Environmental and Experimental Botany 74 (2011) 289295

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Environmental and Experimental Botany

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Heavy metal uptake and stress responses of hydroponically cultivated garlic

(Allium sativum L.)

Petr Soudek, Srka

Petrov, Toms Vanek
Laboratory of Plant Biotechnologies, Joint Laboratory of Institute of Experimental Botany AS CR, v.v.i. and Crop Research Institute, v.v.i., Rozvojov 263, 162 05 Prague 6, Czech Republic

a r t i c l e

i n f o

Article history:
Received 20 April 2010
Received in revised form 28 June 2011
Accepted 28 June 2011
Keywords:
Heavy metals
Uptake
Garlic
Amino acids

a b s t r a c t
The accumulation of toxic metals (Cd, Co, Cu and Ni) by Allium sativum plants was followed under hydroponic conditions. The toxic metals were applied at two concentrations (0.05 and 0.25 mM). The effects of
heavy metals on chlorophyll and carotenoid content, root length and amino acid exudation were examined to evaluate the impact of heavy metal accumulation on plant growth and development. A signicant
hyperaccumulation of cadmium by garlic roots was observed. Cobalt stimulated carotenoid production
in garlic leaves. Copper accumulation was associated with increased exudation of amino acids to the cultivation medium and elevation of chlorophyll content in leaves. Nickel signicantly reduced root growth.

1. Introduction
The study of metal accumulation by plants may be utilized in
two distant research areas food biofortication and soil decontamination. The human body requires a large amount of different
minerals to prevent health problems. Deciency of micronutrients
(like Fe, Zn, Se, Ca, Mg, iodine, riboavin, folic acid, vitamins A,
C, E) affects about 40% of the world population, predominantly
in developing areas (Ramakrishnan and Yip, 2002). Therefore, the
agricultural community is focused on breeding of crop plants,
which contain higher concentrations of micronutrients. New varieties are cultivated and current research is aimed at genetically
modied plants.
Large contamination of the environment by compounds originated from anthropogenic activities has become currently an
extensive problem. These compounds include also heavy metals.
Some plant species are able to grow on contaminated sites and
accumulate high amounts of metals in their body (Baker and Brooks,
1989). Phytoremediation technologies utilize these hyperaccumulation properties to decontaminate polluted sites.
Thus, the heavy metal uptake by crop plants should be extensively studied to solve these two problems (Guerinot and Salt,
2001). For the purpose of soil and water decontamination, plants
with high metal accumulation capacities are required, capable of
a rapid transfer of the metal to the harvestable upper part of the

Corresponding author. Tel.: +420 233022479; fax: +420 233022479.

E-mail address: vanek@ueb.cas.cz (T. Vanek).
0098-8472/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2011.06.011

2011 Elsevier B.V. All rights reserved.

plant. In case of food fortication, the goals are to increase the

uptake of nutrients by plants, promote sustainable agriculture and
improve human health by balanced intake of mineral compounds.
To achieve all these goals, we need more information about heavy
metal uptake, metabolism, transport within the plant body and
storage, including the subcellular localization.
Oxidative stress is one of the symptoms of metal phytotoxicity.
The balance between reactive oxygen species (ROS) and antioxidants is affected in the presence of heavy metals. The organisms
have to mobilize defense system to protect cells against their
destruction. The cell defense system consists of non-enzymatic
compounds, like glutathione, ascorbic acid, -tocopherol, carotene and the antioxidant enzymes, which include superoxide
dismutases, catalases, peroxidases, glutathione reductase and
NADP+ reducing enzymes (Larson, 2005). To prevent the negative
impact of heavy metals on an organism, plants developed detoxication system based on phytochelatins, metalothioneins and other
sulphur-rich compounds. This system also helps to control the concentration of heavy metals in the cells (Cobbett and Goldsbrough,
2002).
Garlic, Allium sativum, is one of the oldest known and cultivated
plants (Jakubowski, 2003). Ancient Egyptians used garlic for medical purposes, such as heart problems, headaches, bites, worms
and tumors. Garlic is known as a source of many different sulphuric compounds, which partially share metabolic pathways with
phytochelatins, employing cysteine as a basic precursor for the synthesis of phytochelatin, thiosulphinate and sulphoxide (Block et al.,
1992; Lancaster and Shaw, 1989; Murasugi et al., 1981). Therefore, high capacity for uptake and storage of heavy metals has

