Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H2O2 during time, or directly with known amounts of H2O2. Data presented here indicate that, in TBY-2 cells, a difference in H2O2 level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H2O2-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H2O2 generation. Under a threshold value of H2O2 overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD