untitled ACTA BOT. CROAT. 75 (1), 2016 25 Acta Bot. Croat. 75 (1), 25–30, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0017 ISSN 0365-0588 eISSN 1847-8476 Impact of nickel on grapevine (Vitis vinifera L.) root plasma membrane, ROS generation, and cell viability Ján Pavlovkin1*, Roderik Fiala1, Milada Čiamporová1, Michal Martinka1,2, Vladimír Repka1 1 Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523, Bratislava, Slovak Republic 2 Department of Plant Physiology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-2, SK-84215 Bratislava, Slovak Republic Abstract – The present study investigated the impact of nickel (Ni2+) on trans-membrane electrical potential (EM) and permeability properties of plasma membrane (PM) in epidermal cells of adventitious grapevine roots. The relationship between disturbances of membrane functionality and the production of superoxide anion, hy- drogen peroxide and cell viability after the exposure of roots to Ni2+ was also studied. Treatments with 0.1–5 mmol L–1 NiCl2 induced a concentration-dependent transient PM depolarization, which was recovered to the initial resting potential within 50–70 min in the presence of Ni2+. Longer (up to 24 h) exposure of roots to 1 mmol L–1 of Ni2+ hyperpolarized the EM by approximately 17 mV. Application of the highest 5 mmol L–1 con- centration of Ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the EM, cell viability, and superoxide content remained unaffected. The increase in the formation of hydrogen peroxide was time- and concentration- dependent and maximum production was recorded after 180 min of Ni2+ treatment. We can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of Ni2+ toxicity. Keywords: cell viability, grapevine, nickel trans-membrane electrical potential, oxidative stress, roots * Corresponding author, e-mail: jan.pavlovkin@savba.sk Introduction Among different environmental heavy-metal pollutants, nickel (Ni2+) has gained considerable attention in recent years, because of its rapidly increasing concentrations in soil, air, and water in different parts of the world. Most agri- cultural soils contain Ni2+ in average of 25 mg kg–1 soil dry weight (DW) but its content is often substantially increased up to 26,000 mg kg–1, by human activities such as mining, emission of smelters, coal and oil burning, sewage, phos- phate fertilizers and pesticides (Holmgren et al. 1993). Many plants that naturally grow on such contaminated soils contain Ni2+ in concentrations exceeding 1000 mg g–1 DW in their tissues (Gonnelli et al. 2001) but they generally pos- sess mechanisms allowing them to tolerate Ni2+ and to de- velop without phytotoxic problems (Gabbrielli et al. 1990). However, many of agriculturally important crops contain less than 5 μg of Ni g–1 DW, and the symptoms of phytotox- icity often become apparent at soil Ni2+ concentrations as low as 25–30 μg g–1 (Khalid and Tinsley 1980). Nickel is now considered an essential mineral nutrient (Brown et al. 1987, Seregin and Kozhevnikova 2006), which in low concentrations fulfi ls a variety of essential roles in plants. Therefore, Ni2+ defi ciency produces an array of ef- fects on growth and metabolism of plants, including re- duced growth, and induction of senescence, leaf chlorosis, alterations in nitrogen metabolism, and reduced iron uptake (Ahmad and Ashraf 2011). According to Bollard (1983) iron defi ciency could explain part of the symptoms induced by Ni2+. In addition, Ni2+ is a constituent of several metallo- enzymes such as urease (Brown et al. 1987). On the other hand excess of Ni2+ in the medium alters various physiolog- ical processes, resulting in detrimental effects on plants and causing diverse toxicity symptoms (Sharma and Dhiman 2013). Among these, iron defi ciency that leads to chlorosis and foliar necrosis and inhibition of nutrient absorption by roots have been widely found in different plant species (Pandey and Sharma 2002, Chen et al. 2009). Ouzounidou et al. (2006) working with wheat plants reported, that long exposure with 1 mM Ni2+ reduced iron content leading to iron and manganese defi ciency. Additionally, excess Ni2+ also can retard shoot and root growth, impair plant metabo- lism, inhibit photosynthesis and transpiration, and cause ul- trastructural modifi cations, which are well documented in the review by Sharma and Dhiman (2013). PAVLOVKIN J., FIALA R., ČIAMPOROVÁ M., MARTINKA M., REPKA V. 26 ACTA BOT. CROAT. 