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been anticipated. Hyperaccumulation of cadmium by A. sativum

was reported by Jiang et al. (2001) and Soudek et al. (2009). Hyperaccumulation of lead by garlic, onion, leek and chive was published
by Soudek et al. (2009). Zhang et al. (2005a) found stimulation of
activities of antioxidant enzymes (SOD, POD, CAT) in leaves of garlic
seedlings cultivated in the presence of cadmium. Liu et al. (2009)
determined oxidative stress in garlic exposed to lead. Xu et al.
(2008) reported the inuence of cadmium on malondialdehyde
content, photosynthetic pigments and chlorophyll uorescence in
garlic seedlings. Garlic was also used for the evaluation of roles
of phytochelatins and metallothioneins in heavy metal tolerance.
GSH and phytochelatin metabolism was followed after exposure to
various metals (and other stressors) including cadmium. Signicant
stress induced changes were also found at the level of expression of
AsPCS1 and AsMT2a genes (Zhang et al., 2005b). Zhang et al. (2006)
also characterized new cadmium-responsive garlic gene AsMT2b.
An over-expression of this gene in Arabidopsis resulted in higher
cadmium tolerance and enhanced cadmium accumulation in comparison with wild-type plants. These results suggest that garlic
could be a promising species in phytoremediation research.
In this paper, determination of metal accumulation was complemented with a thorough evaluation of the stress responses of A.
sativum. We focused on the determination of toxic metal inuence
on chlorophyll and carotenoid content, on growth and length of
roots and exudation of amino acids to the cultivation medium. We
observed signicant differences for various toxic metal treatments.

(15/85%, v/v) using digestion glass tubes. Digestion was completed

by gradual increase of temperature from 60 to 195 C according
Zhao et al. (1994). The digestion protocol was as follows: 3 h at
60 C; 1 h at 100 C; 1 h at 120 C; 3 h at 195 C. After cooling, 20% HCl
(2.5 cm3 ) was added, whirl mixed and warmed to 80 C for 1 h. The
nal volume was brought between 5 and 10 cm3 accurately. Heavy
metal content was measured by AAS (SensAA, GBS, Australia).
2.4. Chlorophyll measurement
For the chlorophyll measurement, small piece of leaf from frozen
samples was used (from 10 to 20 mg), which was extracted one
day in 1 cm3 of methanol. Methanolic extract was transferred to
cuvette and measured on UV mini 1240 Shimadzu spectrophotometer. The absorbance at 470, 652.4 and 665.2 nm was measured
and chlorophyll and carotenoid contents were calculated according
to Lichtenthaler (1987).
2.5. Amino acid determination
Frozen media were lyophilized and the samples were analyzed on Biochrom 20 Amino Acid Analyser (Pharmacia Biotech).
The samples were dissolved in 0.1 cm3 of citrate buffer (pH 2.2),
injected on cation exchanger column (PEEK, 8 m) and eluted by
buffers with different pH and ionic strength. Eluates were mixed
with ninhydrin and colored reaction products were detected at the
wavelength 570 nm and 440 nm.

