Research Article Aluminum Induces Oxidative Burst, Cell Wall NADH Peroxidase Activity, and DNA Damage in Root Cells of Allium cepa L. V. Mohan M. Achary, 1 Narasimham L. Parinandi, 2 and Brahma B. Panda 1 * 1 Department of Botany, Molecular Biology and Genomics Laboratory, Berhampur University, Berhampur, India 2 Davis Heart and Lung Research Institute, Ohio State University College of Medicine, Columbus, Ohio Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O 2 2 , H 2 O 2 ,  OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activa- tion of cell wall-NADH peroxidase (NADH-PX) and/ or plasma membrane-associated NADPH oxidase (NADPH-OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al 31 (800 lM of AlCl 3 ) for 3 or 6 hr without or with the pretreatment of inhibitors spe- cific to NADH-PX and NADPH-OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall-bound protein (CWP) fractions extracted from the untreated control and the Al-treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which meas- ured the extent of activation of peroxidase/oxidase, generation of  OH, and DNA damage. Overall, the present study demonstrates that the cell wall-bound NADH-PX contributes to the Al-induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH-PX in the stress response. Altogether, this study under- scores the crucial function of the cell wall-bound NADH-PX in the oxidative burst-mediated cell death and DNA damage in plants under Al stress. Environ. Mol. Mutagen. 53:550–560, 2012. V V C 2012 Wiley Periodicals, Inc. Key words: abiotic stress; ROS; NADH peroxidase; cell death; DNA damage INTRODUCTION Oxidative burst, a rapid and transient production of ele- vated levels of reactive oxygen species (ROS), including the superoxide anion (O 2 2 ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (  OH), has been recognized as the most common physiological phenomenon associated with plant responses to a variety of environmental, abi- otic, and biotic stresses, such as drought, temperature, salinity, UV, ionizing radiation, metals, ozone, wounding, and infection [Bolwell et al., 1995; Torres and Dangl, 2005; Gill and Tuteja, 2010]. Enzymes, such as the cell wall-bound NADH peroxidase (NADH-PX) and plasma membrane-localized NADPH oxidase (NADPH-OX), have been shown to be main contributors to the oxidative burst in plants [Apel and Hirt, 2004]. ROS arising from such an enzymatically induced oxidative burst have been estab- lished to play crucial roles in plant development and defense mechanisms [Liszkay et al., 2004; Gapper and Dolan, 2006; Swanson and Gilroy, 2010]. An overwhelm- ing production of ROS is known to cause DNA damage that leads to genotoxic stress and genomic instability [Roldan-Arjona and Afriza, 2009; Balestrazzi et al., 2011]. The relative involvement of the two oxidases in the oxidative burst phenomenon has been determined in a few plant species by employing inhibitors specific to NADH-PX, such as salicylhydroxamic acid (SHAM), sodium azide (NaN 3 ), and potassium cyanide (KCN), and to NADPH-OX, including diphenyleneiodonium chloride (DPI) and imidazole (IMZ). Studies indicate that NADH- PX is involved in the generation of ROS (O 2 2 ,H 2 O 2 , and  OH) in response to biotic or abiotic stress in Pisum sati- *Correspondence to: Brahma B. Panda, Department of Botany, Molecu- lar Biology and Genomics Laboratory, Berhampur University, Berham- pur 760007, India. E-mail: panda.brahma@gmail.com Grant sponsor: Senior Research Fellowship from CSIR, New Delhi (to V.M.M.A.). Received 17 March 2012; provisionally accepted 28 June 2012; and in final form 28 June 2012 DOI 10.1002/em.21719 Published online 31 July 2012 in Wiley Online Library (wileyonlinelibrary.com). V V C 2012 Wiley Periodicals, Inc. Environmental and Molecular Mutagenesis 53:550^560 (2012)