75 (1), 2016 Other symptoms observed in Ni2+-treated plants are re- lated to oxidative stress (Gajewska et al. 2006, Sharma and Dietz 2009). When generation of reactive oxygen species (ROS) such as superoxide and peroxide exceeds their re- moval, injuries may occur in the cells (Agrawal et al. 2013). The most common indicator of oxidative stress is lipid per- oxidation resulting in disturbances of membrane integrity and consequently its enhanced permeability (Lukatkin et al. 2013). Baccouch et al. (2006) and Hao et al. (2006) showed enhanced lipid peroxidation in Ni2+-treated wheat plants. By contrast, Ni2+ effects on wheat plants were not caused by lipid peroxidation (Gajewska et al. 2006). The changes in lipid and protein composition might disturb plasma mem- brane (PM) functions and, consequently the ion balance in the cytoplasm (Llamas et al. 2008). Several authors have shown that impairment of nutrient balance may result not only from the changes of plasmalemma lipid composition, but also that Ni2+ affected plasma membrane H+-ATPase ac- tivity (Morgutti et al. 1981). Llamas et al. (2008) and Sanz et al. (2009) reported that Ni induced a concentration-de- pendent PM depolarization in rice and barley but the activ- ity of the PM H+-ATPase was not affected. However, in the long term experiments a drastic loss of potassium was re- corded. The fi rst plant organ facing the elevated Ni2+ concentra- tions is the root system and especially the root cell plasma membrane being not only the site of Ni2+ entry but also a target of its toxic impact. The use of electrophysiological technique allowed us to record instant changes in electro- physiological parameters of individual epidermal cells of the adventitious grapevine roots during Ni2+ treatment. The trans-membrane electrical potential (EM) and permeability measurements have been supplemented with determination of superoxide and hydrogen peroxide generation as well as cell viability in the roots exposed to various Ni2+ concentra- tions for up to 24 h. Material and methods Plant material and growth conditions Grapevine (Vitis vinifera L., cv. Limberger) shoot cut- tings were taken from the production vineyards of the re- gion Rúbaň, Slovakia. After stratifi cation in cold room (4 °C) for one month, nodal explants (10 cm) with single axil- lary bud were used for hydroponic cultivation. The explants were grown in magenta jars fi lled to 60 mL with aerated half strong MS medium (Murashige and Skoog 1962) at 25 ± 1 °C under 14 h photoperiod. Ni2+ treatments Based on published research of other authors (Pandey and Sharma 2002, Llamas et al. 2008), and our preliminary experiments, the concentrations of NiCl2 that have caused the clear effects within short (48 h) treatments were chosen herein (1–5 mmol L–1). Although such concentrations are not common in vineyards soils, they were not lethal for Vitis root cells. Two-month old explants with adventitious roots were transferred to aerated solutions containing 0.1 mmol L–1 KCl and 0.1 mmol L–1 CaCl2, pH 5.8 supplemented with 0 (control), 1, 2 or 5 mmol L–1 NiCl2 for 24 h in the concen- tration-dependence experiments, and with 0 (control) and the highest concentration 5 mmol L–1 NiCl2 for 10, 30, 60 or 180 min in the time-dependence experiments. The roots were then stained for confocal microscope analyses as de- scribed later. Electrophysiology Measurements of trans-membrane electrical potential (EM) were performed on single epidermal cells within the root zone located 0.5–0.9 mm from the root tip of 20 mm long root segments. The root epidermal cells being in direct contact with the environment are more sensitive comparing to the internal root tissues as shown in our previous re- search on grapevine root cells exposed to cadmium (Fiala et al. 2015). The EM was measured using standard microelec- trode technique as described in detail by Kenderešová et al. (2012). After rinsing with 0.5 mmol L–1 CaSO4, the roots were mounted in a 5-mL volume