2. Materials and methods

2.6. Roots length measurement
2.1. Plant material
Commercially produced bulbs of A. sativum Dukt (from Nohel
Garden, Ltd., Czech Republic), were precultivated in substrate
Experlit (Perlit Praha, Ltd., Czech Republic) and supplemented by
half strength Hoaglands liquid solution (Hoagland, 1920) at pH 5.5.
Four-week old seedlings were used for experiments.
2.2. Accumulation experiment
For a short-term experiment, garlic seedlings were cultivated
in Erlenmeyer asks (one plant per ask) in 300 cm3 of deionized
distilled water as a control or in water with heavy metals in two
concentrations (50 M and 250 M). Four replications were tested
for each treatment and each concentration. Heavy metal treatments
were performed using Cd(NO3 )2 , Co(NO3 )2 , Cu(NO3 )2 and NiSO4 .
Samples were taken at 0, 12, 24 and 48 h.
For a long-term experiment, seedlings were cultivated in Erlenmeyer asks (one plant per ask) in 300 cm3 of aerated half strength
Hoaglands liquid medium as a control or in this solution supplemented with heavy metals in two concentrations (50 M and
250 M). Four replications were tested for each treatment and
each harvest time. As heavy metal treatment we used Cd(NO3 )2 ,
Co(NO3 )2 , Cu(NO3 )2 and NiSO4 . Samples were taken at 0, 7 and 14
days.
The roots of plants were washed subsequently with deionized,
distilled water, a 0.1 M EDTA solution and nally with distilled
water. The plant samples were separated into three parts (leaves,
bulbs and roots) and then weighed. The samples were frozen in
liquid nitrogen and stored at 70 C, then lyophilized. Lyophilized
plant samples were used for heavy metal determinations.
2.3. Heavy metal determination
The freeze-dried plant tissues were ground to powder with
Wiley mill or mortar and pestle and samples (ca. 0.125 g) were
digested overnight in 5 cm3 acid mixture of HClO4 and HNO3

Root length was measured by counting the numbers of intercepts of roots in regular area (25 cm 35 cm, 1 cm 1 cm grid
squares) with randomly located and oriented lines of total lengths
(Tennant, 1973).
2.7. Data analyses
The presented data are the mean values of four repeats and
are expressed as the mean SD. The statistical analysis was performed by one-way ANOVA analysis, taking P < 0.05 as signicant
or the Students t-test for 0.05. Results of amino acid concentrations were evaluated by multidimensional statistical method PCA
(principal analysis of main components). Statistical analysis was
performed based on STATISTICA (StatSoft, Inc., Tulsa, Oklahoma,
USA) program.
3. Results
3.1. Short-term accumulation experiment
Short-term experiment was carried out with metals dissolved
in distilled water to avoid the potential inuence of cultivation
medium on heavy metal uptake and on subsequent determination
of exuded amino acid in cultivation medium. During this experiment (48 h), the signicant differences in metal accumulation in
roots and bulbs were found. The concentrations of heavy metals
in leaves of treated plants were close to the levels in control samples (close to the detection limit) and thus they were not presented
here.
Cadmium was rapidly accumulated in roots and bulbs during
the rst 12 h in both treatments. In case of 0.05 mM Cd, the gradual increase between 12 and 48 h was observed (Figs. 1 and 3). In
case of 0.25 mM Cd, the roots and bulbs were saturated very quickly
and the amount of up-taken cadmium did not change signicantly
between 12 and 48 h (Figs. 2 and 4). The nal cadmium concentration in roots was the same for both applied concentrations (ca.

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291

Fig. 1. Heavy metals concentration in root of Allium sativum during short-term

experiment at 0.05 mM concentration. Bars represent means SD (n = 4). Treatment
means with common letters are not signicantly different (P < 0.05).

Fig. 3. Heavy metals concentration in bulb of Allium sativum during short-term

experiment at 0.05 mM concentration. Bars represent means SD (n = 4). Treatment
means with common letters are not signicantly different (P < 0.05).

2.5 mg g1 DM). In bulbs, higher Cd accumulation was achieved at

0.05 mM metal concentration (0.15 mg g1 DM).
In case of cobalt uptake, strong elevation was found in roots and
bulbs after 12-h cultivation for both tested Co treatments. Steady
increase of cobalt concentration between 12 and 48 h was observed.
The amount of cobalt accumulated in roots at the end of the experiment (after 48 h) at both Co concentrations tested (0.05 mM and
0.25 mM) was practically the same (2.2 and 2.0 mg g1 DM, respectively) (Figs. 1 and 2). The Co accumulation in bulbs exhibited the
same tendency (Figs. 3 and 4).
The accumulation of copper was the lowest in comparison with
the other tested metals. The copper concentration in roots was
increasing during the whole experiment. The higher exogenous Cu
concentration in the cultivation medium led to the higher amount
of copper accumulated in roots (Figs. 1 and 2). At both applied
concentrations, Cu accumulation in roots reached the statistical
signicance in comparison with the control. On the contrary, the
copper accumulation in bulbs of garlic cultivated at 0.05 mM Cu did
not show any signicant difference in comparison with the control
(ca. 0.01 mg g1 DM) (Fig. 3). In case of 0.25 mM of Cu in the cultivation medium, signicant accumulation was detected in bulbs
(ca. 0.15 mg g1 DM), with the exception of 24 h (Fig. 4).
The amount of nickel accumulated in roots and bulbs exposed
to 0.25 mM Ni concentration in the medium was the highest in
comparison with other tested metals. Nickel uptake during 12-h

