Figure 5.

Effects of transgenic phytaspase deficiency or overproduction on PCD in tobacco (Samsun NN) plants induced by abiotic stresses. (A) Loss of viability (bleaching) of leaf discs, (B) generation of H₂O₂ and (C) cytochrome c release from mitochondria (M) to cytosol induced by MV or NaCl (versus untreated controls) are facilitated in leaf discs overexpressing phytaspase (OE, OE-1 line) and are suppressed in phytaspase-silenced leaf discs (KD, KD1-1 line) compared with wild type (wt). (A) Leaf discs were immersed in the aqueous solutions of MV or NaCl, or control solution (lacking MV or NaCl) and photographed 24, 48 and 96 h after treatment. (B) H₂O₂ production was determined 24 h after treatment; data are means±s.d. from three experiments with three independent replicates in each. (C) Western blot analysis of mitochondrial (M) and cytosolic (C) fractions was performed with the cytochrome c monoclonal antibody 24 h after treatment. As a control for cell fractionation, western blots were probed with an anti-VDAC monoclonal antibody. VDAC is localised in the outer membrane of mitochondria (Tsujimoto and Shimizu, 2002). Concentrations of MV and NaCl are indicated. Data (including photographs) were typical of all generated KD and OE transgenic lines, respectively, tested in, at least, three experiments with three replicates in each.