Plexiglas chamber and constantly perfused (5 mL min–1) with bathing solution con- taining 0.1 mmol L–1 KCl and 0.1 mmol L–1 CaSO4. The initial maximum depolarization (ΔEM) induced by 0.1– 5 mmol L–1 Ni2+ concentrations was measured by addition of NiCl2 to the perfusion solution. Subsequently, the effect of short-term treatments with Ni2+ was registered after the cells attained the resting potential with an equimolar CaCl2 solution by exchanging CaCl2 with NiCl2 in the perfusion solution, to avoid the effect of the counterion. The EM of roots treated for several hours (up to 24 h) with Ni2+ was also measured using the same solution, containing 1 and 5 mmol L–1 Ni2+. Membrane permeability Changes in electrical conductivity of the solution sur- rounding the adventitious roots of 12 nodal explants were measured to assess the changes of membrane permeability caused by Ni2+ treatments. The 0.5 cm long apical segments of approximately 2 cm long roots (0.4–0.5 g) were trans- ferred to 0.5 mmol L–1 CaSO4 solution for 2 h in order to wash out the nutrient solution from the apoplast. After this time the segments were transferred into 10 mL of distilled water or 1 and 5 mmol L–1 NiCl2 and incubated in a shaking water bath in the dark at 25 °C. Effl ux of electrolytes from roots was determined within 48 h by conductivity meter OK-109-1 (Radelkis, Hungary). The conductivity was ex- pressed in μS cm–1 g–1 fresh weight (FW). The changes in conductivity were expressed as differences between the values of particular conductivity measured, and the initial conductivity. Confocal laser scanning microscopy Propidium iodide (PI, Fluka, Switzerland) was used to counterstain the cell wall and nuclei of ruptured cells. 2’, 7’-dichlorodihydrofl uorescein diacetate (H2DCFDA, Ser- va) was used as indicator for hydrogen peroxide accumula- tion in cells. To monitor real time superoxide production in the root tips we used superoxide detection kit (Enzo Life Sciences, USA). Apical 0.5 cm long root segments were NICKEL EFFECTS ON GRAPEVINE ROOTS ACTA BOT. CROAT. 75 (1), 2016 27 stained 2 min with 10 μg mL–1 PI in water, 15 min with 50 μmol L–1 H2DCFDA in 50 mmol L–1 phosphate buffer pH 7.5 or 15 min with superoxide staining solution (following the manufacturer’s manual), washed for 2 min in distilled water (for superoxide just briefl y) and observed in confocal microscope Olympus FV1000 (Olympus, Japan). PI and H2DCFDA were excited at 488 nm and fl uorescence was detected using emission barrier fi lters 560–660 nm for PI or 505–550 nm for H2DCFDA. The superoxide stain excita- tion wavelength was 543 nm and the emission was detected using 560–660 nm barrier fi lter. The confocal microscopy images represent at least three roots and were selected from at least three different images of each root. Fluorescence signal intensity (CTCF, corrected total cell fl uorescence) was measured and calculated with the open source analys- ing software Image-J2/Fiji (http://imagej.net/Fiji). Statistical analysis Each experiment was repeated at least three times. Data on EM and membrane conductivity were expressed as mean ± standard deviation (SD) with the number of samples (n). Data on cell viability, hydrogen peroxide accumulation and superoxide production were analysed using one-way ANO- VA with P < 0.05 (Prism 5, GraphPad Software Inc.). Means and standard deviations were calculated from three independent experiments (n = 10 apical root segments). As for confocal microscopy, only representative images are shown. Results Trans-membrane electrical potential Under control conditions the EM values of epidermal cells located within the distance of 0.5–0.9 mm from the root tip varied between –121 and –133 mV (–126 ± 5.9 mV, mean ± SD, n = 39). In short term experiments (up to 70 minutes) the application of different Ni2+ concentrations in- duced immediate changes in the EM values of root epider- mal cells (Fig. 1). Both ions, Ni2+ and Cl–, contributed equally to the EM depolarization. Thus, for adequate balanc- ing of high concentrations of Ni2+ and Cl–, the resting po- tential was fi rst measured at corresponding concentrations of CaCl2, which were then replaced by the same concentra- tions of NiCl2, with