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PHYSIOLOGICAL RESPONSES IN TWO RADISH CULTIVARS EXPOSED TO
COPPER AND LEAD STRESS
M. Lisjak1, T. Teklic1, M. Engler1, N. Paradjikovic1, V. Cesar2, H. Lepedus2, I. Stolfa2, D.
Beslo1, Z. Loncaric1, J. T. Hancock3
1
Faculty of Agriculture of University J. J. Strossmayer in Osijek, Trg Svetog Trojstva 3,
31000 Osijek, Croatia; e-mail: [email protected]
2
Department of biology, University of J. J. Strossmayer, Trg Lj. Gaja 6, HR-31000 Osijek,
Croatia; e-mail: [email protected]
3
Centre for Research in Plant Science, University of the West of England, Bristol, Coldharbour
Lane, Bristol BS16 1QY, UK; e-mail: [email protected]
Abstract
Shallow-rooted vegetable species such as radish (Raphanus sativus L.) are often grown in urban
and suburban areas, where garden soils might be polluted through long-lasting exposure to
traffic-induced Pb accumulation and by the application of Cu-based fungicides. This
investigation showed that the exposure of two radish cultivars (Saxa 2 and Saxa Treib) to 0.5
mM copper or lead in nutrient solution for only two days, significantly inhibited plant growth,
stimulated lipid peroxidation and elicited the antioxidative response in radish hypocotyls and
leaves. The established connections among guaiacol peroxidase and catalase total or specific
activities, protein and free proline content in radish tissues, indicate synergistic effect of
enzymatic and non-enzymatic antioxidative mechanisms in this plant species. The interactions of
plant part, cultivar and heavy metal treatment, influenced significantly on tested parameters of
plant response to copper and lead toxicity. It has to be emphasized that a significantly different
assessment of plant antioxidative response can be obtained if defined by total or specific enzyme
activity, depending also on plant part and genotype. Regardless of genotype and heavy metal
applied, very significant positive linear correlations were established between guaiacol
peroxidase and catalase total activities in hypocotyl and their specific activities in leaves.
Introduction
Heavy metal toxicity is one of the major environmental problems resulting with hazardous
consequences in all living organisms. Because of their high reactivity they can directly influence
growth, senescence and energy synthesis processes which are intriguing effects, more so, as the
knowledge of their mechanisms can have a great significance in ecophysiology and medicine
(Maksymiec, 2007). As stated by Sharma and Dietz (2006), metal ions turn toxic as soon as their
concentration exceeds a metal-specific threshold which varies strongly among plant species and
ecotypes, and also with metal properties. Generally, it is known that growth inhibition, noted in
plants under heavy metals uptake, is related to some physiological process alterations owing to
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generated oxidative stress (Jouili and El Ferjani, 2003). The activity of antioxidative enzymes
like catalase and peroxidase can prevent ROS overproduction and oxidative destruction of
essential biomolecules, which commonly occurs in abiotic stress conditions and may lead to fatal
damage at cellular and whole plant level. Beside the enzymatic defense systems, plant
physiological response comprehends a range of protective metabolites that can contribute to
plant tolerance to heavy metal overload as well as to other environmental stress factors. After
Kaul et al. (2008), it is quite likely that the elevated tissue proline levels under stressful growth
conditions constitute a component of cellular antioxidative network involved in mitigation of
stress effects. This proteinogenic amino acid functions as an osmolyte, radical scavenger,
electron sink, stabilizer of macromolecules, cell wall component (Matysik et al., 2002), acts as a
reserve source of carbon, nitrogen and energy during recovery from stress and is essential for
buffering cellular redox potential (Kavi Kishor et al., 2005). Demirevska-Kepova et al. (2004)
stated that data comparing the influence of heavy metals with different chemical properties in a
single plant species are scarce and that such comparison may give some insight into the
biochemical mechanisms underlying the metal toxicity symptoms. Therefore, this research deals
with toxicity of Cu, an essential microelement, and Pb that has not any metabolic function.
