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Journal of Stress Physiology & Biochemistry, Vol. 9 No. 1 2013, pp. 66-73 ISSN 1997-0838 Original Text Copyright © 2013 by Hosseini and Poorakbar
ORIGINAL ARTICLE
Zinc toxicity on antioxidative response in (Zea mays L.) at two different pH
Zahra Hosseini and Latifeh Poorakbar*
Department of Biology, Faculty of Sciences, Urmia University, Iran
Tel.: +989144400987; fax: +98 (441) 2776707 *E-Mail: l.pourakbar@urmia.ac.ir
Received September 22, 2012
Zn is the second most abundant transition metal after iron (Fe). Excess Zn can have negative effects on plants. The effect of Zn at two different pH on lipid peroxidation (MDA), membrane permeability (EC), hydrogen peroxide (H2O2), non-protein thiols (NPT) and the activities of major antioxidant enzymes Zea mays were investigated under controlled growth conditions. Zn-excess conditions increased the EC, MDA, H2O2 content and non-protein thiols and also activities of antioxidant enzymes were increased. Also zinc toxicity was higher in 4.5 pH than
7.5 pH.
Key words: Zinc, pH, Antioxidant enzymes, MDA, H2O2, EC, NPT
ORIGINAL ARTICLE
Zinc toxicity on antioxidative response in (Zea mays L.) at two different pH
Zahra Hosseini and Latifeh Poorakbar*
Department of Biology, Faculty of Sciences, Urmia University, Iran
Tel.: +989144400987; fax: +98 (441) 2776707 *E-Mail: l.pourakbar@urmia.ac.ir
Received September 22, 2012
Zn is the second most abundant transition metal after iron (Fe). Excess Zn can have negative effects on plants. The effect of Zn at two different pH on lipid peroxidation (MDA), membrane permeability (EC), hydrogen peroxide (H2O2), non-protein thiols (NPT) and the activities of major antioxidant enzymes Zea mays were investigated under controlled growth conditions. Zn-excess conditions increased the EC, MDA, H2O2 content and non-protein thiols and also activities of antioxidant enzymes were increased. Also zinc toxicity was higher in 4.5 pH than
7.5 pH.
Key words: Zinc, pH, Antioxidant enzymes, MDA, H2O2, EC, NPT
Soil pollution by toxic metals is a serious problem for the environment and is also one of the environmental stresses for higher plants. Evidence has suggested that heavy metal toxicity is manifested as oxidative stress, which is caused by the stimulation of free oxygen radical production (Gao et al., 2011). Zinc is a heavy metal. It acts as a plant nutrient and plays an important role in many metabolic reactions but it is toxic at higher concentrations and causes growth inhibition (Vaillant et al., 2005) and induction of oxidative damage in various plant species (Panda et al., 2003). At the cellular level, excess Zn can significantly alter mitotic activity (Rout and Das,
2003), affect membrane integrity and permeability (Stoyanova and Doncheva, 2002), and even kill cells (Chang et al., 2005). Various studies indicated that the availability of elements for leaching and plant uptake depend on their solubility in the soil solution (Gao et al., 2010). As a result of the strong influence of pH on metal solubility (McBride et al., 1997), anthropogenic processes which result in the lowering of substrate pH can cause metal toxicities, even if no extra metal has been added to the system (Robinson et al., 1995). The goals of the recent study were to investigate the effect of zinc toxicity on antioxidative response and role of pH in toxicity on Zea mays plants.
MATERIALS AND METHODS
Seeds were surface sterilized in 5% sodium hypochlorite solution for 10 minutes before use, to avoid fungal contamination. Seeds were then washed thoroughly with deionized, imbibed overnight in distilled water and germinated on moistened filter paper in trays for 5 days in darkness at 25°C. After 5 days, uniformly germinated seedlings were transferred to plastic cups. The plants were irrigated over a period 10 days with nutrient solution containing zinc between 0, 400 and 600 nM as sulfate (ZnSO4) and two pH
4.5 and 7.5. After 12 days of Zn treatment, 9 plants from each group (control, Zn 400-pH4.5, Zn 400-pH7.5, Zn 600-pH4.5, Zn 600-pH7.5) were taken and divided into shoot and root fractions and analyzed.