simultaneous presence of 0.1 mmol L–1 CaSO4. The Ni2+-induced rapid and transient depolarization was concentration-dependent (Fig. 2). The initial EM depo- larization induced with different Ni2+ concentrations repo- larized to the initial resting potential values within 50–70 min (Fig. 1). After transient depolarization, the Ni2+ applied at fi nal 1 mmol L–1 concentration caused a slow membrane hyper- polarization. Its magnitude reached the maximum value 8 h after the metal application and, in comparison with the val- ues of control cells it was more negative (by ∆mV 17.6 ± 3.3 mV, mean ± SD, n = 13). After withdrawal of Ni2+ from the perfusion solution the EM repolarized to the value of control cells. Compared to control, the EM was more nega- tive after 1 mmol L–1 Ni while there was no difference after 5 mmol L–1 Ni2+ treatment (Fig. 3). Fig. 1. Changes of the trans-membrane electrical potential (EM) induced by increasing Ni2+ concentrations in epidermal cells of grapevine adventitious roots. The time of Ni2+ application to the perfusion solution is indicated by arrow. Representative curves of individual cells (n = 2–4) are shown. Fig. 2. Initial trans-membrane electrical potential depolarization (ΔEM, mV) induced in epidermal cells of grapevine adventitious roots, by increasing concentrations of NiCl2 added to the perfusion solution bathing the roots. Results are shown as mean values ± standard deviations, n = 3. Fig. 3. Transmembrane potential difference (∆EM, mV) recorded in epidermal cells of grapevine adventitious roots treated with 1 and 5 mmol L–1 NiCl2 for 0–24 h. Results are shown as mean val- ues ± standard deviations, n = 3–8. PAVLOVKIN J., FIALA R., ČIAMPOROVÁ M., MARTINKA M., REPKA V. 28 ACTA BOT. CROAT. 75 (1), 2016 Membrane permeability The treatment of roots with 1.0 mmol L–1 Ni2+ concen- tration did not change the root cell membrane permeability. Only the exposure to the highest Ni2+ concentration 5 mmol L–1 for 24 and 48 h resulted in membrane permeability in- crease comparing to control roots (Fig. 4). Cell viability, superoxide, hydrogen peroxide In all experiments only a weak PI fl uorescence was lo- calized in the walls of the root tip cells indicating that the cells were viable, with intact cell membranes. Superoxide- specifi c staining did not reveal an increased orange fl uores- cence at any Ni2+ concentration or time used in the experi- ments (Figs. 5A, 6A). Only slightly elevated superoxide level occurred in the roots treated with 5 mmol L–1 Ni for 30 min (Fig. 6A). The dynamics of hydrogen peroxide ac- cumulation monitored in the grapevine root cells with H2D- CFDA showed concentration-dependence of the Ni-induced hydrogen peroxide formation as indicated by green cyto- plasmic staining (Figs. 5A, B). The time course of hydro- gen peroxide accumulation in grapevine root cells treated with 5 mmol L–1 Ni demonstrated that the highest hydrogen peroxide production occurred mainly after 180 min (Figs. 6A, B). Discussion Structural, physical and chemical properties of the PM itself as well as any effects of metal ions at the cell surfaces in general have an impact on transport processes. Altera- tions of the PM-H-ATPase activity can be assessed by Fig. 4. Time-course of electrolyte leakage, measured as electrical conductivity, from the segments of grapevine adventitious roots treated with 1 and 5 mmol L–1 NiCl2. Results are shown as mean values ± standard deviations, n = 3. Fig. 5. Concentration-dependent effects of Ni-treatment on cell vi- ability, superoxide and hydrogen peroxide accumulation in grape- vine 0.5 cm apical root segments demonstrated with confocal mi- croscope (A) and expressed using fl uorescence signal intensity (CTCF) value (B). Bar represents 1 mm. Different letters indicate signifi cant differences at 5% level. Results are shown as mean val- ues ± standard deviations, n = 3. Fig. 6. Time-dependent responses to 5 mmol L–1 NiCl2 of cell via- bility, superoxide and hydrogen peroxide accumulation in grape- vine 0.5 cm apical root segments demonstrated with confocal mi- croscope (A) and expressed using fl uorescence signal intensity (CTCF) value (B). Bar represents 1 mm. Different letters indicate signifi cant differences at 5% level. Results are shown as mean val- ues ± standard deviations, n = 3. NICKEL EFFECTS ON GRAPEVINE ROOTS ACTA BOT. CROAT. 