Radish was used as the test plant, since this plant species is grown throughout the world,
frequently in suburban gardens, where soil might be polluted with mentioned heavy metals due
to human activities.
Materials and methods
The plantlets of radish (Raphanus sativus L., cultivars Saxa 2 and Saxa Treib) were grown in
glass pots containing Hoagland nutrient solution. The experiment was carried out in three
replicates and each replicate had four plants. The pots were kept for 3 weeks in a growth
chamber at 20oC, 70% relative air humidity and a 12 h photo-period. Light was supplied by cool
white fluorescent lamps providing the photosynthetic photon flux density of 120 μM m-2 s-1 at the
leaf level. Subsequently, the plantlets were treated with 0.5 mM Cu(SO4) or Pb(NO3)2 in nutrient
solution for the next 2 days. Control plants had 0.05 mg L-1 Cu (as essentials) with no additional
copper or lead in the growth media. Fresh and dry weight of leaf and hypocotyl were determined
as growth response parameters.
Lipid peroxidation level, protein content, guaiacol peroxidase and catalase activities were
analyzed in fresh leaves and hypocotyls. Lipid peroxidation was measured as the amount of
thiobarbituric acid (TBA) reactive substances (TBARS-l, leaf; TBARS-h, hypocotyl) as
described by Heath and Packer (1968). Protein content (PROT-l, leaf; PROT-h, hypocotyl) was
estimated using the method of Bradford (1976). Free proline content (PRO-l, leaf; PRO-h,
hypocotyl) was determined after Bates et al. (1973). Peroxidase (EC 1.11.1.7) activity in leaves
and hypocotyls was determined using guaiacol as a substrate (Siegel and Galstone, 1967) and
catalase (EC 1.11.1.6) activity was measured according to Aebi (1984). Total activities of
peroxidase and catalase were expressed as U g-1 tissue fresh weight (GTA-l, leaf; CTA-l, leaf;
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CTA-h, hypocotyl) or U g-1 fresh weight in the case of GTA-h (hypocotyl). Enzyme specific
activities were expressed as U mg-1 protein (GSA-l, leaf; GSA-h, hypocotyl; CSA-l, leaves;
CSA-h, hypocotyl). Data obtained from the measurements and analyses were evaluated
statistically using ANOVA and limited significant difference (LSD) was calculated when
significant F-ratio occurred (P≤0.05). The significance of the established relations among
physiological response parameters was evaluated using t-test (*P≤0.05; ** P≤0.01).
Results and discussion
The applied heavy metal treatment significantly decreased whole plant fresh weight (PFW; Table
1), as well as dry weight although radish plants were exposed only two days. Jouili and El
Ferjani (2003) treated ten-day-old sunflower seedling roots with 50 μM CuSO4 for five days and
observed significant decrease in dry-matter production and protein level, as well as an increase
of lipoperoxidation product rate, whereas catalase and guaiacol peroxidase activities were
significantly enhanced by copper treatment. They concluded that growth delay could be related
to the inhibition of cellular turgor, the reduction of total protein amount and to the generated free
radicals by lipoperoxidation. As stated by Maximiec (2007), formation of lipid peroxides may be
a prolonged consequence of heavy metal-induced oxidative stress and may act as an activation
signal for plant defense genes through increase of the octadecanoid pathways. After prolonged
time, a higher level H2O2 was the result of attenuation of the antioxidative system in the
particular organelles, connected especially with enzyme protein content reduction. Here, the
estimated protein content was in both cultivars and all treatments higher in leaves as compared to
hypocotyls (Table 1), and cultivar x heavy metal treatment determined protein level in both plant
parts. It was significantly higher in stressed hypocotyls of both cultivars, whereas in leaves of
Saxa 2 declined under heavy metal treatment and in Saxa Treib an opposite trend was observed.