treatment rose up markedly in roots and bulbs. After 48 h of Ni

exposure, about 5 mg g1 DM and 0.23 mg g1 DM were found in
roots and bulbs, respectively (Figs. 2 and 4)

Fig. 2. Heavy metals concentration in root of Allium sativum during short-term

experiment at 0.25 mM concentration. Bars represent means SD (n = 4). Treatment
means with common letters are not signicantly different (P < 0.05).

3.2. Long-term accumulation experiment

In the long-term experiment, the garlic seedlings were exposed
to 0.05 mM and 0.25 mM concentration of tested metals. Unlike
the short-term experiment, the plants were cultivated in Hoagland
hydroponic medium supplemented with heavy metals. At 0.05 mM
metal treatment, the accumulation in leaves and bulbs was close to
detection limit, except for cadmium in bulbs. At 0.25 mM metal
concentration, signicant uptake into different garlic parts was
observed (Table 1). Nevertheless, the nal concentration of accumulated metals was about ve times lower than in short-term
experiments. The greatest accumulation (1.83 mg g1 DM) was
found in roots exposed to 0.25 mM cadmium treatment after 14
days of cultivation.
3.3. Chlorophyll and carotenoid content
Total chlorophyll and chlorophyll a content in leaves of control plants increased during the cultivation period. Chlorophyll b
and carotenoid concentrations did not change under control conditions. The plants exposed to 0.05 mM cadmium treatment exhibited

Fig. 4. Heavy metals concentration in bulb of Allium sativum during short-term

experiment at 0.25 mM concentration. Bars represent means SD (n = 4). Treatment
means with common letters are not signicantly different (P < 0.05).

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Table 1
Heavy metals concentration in different part of Allium sativum seedlings during long-term experiment at 0.25 mM concentration [mg g1 (d.m.)]. Means SD, n = 4.
Metal treatment

Cd

Co

Cu

Ni

Time

0h
7d
14 d
0h
7d
14 d
0h
7d
14 d
0h
7d
14 d

50 M

250 M

Roots

Bulbs

Leaves

n.d.
0.58
0.89
n.d.
0.25
0.46
0.02
0.20
0.40
n.d.
0.12
0.23

n.d.
0.14
0.21
n.d.
0.04
0.04
0.01
0.05
0.09
0.01
0.05
0.05

n.d.
0.04
0.05
n.d.
0.02
0.03
n.d.
0.01
0.02
n.d.
0.03
0.04

0.06
0.19
0.02
0.08
0.01
0.02
0.06
0.01
0.02

0.00
0.05
0.01
0.00
0.00
0.00
0.03
0.00
0.01
0.00

decrease in chlorophyll b and total chlorophyll content. At 0.25 mM

cadmium, increase of chlorophyll a and total chlorophyll did not
reach statistical signicance (Fig. 5A). Statistically signicant drop
of chlorophyll b and non-signicant increase in chlorophyll a and
carotenoids were found in garlic leaves at both tested cobalt concentrations. The total chlorophyll gradually decreased during the
rst 7 days of cultivation, and afterwards started increasing for
another 7 days (Fig. 5B). Copper in both tested concentrations led
to the chlorophyll b decrease during cultivation. Chlorophyll a,
total chlorophyll and carotenoids were not affected (Fig. 5C). In the
case of nickel, no signicant effect on chlorophyll and carotenoid
content was observed. The levels of chlorophylls and carotenoids

Roots

Bulbs

Leaves

0.00
0.01

1.58 0.08
1.83 0.36

0.77 0.14
1.12 0.30

0.21 0.05
0.31 0.08

0.01
0.00

0.53 0.07
0.81 0.07

0.26 0.16
0.16 0.02

0.19 0.13
0.19 0.02

0.00
0.01

0.32 0.04
0.63 0.02

0.10 0.02
0.21 0.06

0.01 0.00
0.03 0.01

0.00
0.01

0.47 0.04
0.82 0.04

0.21 0.02
0.24 0.04

0.15 0.02
0.25 0.04

were slightly lower in comparison with the control, but gradually

increasing trend was detected during the cultivation (Fig. 5D).

