poplar leaves
treated with excess Zn, which may be considered a sign
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Figure 6. Light micrographs of cross semithin sections of leaf of B. napus. a and c: Control plants; b and d: plants treated with 350
µM Zn2+. c and d are higher magnifications of the frames in a and b, respectively. ep: epidermal cell; pl: palisade cell; sp: spongy
mesophyll cell.
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Figure 7. TEM micrograph of cross ultrathin sections of the leaf mesophyll cells of B. napus. a, b and c: Control plants; d, e, and
f: plants treated with 350 µM Zn2+; cw: cell wall; mc: mitochondrion; v: vacuole; N: nucleus; nu: nucleolus; Cp: chloroplast; pG:
plastoglobule; s: starch grain; dp: metal deposition
of advanced induced senescence (Todeschini et al., 2011).
Consistent with our study, Todeschini et al. (2011)
reported an increase in the number of plastoglobuli
of chloroplasts induced by high Zn concentration.
Occurring more frequently during senescence, increased
plastoglobuli of chloroplasts is one of the ultrastructural
changes under abiotic stresses such as heavy metals
(Brehelin et al., 2007). Besides sequestration in root cells,
Zn can also be deposited in leaf cells. Zn sequestration in
the vacuoles of leaf epidermal (Thlaspi caerulescens) and
mesophyll (Arabidopsis halleri) cells has been reported
(Todeschini et al., 2011).
Overall, our data revealed that B. napus cultivar
Hayola 401 could not tolerate a high concentration of Zn.
Under 350 µM Zn, B. napus showed many anatomical and
ultrastructural toxicity symptoms. However, some species
accumulate more than 10,000 mg of Zn per kilogram of
dry weight without any toxicity symptoms. For instance,
it has been reported that Arabis paniculata could tolerate
2000 µM Zn, accumulating more than 12,000 and 6000
mg Zn/kg dry weight of root and shoot, respectively.
Interestingly, excess Zn up to 500 µM increased the
biomass of A. paniculata (Zeng et al., 2011). In conclusion,
although B. napus has been reported as a metalaccumulator species (Grispen et al., 2006), we suggest that
B. napus cultivar Hayola 401 is not a good candidate for
the phytoremediation of excess Zn.
Acknowledgments
This work was supported by the Faculty of Sciences,
Ferdowsi University of Mashhad, Iran (Grant No. 24427).
The authors are grateful to Fatemeh Naseri and Roksana
Pesian for their technical help in preparing samples for
TEM and taking TEM micrographs, respectively.
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MOUSAVI KOUHI et al. / Turk J Biol
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