Very significant differences in free proline content between leaves and hypocotyls were noticed,
as well as regarding tested cultivars and heavy metal treatments. In general, proline content was
higher in hypocotyls with the exception of Pb-treated Saxa Treib. This cultivar showed mostly
much lower proline level in comparison with Saxa 2. Taken as an average of two tested cultivars,
Cu treatment increased proline level by 66% in leaves and 298% in hypocotyls, respectively. Pb
effect on proline accumulation was 41% increment in leaves and 31% in hypocotyls. In the
research of Zengin and Munzuroglu (2005), proline content increased by 12.2% (in 0.1 mM Cu),
21.3% (0.2 mM Cu) and 30.9% (0.3 mM Cu) after ten-day exposure of seven-day-old bean
seedlings. As stated by Sharma and Dietz (2006), proline may be involved in plant heavy metal
stress by different mechanisms, i.e. osmo- and redox-regulation, metal chelation, and scavenging
of free radicals, based on its known properties. In our research, proline content in both plant parts
was significantly related to lipid peroxidation level in leaves (rPRO-h: TBARS-l = 0.765**; rPRO-l:
TBARS-l = 0.547*). The established TBARS levels (Table 1) implicate the oxidative stress level,
especially in hypocotyls that were in direct contact with nutrient solution. Lipid peroxidation in
leaf was considerably lower, regardless of cultivar, with higher values observed in Cu treated
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plants. TBARS-h correlated positively with PROT-h (r=0.78**), GTA-h (r=0.888**), CTA-h
(r=0.677**) and GSA-l (r=0.587*).
Table 1. Protein content, free proline and lipid peroxidation level in radish genotypes under
influence of 48-h of heavy metal treatment in nutrient solution (PFW-plant fresh weight; PDWplant dry weight; PROT-h –protein content in hypocotyl; PROT-l –protein content in leaf; PROh –proline content in hypocotyl; PRO-l – proline content in leaf; TBARS-h –lipid peroxidation
level in hypocotyl; TBARS-l –lipid peroxidation level in leaf; Control-untreated plants had 0.05
mg L-1 Cu as essentials with no additional Cu or Pb in growth media; data are means ±S.E. of
three replicates).
Treatment
Control
Cu 0.5 mM
Saxa 2
Saxa
Treib
PFW (g plant-1)
4.59±0.93
6.00±0.84
3.43±0.43 5.01±0.77 3.73±0.94 3.13±0.48
PDW (g plant-1)
0.28±0.06
0.35±0.05
0.28±0.04 0.15±0.02 0.20±0.05 0.19±0.03
PROT-h (mg g-1 FW)
0.64±0.08
0.48±0.02
0.86±0.09 0.76±0.05 1.45±0.11 0.94±0.05
PROT-l (mg g-1 FW)
5.09±0.43
3.55±0.19
3.31±0.43 5.46±0.10 3.06±0.16 4.89±0.18
PRO-h (µM g-1 FW)
2.04±0.13
0.38±0.03
6.10±0.47 1.02±0.11 2.66±0.14 0.50±0.08
PRO-l (µM g-1 FW)
1.02±0.04
1.05±0.19
2.92±0.12 0.53±0.02 1.14±0.09 1.79±0.17
Cultivar
Saxa 2
Saxa
Treib
Pb 0.5 mM
Saxa 2
Saxa
Treib
TBARS-h (nM g-1 FW) 264.7±7.2 226.4±2.4 295.0±5.3 320.9±2.6 413.5±2.3 240.1±3.0
TBARS-l (n Mg-1 FW)
9.1±0.3
8.5±0.3
18.3±0.2
14.4±0.2
10.1±0.2
9.1±0.1
The applied heavy metals doses (50 μM Cd, 20 μM Cu and 500 μM Zn, 8 days in nutrient
solution) enhanced significantly the activity of the enzyme guaiacol peroxidase in the roots of
cucumber, bean and lettuce in the research of Vassilev et al., (2007). Total activity of peroxidase
in our research was strongly influenced by plant part, cultivar and heavy metal treatment. It was
mostly enhanced in both cultivars and both plant parts due to heavy metal stress, the only
exception were leaves of Cu-treated Saxa 2 (Table 2). Depending on protein content, peroxidase
specific activity was higher in leaves than in hypocotyls whereas heavy metal treatment had an
impact through the interactions with cultivar and plant part. As opposite to peroxidase total
activity, its specific activity in both HM treatments was significantly lower as compared to
control plants, in hypocotyls and in leaves as well. As for catalase activity, significantly higher
CTA-h values in both cultivars were observed under influence of the applied heavy metal
treatments. However, CTA-l was significantly higher in Pb stressed plants and the influence of
Cu stress was not significant. CTA-h was correlated to proline content in both plant parts (rPROh:CTA-h=0.784**; rPRO-l:CTA-h= 0.577*), and CTA-l showed very significant negative correlation
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with PRO-h (r=-0.681**). Regarding catalase specific activity, it was higher in hypocotyls of
control and Cu treated plants. Pb-treated plants of both cultivars showed higher CSA in leaves.
