The attenuation during PCD of YFP fluorescence is due to low intracellular pH. (A, B) Epidermal cells were bombarded with p35S::YFP and incubated in the dark for 24 hours. YFP fluorescence was scored three times in the same onion epidermal cells, first untreated (nt) set as 100%, next buffered either at pH 5.5 or pH 7 and finally transferred to either pH 5.5 or pH 7. Modified intracellular pH was obtained by permeabilisation as in figure 1C. (C) Fluorescence images of one representative epidermal cell expressing p35S::YFP for 24 hours, first untreated (nt) and next buffered to pH 5.5; bright field and green channel have been merged. (D) Red and green merged images of the same cell co-expressing BAX::YFP and RFP-td-tomato. Before PCD (12 hr post-bombardment) both YFP and RFP fluorescence are detectable. After PCD (48 hr) only RFP fluorescence is detectable. White arrow shows plasma membrane retraction characteristic of PCD cell collapse. (E) BAX-induced PCD: the same cells co-expressing BAX::YFP and RFP-td-tomato were scored for YFP and RFP fluorescence at 12, 24 and 48 hours post-bombardment. Incubation with FAM-VAD-FMK allowed caspase-like protease activity to be detected in cells transfected with p35S::BAX (values shown are minus the background obtained with an RFP-td-tomato control). (F) At 48 hours, the same cells were buffered to pH 7 (as in A-B) and the YFP and RFP fluorescence was re-scored. Errors bars represent 2× SE of triplicates of 100 cells each.