Concentration of H2O2 was determined following Jana and Choudhuri (1981). Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) content according Heath and Packer (1968). Ascorbate peroxidase activity was measured according to
Nakano and Asada (1981) by monitoring the rate of
ascorbate oxidation at 290 nm. Catalase activity was assayed according to Aebi (1983). Guaiacol peroxidase activity was measured according to
Upadhyaya et al (1985). GR Activity was assayed
according to Foyer (1976). Percentage electrolyte leakage was assayed according to Lutts et al (1996). Non-protein thiols were measured with Edreva et al (1984).
Statistical analysis
Results were based on three replicates and statistical analysis was performed using ANOVA (for P < 0.05). Based on the ANOVA results, a Tukey test for mean comparison was performed, for a 95% confidential level, to test for significant differences among treatments.
RESULTS
In the Zea mays, lipid peroxidation, which is measured as Malondialdehyde (MDA) content, increased at all Zn treatments than control and maximum increase was observed at 600 ^M (pH
4.5) (Fig. 1, A and B). H2O2 content increased at all Zn treatments than those in the control while the maximum increase was observed at 600 ^M (pH
7.5). But no significant difference was observed between treatments and control (Fig. 1, C and D). Effect of zinc on APX and GR activities is shown in Fig. 2. APX and GR activity increased in all Zn treatments in both shoot and root compared with control. The highest value was occurred in the 600 HM (pH 4.5) Zn treatment. APX and GR activity were higher in 600 ^M than 400 ^M concentration of zinc. But no significant difference was abserved between pH 7.5 and pH 4.5 in APX activity while GR activity significantly was higher at pH 4.5 than pH
7.5 in both 400 ^M and 600 ^M concentration. CAT and GPX activity increased in all Zn treatments in both shoot and root compared with control (Fig. 3) and maximum increase was observed at 600 ^M (pH 4.5) treatment of zinc. CAT activity significantly was higher at pH 4.5 than pH 7.5 in shoot and there was significant difference between pH 7.5 and pH
4.5 of 600 ^M concentration in root. GPX activity significantly was higher at pH 4.5 than pH7.5 in 600 HM concentration in root. But no significant difference was abserved between pH 7.5 and pH
4.5 in shoot.
Non-protein thiol compounds are involved in heavy metal detoxification. Non-protein thiols increased in all Zn treatments than those in the control and this increase was higher in 600 ^M than 400 ^M of zinc (Fig. 4). Also Non-protein thiol compounds were significantly higher in pH 4.5 than pH 7.5 in shoot. Electrolyte leakage (Fig. 4, C)
reflected the plasmalemma damage caused by the Zn. Electrolyte leakage percentage in all Zn treatments increased as compared to the control.
But no significant difference was abserved between Zn 400 ^M and Zn 600 nM also between pH 7.5 and pH 4.5.
Figure 1. The effects of Zinc on Malondialdehyde content in shoot (A) and root (B) and H2O2 content in shoot (C) and root (D) of Zea mays. Values are means of three replicates ± S.E.
Figure 2. The effects of Zinc on APX activities in shoot (A) and root (B) and GR activities in shoot (C) and root (D) of Zea mays. Values are means of three replicates ± S.E.
Figure 3. The effects of Zinc on CAT activities in shoot (A) and root (B) and GPX activities in shoot (C) and root (D) of Zea mays. Values are means of three replicates ± S.E.
C
Figure 4. The content of non-protein -SH (NTP) in shoot (A) and root (B) and ELP (C) of Zea mays under Zn treatment. Values are means of three replicates ± S.E.