GTA-h and CTA-h correlated positively with PROT-h (r=0.829** and r=0.0.672**,
respectively), and each other (rGTA-h:CTA-h=0.745**). The specific activities of these two enzymes
were also significantly and positively related in radish hypocotyls (rGSA-h:CSA-h=0.691**) and
leaves (rGSA-l:CSA-l=0.786*) in given experimental conditions.
Table 2. The activities of guaiacol peroxidase and catalase in radish genotypes under influence of
48-h of heavy metal treatment in nutrient solution (GTA-h -peroxidase total activity in
hypocotyl; GTA-l -peroxidase total activity in leaf; GSA-h -peroxidase specific activity in
hypocotyl; GSA-l -peroxidase specific activity in leaf; CTA-h -catalase total activity in
hypocotyl; CTA-l -catalase total activity in leaf; CSA-h -catalase specific activity in hypocotyl;
CSA-l -catalase specific activity in leaf; Control-untreated plants had 0.05 mg L-1 Cu as
essentials with no additional Cu or Pb in growth media; prot – protein; data are means ±S.E. of
three replicates).
Treatment
Control
Cu 0.5 mM
Pb 0.5 mM
Cultivar
Saxa 2
Saxa
Treib
Saxa 2
Saxa
Treib
Saxa 2
Saxa
Treib
GTA-h (Umg-1 FW)
0.46±0.01
0.33±0.01
0.56±0.02
0.48±0.04
0.82±0.06
0.46±0.01
GTA-l (Ug-1 FW)
10.73±0.13
9.93±0.38
10.00±0.75 11.20±0.61 11.97±0.48 11.77±1.48
GSA-h (Umg-1 prot)
0.74±0.10
0.69±0.04
0.67±0.09
0.62±0.03
0.58±0.08
0.49±0.01
GSA-l (Umg-1 prot)
2.15±0.22
2.81±0.13
3.07±0.21
2.05±0.15
3.93±0.21
2.41±0.32
CTA-h (Ug-1 FW)
0.85±0.03
0.80±0.01
1.49±0.01
1.06±0.03
1.40±0.06
1.01±0.01
CTA-l (Ug-1 FW)
3.85±0.03
4.15±0.03
3.45±0.03
4.15±0.08
4.15±0.13
5.30±0.01
CSA-h (Umg-1 prot)
1.35±0.12
1.69±0.07
1.78±0.17
1.39±0.05
0.98±0.10
1.06±0.05
CSA-l (Umg-1 prot)
0.77±0.07
1.18±0.07
1.08±0.15
1.00±0.03
1.36±0.02
1.09±0.04
This investigation showed that the exposure of two radish cultivars to 0.5 mM copper or lead in
nutrient solution for only two days, significantly inhibited plant growth, stimulated lipid
peroxidation and elicited the antioxidative response in radish hypocotyls and leaves. The
established correlations among estimated parameters of plant physiological response indicate
synergistic effect of enzymatic and non-enzymatic antioxidative mechanisms in this plant
species.
It has to be emphasized that significantly different assessment of plant antioxidative response can
be obtained if defined by total or specific enzyme activity, depending also on plant part and
genotype.
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Acknowledgements
This work was an integral part of the MSc thesis of Meri Engler and supported by The Ministry
of science, education and sports, Croatia.
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