1. Pretreatment with H 2 O 2 alleviates aluminum-induced oxidative stress in wheat seedlings Fang Jie Xu 1, Chong Wei Jin 1, Wen Jing Liu 2, Yong Song Zhang 2, Xian Yong Lin 1,2* 1 Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, 2 Zhejiang Key Laboratory of Subtropical Soil Science and Plant Nutrition, College of Environmental & Resources Science, Zhejiang University, Hangzhou 310029, China Hydrogen peroxide (H 2 O 2 ) is a normal reactive oxygen species (ROS) molecule which not only acts as a main cause of oxidative stress, but also involved in signal transduction pathways leading to activation of plant defense against biotic and abiotic stresses. There is growing evidence showed that pretreatment of H 2 O 2 could alleviated the deleterious effects of various stress conditions on plant growth and triggering higher stress tolerance in many plant species. However, to the best of our knowledge, it is still little information about the role of pre-application of H 2 O 2 in activating Al acclimation and its relation to Al tolerant acquisition is still unclear. The present study was aimed at elucidating the effect of H 2 O 2 pretreatment on Al-induced antioxidant responses, including IRE, ROS overproduction, lipid peroxidation, cell death and antioxidant activity increases in two wheat genotypes differing in Al tolerance. This research was financially supported by the National Basic Research Program of China (973 Program, No. 2007CB109305), National Natural Science Foundation of China (grant Nos. 30771292, 30900920, 30270784), the Foundation for University Ph. D. Granting Discipline of the Ministry of Education (grant No. 20060335014) and IPNI. * Corresponding author: xylin@zju.edu.cn The pretreatment of H 2 O 2 at appropriate time and concentration led to a pronounced alleviation of Al- induced deleterious effects in both wheat genotype during the subsequent Al exposure. Al sensitivity is an important factor affecting the function of H 2 O 2 pretreatment in improving Al acclimation. The H 2 O 2 -pretreated increase in Al tolerance is greater in the Al-sensitive wheat genotype (Yangmai-5) as compared to the Al-tolerant wheat genotype (Jian-864). The pre-application of H 2 O 2 may induce a stronger antioxidant capacity involving higher levels of enzymatic and non-enzymatic antioxidants in root tips of wheat seedlings during subsequent Al exposure, thereby reducing endogenous ROS accumulation and improving Al acclimation of the wheat plants. Therefore, it could be demonstrated that the increased antioxidant defense system level induced by exogenously applied H 2 O 2 acts as the primary factor regulating Al acclimation process in wheat plants. INTRODUCTION RESULTS CONCLUSIONS Acknowledgement Figure 1. Relative root elongation of Yangmai-5 and Jian-864 exposed to various concentrations of AlCl 3 for 24 h (A) or exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment (B). * indicates significant difference at P < 0.05 and ** indicates a significant difference at P<0.01 between two treatments at each Al concentration. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=20). Figure 2. MDA concentration (A) and Evan blue uptake (B) in root tips of Yangmai-5 and Jian- 864 exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=3). Figure 3. Concentrations of O 2.- (A) and H 2 O 2 (B) in root tips of Yangmai-5 and Jian- 864 exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=3). Figure 6. DPPH-radical scavenging activity (A) and FRAP assayed total antioxidant capacities (B) in root tips of Yangmai-5 and Jian-864 exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=3). Figure 4. Activities of SOD (A), CAT (B), POD (C), APX (D), MDHAR (E), DHAR (F), GR (G) and GPX (H) in root tips of Yangmai-5 and Jian-864 exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=3). Figure 5. Concentrations of AsA (A), GSH (B), ratios of AsA/ (AsA+DHA) (C) and GSH/GSSG (D) in root tips of Yangmai-5 and Jian-864 exposed to 0 or 30 M AlCl 3 for 24 h, with or without 2 h of 0.6 mM H 2 O 2 pretreatment. Bars (i.e. means) with different letters are significantly different within different genotypes and different treatments (P<0.05, n=3).