Real-time detection of caspase-3-like protease activation in vivo using fluorescence resonance energy transfer during plant programmed cell death induced by ultraviolet C overexposure.
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-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.
Zhang
Lingrui
L
Ministry of Education Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Xu
Qixian
Q
Xing
Da
D
Gao
Caiji
C
Xiong
Hongwu
H
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D017360:Arabidopsis / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D061945:Caspase Inhibitors; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D038761:Photobleaching / Q000187:drug effects / Q000528:radiation effects; D011523:Protoplasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014466:Ultraviolet Rays
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053148:Caspase 3