Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-Cinduced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.

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Activation of heat shock factor (HSF)-1 DNA binding and heat shock protein (hsp)-70 expression enable resistance of cells to various forms of stress and maintain cell survival. Fas, a membrane-bound protein, is a central pro-apoptotic factor. Its activation leads to a cascade of events resulting in programmed cell death. Herein, these two mechanisms with contrary functions, promoting either cell survival or death, were addressed for their potential to inhibit each other's activation. Induction of Fas-mediated signalling was followed by a rapid decrease of HSF1 DNA binding and inducible hsp70 expression. Inhibition of HSF1 DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS-signalling. These effects of Fas-activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1)-inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase of apoptosis rates from 20% to 50% in response to heat stress. When analyzing Fas-mediated apoptosis in the presence of HSF1/hsp70 activation, decreased apoptosis rates were detected with induced expression of hsp70 but not with activation of HSF1-DNA binding alone. Thus, we conclude that inhibition of the HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis.

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