Physiological responses in two radish cultivars exposed to copper and lead stress

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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

¹Faculty of Agriculture of University J. J. Strossmayer in Osijek, Trg Svetog Trojstva 3,

31000 Osijek, Croatia; e-mail: mlisjak@pfos.hr

²Department of biology, University of J. J. Strossmayer, Trg Lj. Gaja 6, HR-31000 Osijek, Croatia; e-mail: vcesarus@yahoo.com

³Centre for Research in Plant Science, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK; e-mail: John.Hancock@uwe.ac.uk

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 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 20^oC, 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^-2s^-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; 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 (r_{PRO-h: TBARS-l} = 0.765**; r_{PRO-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 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; PDW-plant dry weight; PROT-h –protein content in hypocotyl; PROT-l –protein content in leaf; PRO-h –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		Pb 0.5 mM
Cultivar	Saxa 2	Saxa Treib	Saxa 2	Saxa Treib	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^-1FW)	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
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 (r_PRO-h:CTA-h=0.784**;r_PRO-l:CTA-h= 0.577*), and CTA-l showed very significant negative correlation 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 (r_GTA-h:CTA-h=0.745**). The specific activities of these two enzymes were also significantly and positively related in radish hypocotyls (r_GSA-h:CSA-h=0.691**) and leaves (r_GSA-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^-1prot)	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.

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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