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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
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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.
291
292
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
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
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).
293
acids was found (Ser and Arg, Ala and Ile, Met and Thr, Tyr and Val,
or Asp, His and Cys).
4. Discussion
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
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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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).
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
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