Figure 1

BAX expression induces PCD and intracellular acidification. (A) Left panel; a close up of a p35S:: BAX::YFP expressing cell showing typical punctuated subcellular localisation. Centre panel; all BAX::YFP expressing cells were negative for mitotracker (n = 30). Right panel; untransfected cells were positive for mitotracker. Cells were observed 30 h post-bombardment. Scale bar 10 μm. (B) Corpse morphology (cellular collapse) and fluorescent VADase activity in cells expressing BAX 48 h post-bombardment. White arrow shows plasma membrane retraction. For VADase, unlabelled p35S::BAX was co-transfected with p35S::RFP-td-tomato in order to prevent overlap between the fluorescence of YFP and FAM-VAD-FMK. Inset panels show the percentage of BAX cells positive for these two PCD-hallmarks (values shown are minus the background obtained with an RFP-td-tomato control). Scale bar 100 μm. (C) Calibration curve for ptGFP readings in vivo. Cells were transfected with p35S ptGFP and incubated for 30 hours in the dark. Epidermal pieces were then incubated for 1 hour in 3 mL of buffer solution at various pHs with 0.003% Triton-20-X-100. F475/F390 ratiometric readings were then taken using 30 transfected cells for each pH value. Using the standard curve, the pH of control cells expressing ptGFP or of cells expressing BAX/ptGFP was interpolated using readings at 30 h. The average for all control cells or for all low pH BAX cells was plotted on the standard curve. Error bars are for 51 control cells and 15 low pH cells.