3.4. Roots length measurement

The exposure of garlic to any of the tested metals at both applied
concentrations (0.05 and 0.25 mM) resulted in the reduction of the
growth of the root system. After 7 days of cultivation, differences
between the control and metal-treated plants did not reach statistical signicance within the rst 7 days of experiment (Fig. 6).
After 14 days, however, the growth reduction in the medium

Fig. 5. Effects of heavy metal treatment (Cd (A), Co (B), Cu (C) and Ni (D)) on the concentrations of chlorophyll a, b, (a + b) and carotenoids of Allium sativum during a long
term experiment. Treatment means with common letters in the same type of column are not signicantly different (P < 0.05). Bars represent means SD (n = 4).

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293

acids was found (Ser and Arg, Ala and Ile, Met and Thr, Tyr and Val,
or Asp, His and Cys).
4. Discussion

Fig. 6. Effects of heavy metal treatment (Cd, Co, Cu or Ni at the concentration

0.25 mM) on the roots length of Allium sativum during a long term experiment.
Treatment means with common letters are not signicantly different (P < 0.05). Bars
represent means SD (n = 4).

supplemented with 0.25 mM metal was by ca 43, 63, 41 and 71% in

cases of Cd, Co, Cu and Ni, respectively.
3.5. Amino acid exudation
The highest exudation of free amino acids into the cultivation medium was observed in the presence of copper ions. The
values of almost all amino acids were 100 times higher in comparison with other metal treatments or with the control. The highest
concentration was found for serine (813 nmol mg1 DM). The concentration of Asp, Arg, Lys, Val and Glu were lower, reaching up
to 300 nmol mg1 DM. In case of other metals, the excretion of
amino acids did not differ signicantly from the control (Table 2).
The multidimensional statistical method PCA showed no signicant difference among tested amino acids and among tested heavy
metals. Fig. 7 illustrates similarities between plant exudates on
the basis of content of amino acids for all tested heavy metals.
There are ve groups of exudates which correlated with heavy
metal treatment. Root exudates for copper treatment exhibited
farther distance from the other four main groups of tested exudates. Fig. 8 displays correlations between tested amino acids on
the basis of their concentration in the exudates under different
heavy metal treatments. The positive correlation between amino

A. sativum was chosen as a model plant, due to its ability to

produce thiosulnates such as allicine, which contain thiol groups
(Block et al., 1992). SH-group participates in binding of toxic metals in complexes such as phytochelatins (Cobbett and Goldsbrough,
2002). Therefore, we expected thiosulphinates to affect positively
detoxication pathways of toxic metals in garlic. The results show
a rapid uptake of all tested toxic metals within 12 h. The roots cultivated in the presence of 250 M toxic metals contained from 0.9
to 5 mg of metal per g DM. According to Baker and Brooks (1989),
hyperaccumulation is dened as the ability of plants to accumulate
up to 0.1 mg g1 DM Cd or 1 mg g1 DM Co, Cu or Ni. Based on the
denition of hyperaccumulation, A. sativum can be considered as
hyperaccumulator of cadmium (ca. 400 mg g1 DM). The accumulation of copper and nickel in the whole garlic plants was close to
the hyperaccumulation level (ca. 0.65 mg g1 DM and 0.74 mg g1
DM, respectively). In the long term of experiment, the heavy metal
accumulation was lower in comparison with the short-term experiment. These differences could be attributed to different medium
composition. The competition of other ions in the solution lowered the uptake of heavy metals by the plants. Nevertheless, the
accumulation of cadmium was still on the hyperaccumulation level.
Jiang et al. (2001) also reported hyperaccumulation properties for
garlic A. sativum, which was able to accumulate more than 1826
times higher amount of cadmium in comparison with the control.
One of the explanations of the garlic ability to accumulate such
high concentrations of toxic metals may be its production of thiolcompounds, like thiosulnates.
Toxic metals represent generally a signicant stress for plants.
Severity of the stress may be indicated by changes in chlorophyll
and carotenoid contents in plant leaves. Chlorophyll participates in
photosynthesis and correlates well with vitality of plants. Besides
others, carotenoids function in the protection of photosynthetic
apparatus. Chlorophyll and carotenoid contents were in our experiment signicantly lower in plants cultivated in the presence of
cobalt and copper ions, especially at 0.25 mM metal concentration. Cadmium affected chlorophyll and carotenoid contents at
0.05 mM concentration, but not at 0.25 mM, when the plants did not
exhibit any phytotoxicity symptoms. It is possible to conclude that
cobalt, copper and cadmium are signicant stress factors for garlic

