Vitis 54, 175–182 (2015) Early cadmium-induced effects on reactive oxygen species production, cell viability and membrane electrical potential in grapevine roots R. Fiala 1) , V. Repka 1) , M. ČiaMporová 1) , M. MaRtinka 1), 2) and J. pavlovkin 1) 1) Department of Plant Physiology, Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovak Republic 2) Department of Plant Physiology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic Summary Cadmium (Cd) is one of the most worldwide con- cerned metal pollutants. It is able to induce reactive ox- ygen species production through indirect mechanisms causing oxidative stress. Vitis vinifera roots were treat- ed with 100 μM Cd for 0-180 min or 20-100 μM Cd for 24 h. Fluorescence confocal microscopy showed elevat- ed hydrogen peroxide and superoxide levels in the api- cal root segments. Two phases (after 30 min and 24 h) of the superoxide raised levels were observed. This was accompanied by the decrease in root cell viability. Cd in concentrations between 0.005-10 mM induced sig- nifcant, but different changes in membrane electrical potential (E M ) of the root epidermal cells. The low con- centrations of Cd (0.005-0.01 mM) caused transient E M hyperpolarization followed by depolarization, whereas by higher concentrations (0.05-5.0 mM) E M was depo- larized. In any case, the depolarization or hyperpolari- zation were only transient up to 5 mM Cd concentra- tion indicating that the plasma membrane function was not irreversibly destroyed. Hyperpolarization of E M induced by fusicoccin (FC) was completely suppressed only in the presence of 10 mM Cd pointing to the in- hibition of H + -ATPase. The results suggest that the Cd interactions, depending on cellular development, result in activation of a complex of various mechanisms such as peroxide and hydrogen peroxide production, which in turn may be a more probable reason for the root cell responses to Cd toxicity than the transient E M changes. K e y w o r d s : grapevine; 'Limberger'; cadmium; reactive oxygen species; cell viability; membrane electric potential. Introduction Cadmium, although a non-essential element, can be accumulated by plants from mineral fertilizers, pesticides, sewage sludge or as an environmental pollutant resulting from various industries, e.g. mining and smelting (nazaR et al. 2012). Cd is considered to be highly toxic (pál et al. 2006), easily taken up by plants (Lee et al. 1998), then en- tering the food chain and resulting in serious health issues for animals and humans (Horiguchi et al. 2010). In plants Cd can induce complex changes at the genetic, biochemi- cal and physiological levels (gallego et al. 2012) leading to a number of phytotoxic effects including inhibition of plant growth and development, chlorosis, necrosis, reduc- tion of chlorophyll content and photosynthetic rate, imbal- ance in mineral nutrient uptake and affection of enzymatic activities (TRan and Popova 2013). Rupp et al. (1985) found transport of iron from roots to leaves to be nearly completely inhibited by Cd (1-10 ppm), forcing the iron accumulation in roots. Cd thus may cause iron defciency chlorosis in grapevines. Vitis vinifera cell suspension cul- tures grown in the presence of 1 mM CdCl 2 were found to have increased (in a time and concentration dependent manner) contents of α‑tocopherol, which is the major com- pound of vitamin E found in leaf chloroplasts and may be involved in Cd tolerance and hyperaccumulation (cetin et al. 2014). When released in plant environment, Cd is predomi- nantly accumulated by plant roots. Yuan-peng et al. (2012) showed that almost all of the absorbed Cd remains in the roots of grapevine, with a majority accumulated in the cell wall fraction. Du et al. (2012) showed in grapevine that absorbed Cd was mostly distributed to underground organs (roots and rhizomes) or below a graft position. It is well established that Cd induces reactive oxygen species (ROS) generation in many plant species. Briefy, ROS such as superoxide anion (O·̄ 2 ) and hydrogen perox- ide (H 2 O 2 ), were initially recognized as toxic by-products of aerobic metabolism (particularly formed in the electron transport chain in mitochondria), removed by means of antioxidants and antioxidative enzymes (roMero-puertas et al. 2007, paraDiso et al. 2008). It is known that various environmental stress stimuli induce excess ROS production causing widespread damage to cells. On the other hand, O·̄ 2 plays a role in the immune system (guzik et al. 2003). H 2 O 2 has important roles as a signalling molecule involved in regulating a variety of biological processes such as root growth, programmed cell death and response to biotic and abiotic environmental stimuli in plants (Veal et al. 2007, Dat et al. 2000). The controlled, harmless accumulation of specifc ROS, like H 2 O 2 , acts as a cellular signal for the onset of grapevine berry ripening (pilati et al. 2014). H 2 O 2 is mainly detoxifed by catalase in glyoxysomes and per- oxisomes and by ascorbate peroxidase in chloroplasts, mi- tochondria and peroxisomes (shigeoka et al. 2002). When Correspondence to: Dr. J. pavlovkin, Department of Plant Physiology, Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, 84523 Bratislava, Slovakia. E‑mail: jan.pavlovkin@savba.sk © The author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative-commons.org/licenses/by-sa/4.0/). DOI: http://dx.doi.org/10.5073/vitis.2015.54.175-182