DISCUSSION
Heavy metal accumulation in plant lead to some physiological and biochemical changes. Production of free radicals in plants under heavy metal stress has been documented (McGeer et al., 2000). Like other metals zinc also induces free radical generation in plants (Rout and Das, 2003). During present study zinc stimulated MDA production in Zea mays. As a product of membrane lipid peroxidation, MDA is used to assess the extent of oxidative stress in plants, and its level is increased under stress conditions (Liu et al., 2009). In plants, heavy metals induce oxidative stress by generation of superoxide radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO^), and singlet oxygen (1O2), collectively termed ROS (Jain et al., 2010). These ROS are highly reactive and cause the death of plants by damaging membrane lipids, proteins, pigments and nucleic acids. To cope up with the damages caused by the ROS, cells possess their own comprehensive and integrated endogenous antioxidant defense system composed of both enzymatic as well as non-enzymatic components (Miller et al., 2008). Similar to our results an increase in MDA and H2O2 content under excess zinc has been reported in sugarcane (Jain et al., 2010). In the present study increased electrolyte leakage under zinc stress. Generation of free radicals and increased lipid peroxidation under zinc stress may have resulted in an increase in membrane permeability and loss of membrane integrity (Rout and Das, 2003). Jain et al. (2010) reported that elevated levels of H2O2 and O2^- caused by stress facilitate the formation of highly active hydroxyl radicals (OH^). These hydroxyl radicals are generally considered to be the most likely ROS to initiate the peroxidation destruction of lipids that lead to membrane damage. Non-protein thiols, including
glutathione, thiol-rich peptides and other SH compounds, fulfill an important role in the detoxification of heavy metals in plants (Tiryakioglu et al., 2006). In our study, an increase in the level of non-protein thiols observed with the increase Zn. Similarly, a marked increase was seen in nonprotein thiols as a response to different metal stress in a number of plants (Tiryakioglu et al., 2006). The induction of certain enzymes that detoxify ROS is considered to play an important role in the defense against oxidative stress caused by toxic metal concentrations (Miller et al., 2008). In plants, a number of enzymes regulate H2O2 intracellular levels, but CAT, APX and GPX are considered the most important (Liu et al., 2009). These enzymes work independently in different parts of the plants to break up H2O2. The results of the present study show that the plants treated with Zn had high GPX (Fig. 3), CAT (Fig. 3), APX (Fig. 2) and GR activity (Fig. 2), indicating that there was efficient ROS scavenging activity in the system. Previous studies have also shown that induction of GPX activity has been reported in many plant species, when exposed to zinc stress (McGeer et al., 2000).
The results also showed that at the low pH zinc toxicity higher than that at high pH in Zea mays because MDA content, electrolyte leakage, nonprotein thiol and antioxidant enzymes activity was higher at pH 4.5 in comparison with that at pH 7.5. High toxicity in low pH might be the resulting in enhance zinc bioavailability in Zea mays. Lowering pH increased easily available Zn concentrations and enhanced metal uptake. Zn deficiency is a common problem in plants grown in high pH, calcareous soils (Cakmak et al. 1996), whereas in low pH soils Zn availability is generally high (Chaney, 1993). Autumn et al. (2006), reported that lowering pH is
an effective method to enhance metal
bioavailability and Thlaspi caerulescens uptake for both Cd and Zn. The most obvious method for reducing plant Zn toxicity is to raise soil pH. Havlin and co-authors (2005) reported that available Zn and Mn concentrations decreased with raising the soil pH.
In conclusion, our results have demonstrated Zn phytotoxicity in Zea mays. Higher H2O2, MDA contents and increased activity of antioxidant
enzymes in Zea mays indicating oxidative damage by zinc toxicity. Also at the low pH zinc toxicity was higher in Zea mays.
REFRENCES
Aebi, H.E. (1983) Methods of Enzymatic Analysis Verlag Chemic: Deer_eld Beach, FL, 3rd ed. 3, pp. 273-286 .
Autumn, S., Wang, J., Scott, A., Rufus, L., Chaney Thiery, A., Dolorme and Ronger, D. (2006) Reeves soil pH effect on uptake of Cd and Zn by
Thlaspi caerulescens. Plant and soil., 281, 325-
337.
Cakmak, I., Yilmaz, A., Ekiz, H., Torun, B., Erenoglu, B. and Braun, H.J. (1996) Zinc deficiency as a critical nutritional problem in wheat production in Central Anatolia. Plant and Soil., 180, 165-172.
Chang, H.B., Lin, C.W. and Huang, H.J. (2005) Zinc-induced cell death in rice (Oryza sativa L.) roots. Plant Growth Regul., 46, 261-266. Chaney, R.L. (1993) Zinc phytotoxicity. In: Robson A.D. (Ed.), Zinc in soil and plants. Kluwer Academic Publishers, Dordrecht, the Netherlands: 135-150.
Edreva, A. and Hadjiiska, E. (1984) About the determination of sulfhydril(thiol) group
content in plant material. Plant Physiol. (Bulg), 10, 73-83.