Table 2
Content of amino acid exudates in the medium after 48 h cultivation of garlic with different heavy metal treatments (concentration 250 M) Means SD (n = 4) [nmol mg1
(d.m.)].
Amino acids

Heavy metal treatment

Control

Asp
Thr
Ser
Glu
Gly
Ala
Cys
Val
Met
Ile
Leu
Tyr
Phe
His
Lys
Trp
Arg
Pro

1.04
0.94
2.83
2.11
2.16
2.09
0.64
2.01
0.61
0.34
0.28
2.16
0.97
0.38
0.88
0.34
0.32
0.49

Cd
0.13
0.12
0.30
0.10
0.35
0.31
0.05
0.59
0.06
0.18
0.16
0.22
0.27
0.09
0.07
0.04
0.09
0.04

0.95
0.52
4.13
0.80
5.54
1.48
0.23
1.19
1.18
1.01
0.19
3.44
0.95
0.87
2.95
3.04
0.44
0.33

Co

0.19
0.16
0.40
0.09
0.43
0.30
0.08
0.31
0.17
0.22
0.02
0.58
0.32
0.27
0.29
0.20
0.13
0.06

3.74
1.53
6.79
3.29
3.48
2.97
1.81
4.12
0.69
0.42
0.25
4.67
2.60
2.56
5.74
3.58
1.54
0.60

Cu

0.50
0.38
0.81
0.32
0.50
0.25
0.13
0.22
0.14
0.14
0.10
0.28
0.84
0.34
0.53
0.43
.0.22
0.14

503.82
168.80
812.90
384.79
50.23
109.51
76.79
360.15
62.30
69.17
89.95
138.90
155.74
295.22
422.35
212.42
796.35
25.11

Ni

56.60
55.40
91.05
85.79
4.50
23.34
32.19
55.20
12.14
11.20
9.99
15.67
21.82
70.33
34.68
51.56
93.71
2.88

1.80
2.06
6.54
6.21
3.88
3.77
0.21
1.47
0.82
0.47
0.38
1.37
3.08
3.89
2.67
0.70
0.52
0.69

0.38
0.13
0.76
0.59
0.30
0.67
0.01
0.03
0.18
0.08
0.07
0.20
0.25
0.40
0.17
0.19
0.17
0.21

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P. Soudek et al. / Environmental and Experimental Botany 74 (2011) 289295

Fig. 7. PCA centered score plot of garlic root exudates on the basis of amino acid content values for all tested toxic metals. Projection of the cases, where factors 1 and 2
explain 26.76% and 24.97% of total variability of the data. Plant exudates are distributed to the ve groups (in ellipses) according to their response (amino acid content) after
48 h of garlic cultivation in medium with different metal treatment.

seedlings. Changes in chlorophyll and carotenoid contents may correspond to the generation of free radicals, induced by toxic metals
(Baccouch et al., 1998; Richards et al., 1998), and with the function
of carotenoids in the photosystems, particularly with its protection against irreversible photooxidation (Prochzka et al., 1998). A
decrease in chlorophyll content in plants cultivated in high concentrations of toxic metals was reported also by Bachir et al. (2004)
(Cd in Gossypium hirsutum L.), Abou Auda et al. (2002) (Ni and Mn
in Alyssum murale), Gajewska et al. (2006) (Ni in Triticum aestivum
L.) and Symeonidis and Karataglis (1992a,b) (Pb and Zn in Holcus
lanatus L.).

