Pak. J. Bot., 43(3): 1521-1525, 2011. PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF THE LEAVES OF VERBASCUM WIEDEMANNIANUM FISCH. & MEY. TO CADMIUM YASEMİN OZDENER* AND H. GÜRAY KUTBAY 1 * *Department of Biology, Faculty of Sciences and Arts, University of Ondokuz Mayıs, 55139, Kurupelit-Samsun, Turkey Telephone :90 362 312 19 19-5498; Fax: 90 362 457 60 81 1 Corresponding author: H.Güray Kutbay; Corresponding author e-mail: guraykutbay@gmail.com Abstract The effect of cadmium (Cd) on the root and seedling lengths, chlorophyll a, b and proline content, lipid peroxidation and peroxidase activity of Verbascum wiedemannianum has been examined. Plants grown in Van Waes-Deberg culture medium were treated with 0.00 (control), 0.01 mM and 0.025 mM cadmium root and shoot growth was remarkably decreased against 0.01 and 0.025 mM Cd as compared to control group. Malondialdehyde (MDA) content, which is an index of lipid peroxidation, and guaiacol peroxidase activity (POD) increased approximately as twice as control group in leaves of plants treated with 0.025 mM Cd. Although chlorophyll a and b contents were decreased in response to the increasing of Cd concentration but statistically significant differences were not found in this respect. However, proline content was significantly increased in response to increasing Cd. Introductıon Metals in the environment may present a more insidious problem than organic chemicals because they cannot be degraded to innocuous products, such as carbon dioxide and water. Because metals are transported very well by the atmosphere, many urban areas have been loaded with considerable amounts of toxic metals from point and non-point sources due to human activity (Fargašová, 1998; Mishra & Choudhury, 1999). It is a known fact that the widespread accumulation of metals in the environment is increasingly becoming a problem for organisms of every kind. Metals are continuously released into the biosphere by volcanoes, natural weathering of rocks, and by industrial activities such as mining, the combustion of fossil fuels and the release of sewage. Therefore, they present a risk for primary and secondary consumers and ultimately humans (Munzuroglu & Geckil, 2002). Cadmium (Cd) is of particular concern to human health as it can be readily absorbed by roots and be concentrated by many cereals, potatoes, vegetables, and fruits. Elevated levels of Cd generally inhibit seed germination, cell growth as well as whole plant growth, nutrient uptake, distribution and photosynthesis. The photosynthetic process has been reported to be very sensitive to Cd. Experiments have shown the effect of Cd on stomatal function, on chlorophyll biosynthesis, on electron transport, and on the Calvin cycle as well as on the ultrastructure of chloroplasts (Catak et al., 2000; Oncel et al., 2000; Zhang et al., 2003; Iqbal & Shazia, 2003; Jeliazkova & Cracker, 2003; Gür et al., 2004; Wang & Zhou, 2005; Farooqi et al., 2009). Cd has been found to generate free radicals that may damage plant tissues. Reactive oxygen species (ROS) are a main part of free radicals. They can lead to oxidative stress. ROS cause lipid peroxidation, membrane damage and inactivation of enzymes (Sanitá di Toppi & Gabrielli, 1999; Skórzyńska-Polit et al., 2003/2004; Zhang et al., 2003; Monterio et al., 2009). In cellular level, lipid peroxidation is the most significant damage caused by ROS. The MDA (product of lipid peroxidation) level is regarded as a biochemical marker for injury mediated by ROS (Palma et al., 2002; Verma & Dubey, 2003; Sinha et al., 2005; Monterio et al., 2009). Many environmental stresses have been reported to increase the level of proline in plants, such as heavy metals, temperature and drought (Saradhi & Saradhi, 1991, Rai et al., 2004; Claussen, 2005). Hayat et al., (2007) determined that proline level increased the physiological drought stress generated by cadmium. Also the similar results were found by Dhir et al., (2004) and the levels of proline of leaves of Brassica juncea increased with increasing Cd concentration. Peroxidases are known to play a significant role in oxidative stress conditions and it has been shown that peroxidase activity can be used as a potential biomarker for sublethal metal toxicity in plants (Radotic et al., 2000; Sinha et al., 2005). Since peroxidase activity is related to ROS formation, toxic heavy metals cause stimulation in activity of peroxidase (Stoeva et al., 2005; Ganesh et al., 2008). Verbascum wiedemannianum is an endemic and medicinal plant distributed in Turkey. This species is different from the other Verbascum species with its red-dark purple flowers and it has been clasified as a threatened species (Ekim et al., 2000). It was known that the various parts of V. wiedemannianum (leaves, stem, flowers etc.) had antioxidative and antimicrobial activity (Tepe et al., 2006). This endemic species has useful properties. This study is aimed to determine biochemical changes on leaves of V. wiedemannianum after exposure to Cd which is one of the most toxic heavy metals. In our study, the effect of cadmium (Cd) root length and seedling growth, lipid peroxidation, GPOD (guaiacol peroxidase) activity, contents of proline, chlorophyll a and b leaves of V. wiedemannianum have been examined. It was determined that how this parameter changed due to concentration of the heavy metal. Materıals and Methods The seeds of V. wiedemannianum were collected from Çorum- Turkey. Seeds were air dried and stored at room temperature. They were sterilized in a 1.5% NaOCl for 30 min., then washed three times with sterilized ddH 2 O and sown in 9.5cm Petri dishes on ½ strength Van Waes-Deberg culture media (Van Waes & Debergh, 1986) supplemented with 10 g L -1 sucrose, 7% agar and with or without 0.00; 0.01; 0.025 mM Cd. Metal solutions were added to the medium after autoclaving in sterilised condition. Cd concentration was determined as a result of pre-treatment studies. Plant growth was completely inhibited by using >0.025 mM Cd. Cultures were incubated in a growth chamber at 20 ± 2ºC under full dark condition. Germinated seeds (with 3-5 mm radicle) on control medium were transferred to media containing no metal and Cd at different concentrations. The Petri dishes were incubated in 16:8 h (light: dark) photoperiod and at 20 ± 2ºC. Length of root and seedling and parameters of physiological and biochemical were measured after 20 days from incubation. Fresh and dried weight of leaves and roots of 25 seedlings was measured.