75 (1), 2016 29 studying changes in the EM. According to our results, the effect of Ni on EM of grapevine adventitious root epidermal cells differed from that reported for the other divalent cat- ion, cadmium (Llamas et al. 2000, Pavlovkin et al. 2006, Fiala et al. 2015) and mercury (Repka et al. 2013). In short- term experiments, Ni induced rapid and concentration-de- pendent transient depolarization of the PM in the grapevine roots indicating its entry into the cells. But after this initial depolarization the EM of Ni-treated roots reached the values similar or slightly higher than those of the control in less than 70 min. Llamas et al. (2008) working with rice plants suggested that such effect may be due to a stimulation of H+ effl ux, as demonstrated for Ni in maize roots (Morgutti et al. 1981). According to these authors, the entry of Ni into the cells occurs downhill the electrochemical gradient by a mechanism of uniport, inducing an immediate H+ effl ux for charge compensation, followed by K+ effl ux. Their results may explain the more negative values of EM in comparison to the control, in the grapevine roots treated 24 h with 1 mmol L–1 Ni. In addition to the initial effect on the active component of EM, the effect of Ni2+ on the passive component of EM cannot be ruled out. There is evidence that the PM permea- bility alterations might be involved in plant heavy metal tolerance (Llamas et al. 2008) and its disruption can be a consequence of increased peroxidation of unsaturated fatty acids in the cell membranes (Lukatkin et al. 2013). Accord- ing to our results, no signifi cant changes of the membrane permeability comparing to controls were found in grape- vine roots treated with 0.5 mmol L–1 Ni2+ up to 24 h. How- ever, when the roots were treated with 5 mmol L–1 Ni2+, a progressive increase in membrane permeability was mea- sured after 16 h of treatment. Taken together, the changes induced by Ni2+ stress were not signifi cant in EM (Figs. 1–3), superoxide production or cell viability (Figs. 5B, 6B). However, hydrogen peroxide concentration signifi cantly increased in the adventitious roots of grapevine exposed to 1, 2, and 5 mmol L–1 Ni2+ (Figs. 5B, 6B). At the highest 5 mmol L–1 concentration of Ni2+ a signifi cant increase in membrane permeability oc- curred (Fig. 4), which could be responsible for the changes in water content, as was determined by Llamas et al. (2008). According to Gajewska et al. (2006), Ni2+ stress in roots is more related to the accumulation of hydrogen peroxide in root tissues than to enhanced lipid peroxidation. Further- more, the elevated levels of hydrogen peroxide may be a consequence of its inappropriate removal. In line with this statement, a signifi cant decrease of catalase activity was ob- served in Ni-treated wheat leaves (Gajewska and Sklodows- ka 2007). Our results suggest than Ni2+ does not directly af- fect plasma membrane ATPase and superoxide production. However, it increases plasma membrane permeability and hydrogen peroxide production, which can be related with Ni2+ toxicity in grapevine roots. Acknowledgement The work was supported by Slovak Grant Agency VEGA, Project 02/0023/13. References Agrawal, B., Czymmek, K. J., Sparks, D. L., Bais, H. P., 2013: Transient infl ux of nickel in root mitochondria modulates or- ganic acid and reactive oxygen species production in nickel hyperaccumulator Alyssum murale. Journal of Biological Chemistry 8, 7351–7362. Ahmad, M. S., Ashraf, M., 2011: Essential roles and hazardous effects of nickel in plants. Review of Environmental Contami- nation and Toxicology 214, 125–167. Baccouch, S., Chaoui, A., El Ferjani, E., 2006: Nickel toxicity in- duces oxidative damage in Zea mays roots. Journal of Plant Nutrition 24, 1085–1097. Bollard, E. G., 1983: Involvement of unusual elements in plant growth and nutrition. In: Läuchli, A., Bieleski R. L. (eds.), En- cyclopedia of plant physiology, New Series, Vol. 15B, Inor- ganic plant nutrition, Springer, pp. 695–744. Brown, P. H., Welch, R. M., Cary, E. E., 1987: Nickel: a micronu- trient