H2O2 involvement in polyamines-induced cell death in tobacco leaf discs

Abstract

The response of tobacco (Nicotiana tabacum L.) wild-type SR1 leaf discs in terms of reactive oxygen species (ROS) formation and cell death occurrence was evaluated after exposure to the polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm). Although NADPH oxidase-like enzyme activity was inhibited by all PAs at 3 or 21 h of treatment, H2O2 content increased significantly in a time- and concentration-dependent manner, suggesting that H2O2 accumulation was linked to the activity of other ROS-generating enzymes. Polyamine oxidase (PAO) activity, which increased markedly upon application of Spd or Spm, is a prime candidate for the increased H2O2 accumulation. Except for 0.1 mM Put, which maintained guaiacol peroxidase (GPOX) and catalase (CAT) activities at the same level as the control, the other PA treatments decreased CAT, ascorbate peroxidase, and GPOX activities at 21 h, contributing to the H2O2 increase. Esterase activity and Evans blue staining, two cell death parameters, were negatively affected at 3 h of treatment with 1 mM Spd and with both concentrations of Spm, whereas at 21 h there was an increase in cell death with both concentrations of the three PAs, except for 0.1 mM Put, which did not alter those parameters. The expression of the senescence-associated cysteine protease gene CP1 was measured to monitor senescence, a physiological cell death process. Application of all PAs increased the expression of the gene, except for 0.1 mM Put, which decreased its expression at 21 h. This result was in agreement with the prevention of cell death exerted by Put and evidenced by Evans blue staining, esterase activity, and electrolyte release.