Fig. 8. PCA loading plot of amino acids on the basis of amino acid content values
for all tested metal treatments. Amino acids with positively correlated amino acid
content values are showed close to each other (Trp, Lys), whereas the negatively
correlated amino acids are oriented to the opposite course (Ala, Ile and Cys).

Root length is a parameter which affects plant ability to uptake

compounds from its surroundings. The bigger the root system, the
higher uptake may occur. We monitored the root system development in the presence or absence of toxic metals. We found
signicant differences in root lengths of plants grown on cadmium,
copper, cobalt in comparison with the controls. These differences
may be caused by the toxicity of heavy metals. Metals are accumulated mainly in root tips, place of high metabolic activity. Our data
are in accordance with other studies which reported the inuence
of toxic metals in different plant species (Corylus avellana, Zea mays,
Helianthus annuus, Gossypium hirsutum, Alyssum murale, Holcus
lanatus, Brassica pekinensis, Brassica chinensis) and for different toxic
metals (Ni, Mn, Cd, Cu, Cr, Pb, Zn). All authors determined negative
correlation between the root length and increasing concentration
of toxic metal in the medium (Symeonidis and Karataglis, 1992a,b;
Ouzounidou et al., 1995; Abou Auda et al., 2002; Bachir et al., 2004;
Lin et al., 2003; Doncheva, 1998; Strati et al., 1999; Liu et al., 2007).
The last experiment was analysis of roots exudates. It is well
known that plants exude organic compounds from their root systems. These compounds facilitate or block the uptake of certain
substances such as metals from root surrounding (Dakora and
Phillips, 2002). The mechanisms of exudates-mediated modulation of metal uptake vary depending on the type of metal or plant
species. Some plants use amino acids as defense against heavy metals (Dakora and Phillips, 2002). The aim of this experiment was to
determine the content of free amino acids in the medium supplemented with toxic metals. To eliminate the disturbing inuence
of inorganic salts, distilled water was utilized as the medium. The
results showed an increase (100 times) in amino acid contents in
garlic plants exposed to copper treatment. For the other heavy metals, the changes were not signicant. We found only mild increase
of asparagine in the presence of cobalt, serine in case of nickel and
cobalt, and glutamine, histidine and phenylalanine in the presence
of nickel. Oven et al. (2002) reported an increase of free cysteine and
ascorbic acid in plant cells of Crotalaria cobalticola, Silene cucubalus
and Rauvola serpentina after exposition to cobalt ions. Krmer
et al. (1996) reported a rapid increase of histidine in Alyssum lesbiacum after exposure to nickel ions. Mazen (2004) treated Corchorus

P. Soudek et al. / Environmental and Experimental Botany 74 (2011) 289295

olitorius plants with heavy metals (Cd, Pb, Al and Cu) using hydroponic medium and determined total contents of free amino acids in
leaf tissues. Grill et al. (1987) detected PCs as a response to the presence of lead, zinc, nickel, copper and cadmium ions in cell culture
of Rauvola serpentina. The authors of these papers did not mention
the potential exudation of these compounds to the medium. Potential function of exuded amino acids, especially in case of copper, will
be studied in the future.
5. Conclusion
The accumulation of all tested heavy metals was fast and efcient and the heavy metals were taken up within 48 h. The highest
accumulation was found in the roots of tested plants, the translocation to the upper parts was very low. Our results indicate that
garlic is hyperaccumulator of cadmium. Plants cultivated in the
medium with copper and cobalt exhibited decrease in chlorophyll
and carotenoid contents. Plants exposed to cobalt- and nickelenriched medium had signicantly reduced root growth. Plants
cultivated with copper exhibited even 100 times higher amino acid
exudation to the medium in comparison with the control or other
tested metals. Generally, garlic has the potential for high heavy
metal uptake and can be a good model for phytoremediation studies. The high accumulation of micronutrient elements (Co, Cu) in
bulbs of garlic indicates the possibility to use the plant for biofortication purposes.
Acknowledgements
This work was supported by project COST FA0605
(COST.FA0605OC9082) and project NPV II (2B08058). The authors

are grateful to Dr. R. Vankov

for her nal linguistic revision of the
English text.
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