Foyer, C.H. and Halliwell, B. (1976) The Fresence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta., 133, 21-25.
Gao, Y., Miao, C.Y., Xia, J., Mao, L., Wang, Y.F. and Zhou, P. (2011) Effects of coexisting plant species on soil enzyme activities and microbial community structure in multiple heavy metal contaminated soils: PCR-RAPD method. Front Environ Sci Eng China.
Gao, Y., Zhou, P., Mao, L., Shi, W.J. and Zhi, Y.E. (2010) Phytoextraction of Cd and physiological changes in Solanum nigrum as a novel
hyperaccumulator. Russ J Plant Physiol., 57(4), 538-546.
Havlin, J.L., Beaton, J.D., Tisdale, S.L. and Nelson, W.L. (2005) Soil fertility and fertilizer: An
Introduction to Nutrient Management. Upper Saddle River, Newjersey. 515 pp.
Heath, R.L. and Pacher, L. (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and
stoichiometery of fatty acid peroxidation. Archives of Biochemistry and Biophysics., 125, 189-198.
Jana, S. and Choudhuri, M.A. (1981) Glycolate metabolism of three submerged aquatic
anagiosperms during aging. Aquatic Botany., 12, 345-354.
Jain, R., Srivastava, S., Solomon, S., Shrivastava, A.K., Chandra, A. (2010) Impact of excess zinc on growth parameters, cell division, nutrient accumulation, photosynthetic pigments and oxidative stress of sugarcane (Saccharumspp.). Acta Physiol. Plant., 32, 979-986.
Liu, Z.J., Zhang, X.L., Bai, J.G., Suo, B.X., Xu, P.L. and
Wang, L. (2009) Exogenous paraquat changes antioxidant enzyme activities and lipid peroxidation in droughtstressed cucumber leaves. Sci. Hortic., 121, 138-143.
Lutts, S., Kinet, J.M., and Bouharmont, J. (1996) NaCl-induced senescence in leaves of rice (Oriza sativa L.) cultivar differing in salinity resistance. Annal of Botany., 78, 389-398.
McBride, M., Sauve, S. and Hendershot, W. (1997) Solubility control of Cu, Zn, Cd and Pb in contaminated soils.European Journal of Soil Science., 48, 337-346.
McGeer, J.C., Szebedinszky, C., McDonald, D.G. and Wood, C.M. (2000) Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout. 1: Lono-regulatory disturbance and metabolic costs. Aqua. Toxicol., 50, 231243.
Miller, G., Shulaev, V. and Mitter, R. (2008) Reactive oxygen signaling and abiotic stress. Physiol. Plant., 133, 481-489.
Nakano, Y. and Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol., 22, 867-880.
Panda, S.K., Chaudhury, I. and Khan, M.H. (2003) Heavy metals induced lipid peroxidation and effect antioxidants in wheat leaves. Biol.
Plant., 46, 289-294.
Rout, G.R. and Das, P. (2003) Effect of metal toxicity on plant growth and metabolism: I. Zinc. Agronomie., 23, 3-11.
Robinson, B., Malfroy, H., Chartres, C., Helyar, K. and Ayers, G. (1995) The sensitivity of ecosystems to acid inputs in the Hunter Valley. Australia. Water, Air and Soil Pollution., 85, 1721-1726.
Stoyanova, Z. and Doncheva, S. (2002) The effect of zinc supply and succinate treatment on plant growth and mineral uptake in pea plant. Braz. J. Plant Physiol., 14, 111-116.
Tiryakioglu, M., Eker, S., Ozkutlu, F., Husted, S. and Cakmak, I. (2006) Antioxidant defence system and cadmium uptake in barley genotypes differing in cadmium tolerance. J Trace Elem Med Biol., 20(3), 181-189.
Updhyaya, A., Sankhla, D., Davis, T.D., Sankhla, N. and Smidth, B.N. (1985) Effect of paclobutrazol on the activies of some enzymes of activated oxygen metabolism and lipid peroxidation in senescing soybean leaves. Plant Physiol., 121, 453-461.
Vaillant, N., Monnet, F., Hitmi, A., Sallanon, H. and Coudret, A. (2005) Comparative study of responses in four Datura species to a zinc stress. Chemosphere., 59, 1005-1013.