essential for higher plants. Plant Physiology 85, 801– 803. Chen, C., Huang, D., Liu, J., 2009: Functions and toxicity of nick- el in plants: Recent advances and future prospects. Clean Soil, Air, Water 37, 304–313. Fiala, R., Repka, V., Čiamporová, M., Martinka, M., Pavlovkin, J., 2015: Early cadmium-induced effects on reactive oxygen spe- cies production, cell viability and membrane electrical poten- tial in grapevine roots. Vitis 54, 175–182. Gabbrielli, R., Pandolfi ni, T., Vergnano, O., Palandri, M. R., 1990: Comparison of two serpentine species with different nickel tolerance strategies. Plant and Soil 122, 271–277. Gonnelli, C., Galardi, F., Gabbrielli, R., 2001: Nickel and copper tolerance and toxicity in three Tuscan populations of Silene paradoxa. Physiologia Plantarum 113, 507–514. Gajewska, E., Slaba, M., Andrzejewska, R., Skłodowska, M., 2006: Nickel-induced inhibition of wheat root growth is relat- ed to H2O2 production, but not to lipid peroxidation. Plant Growth Regulation 49, 95–103. Gajewska, E., Skłodowska, M., 2007: Effect of nickel on ROS content and antioxidative enzyme activities in wheat leaves. BioMetals 20, 27–36. Hao, F., Wang, X., Chen, J., 2006: Involvement of plasma-mem- brane NADPH oxidase in nickel-induced oxidative stress in roots of wheat seedlings. Plant Science 170, 151–158. Holmgren, G. G. S., Meyer, M. W., Chaney, R. L., Daniels, R. B., 1993: Cadmium, lead, zinc, copper and nickel in agricultural soils of the United States of America. Journal of Environmen- tal Quality 22, 335–348. Khalid, B. Y., Tinsley, J., 1980: Some effects of nickel toxicity on rye grass. Plant and Soil 55, 139–144. Kenderešová, L., Staňová, A., Pavlovkin, J., Ďurišová, E., Nadu- binská, M., Čiamporová, M., Ovečka, M., 2012: Early Zn2+- induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidop- sis species. Annals of Botany 110, 445–449. Llamas, A., Ullrich, C. I., Sanz, A., 2000: Cd2+ effects on trans- membrane electrical potential difference, respiration and PAVLOVKIN J., FIALA R., ČIAMPOROVÁ M., MARTINKA M., REPKA V. 30 ACTA BOT. CROAT. 75 (1), 2016 membrane permeability of rice (Oryza sativa L) roots. Plant and Soil 219, 21–28. Llamas, A., Ullrich, C. I., Sanz, A., 2008: Ni2+ toxicity in rice: Ef- fect on membrane functionality and plant water content. Plant Physiology and Biochemistry 46, 905–910. Lukatkin, A. S., Kashtanova, N. N., Duchovskis, P., 2013: Chang- es in maize seedlings growth and membrane permeability un- der the effect of epibrassinolide and heavy metals. Russian Agricultural Sciences 39, 307–310. Morgutti, S., Ferrari-Bravo, P., Marre, M. T., Cocucci, S. M., 1981: Effects of Ni2+ on proton extrusion and related transport processes and on the transmembrane electrical potential in maize roots. Plant Science Letters 23, 123–128. Murashige, T., Skoog, F., 1962: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiolo- gia Plantarum 15, 473–97. Ouzounidou, G., Moustakas, M., Symeonidis, L., Karataglis, S., 2006: Response of wheat seedlings to Ni stress: Effects of supplemental calcium. Archives of Environmental and Con- tamination Toxicology 50, 346–352. Pandey, N., Sharma, C. P., 2002: Effect of heavy metals Co2+, Ni2+, and Cd2+ on growth and metabolism of cabbage. Plant Science 163, 753–758. Pavlovkin, J., Luxová, M., Mistríková, I., Mistrík, I., 2006: Short- and long-term effects of cadmium on transmembrane electric potential (EM) in maize roots. Biologia 61, 109–114. Repka, V., Fiala, R., Čarná, M., Pavlovkin, J., 2013: Membrane potential differences and viability of grapevine root cells treat- ed with HgCl2. Plant, Soil and Environment 59, 353–358. Sanz, A., Llamas, A., Ullrich, C. I., 2009: Distinctive effects of Cd and Ni on membrane functionality. Plant Signalling and Be- haviour 4, 980–982. Seregin, I. V. Kozhevnikova, A. D., 2006: Physiological role of nickel and its toxic effects on higher plants. Russian Journal of Plant Physiology 53, 257–277. Sharma, S. S., Dietz, K. J., 2009: The relationship between metal toxicity and cellular redox imbalance. Trends in Plant Science 14, 43–50. Sharma, A., Dhiman, A., 2013: Nickel and cadmium toxicity in plants. Journal of Pharmaceutical and Scientifi c Innovation 2, 20–24.