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

Abstract

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.

Figure 1.

Schematic representations of ECFP-DEVD-EYFP (A) and ECFP-DEVG-EYFP (B) fusion proteins. The fusion proteins contain an 18-amino acid linker possessing the caspase-3 cleavage sequence DEVD and the mutation sequence DEVG, respectively. The arrow indicates the cleavage site by plant caspase-3-like protease. [See online article for color version of this figure.]

Figure 2.

Expression of the chimeric fusion protein ECFP-DEVD-EYFP in protoplasts. Control protoplast without transfection and the transfected protoplasts were excited using a 458-nm laser; the emission fluorescence was divided into chlorophyll autofluorescence (red), ECFP (blue), and EYFP (yellow). Representative images from one experiment are presented. Repetition of experiments led to results very similar to those shown. Bars = 5 μm. [See online article for color version of this figure.]

Figure 3.

Spectral characteristics and distribution profile of the fluorescence in transfected protoplasts (excitation at 458 nm). A, The emission spectra were recorded between 470 and 600 nm from different areas of the transfected protoplasts. B, The fluorescence emission intensity of CFP at 495 nm (blue curve), YFP at 545 nm (yellow curve), and the chlorophyll autofluorescence at 685 nm (red curve) along the arrow represents their distribution profile inside the transfected protoplast. Representative images and data from one experiment are presented. Repetition of experiments led to results very similar to those shown. a.u., Arbitrary units. Bars = 5 μm. [See online article for color version of this figure.]

Figure 4.

Acceptor photobleaching analysis of protoplasts expressing ECFP-DEVD-EYFP. A, EYFP was selectively bleached by repeated scanning of the indicated cell area at a high laser power of 514 nm. B, The emission spectrum from the indicated bleach area was recorded before and after photobleaching. Representative images and data from one experiment are presented. Repetition of experiments led to results very similar to those shown. a.u., Arbitrary units; DIC, differential interference contrast. Bar = 5 μm. [See online article for color version of this figure.]

Figure 5.

Dynamics of caspase-3-like activation in response to UV-C treatment. A and B, The transfected protoplasts without UV-C treatment in light for 20 min were excited by a 458-nm laser. ECFP, EYFP, and EYFP/ECFP FRET images were recorded, and the fluorescence intensity of ECFP, EYFP, and chlorophyll as well as the EYFP/ECFP ratio were measured during a time lapse. C and D, After treatment with 15 kJ m⁻² UV-C and constant light for 20 min, the transfected protoplasts were excited by a 458-nm laser. ECFP, EYFP, and EYFP/ECFP FRET images were recorded, and the fluorescence intensity of ECFP, EYFP, and chlorophyll as well as the EYFP/ECFP ratio were measured during a time lapse. The red arrow and the black arrow indicate the start and end of the decrease of EYFP/ECFP ratio, respectively. Representative images and data from one experiment are presented. Repetition of experiments led to results very similar to those shown. a.u., Arbitrary units; DIC, differential interference contrast. Bars = 5 μm.

Figure 6.

Changes in the fluorescence of ECFP and EYFP as well as the EYFP/ECFP FRET ratio in protoplasts transfected with the plasmid containing ECFP-DEVG-EYFP in response to UV-C treatment. A and B, After treatment with 15 kJ m⁻² UV-C and constant light for 20 min, the transfected protoplasts were excited by a 458-nm laser. Chloroplasts, ECFP, and EYFP were recorded, and the fluorescence intensity of ECFP, EYFP, and chlorophyll as well as the EYFP/ECFP ratio were measured during a time lapse. Representative images and data from one experiment are presented. Repetition of experiments led to results very similar to those shown. a.u., Arbitrary units. Bars = 5 μm.

Figure 7.

Changes in the fluorescence of ECFP and EYFP as well as the EYFP/ECFP FRET ratio in transfected protoplasts preincubated with the caspase-3 inhibitor Ac-DEVD-CHO in response to UV-C treatment. A and B, The transfected protoplasts were preincubated with the caspase-3 inhibitor Ac-DEVD-CHO (100 μm), exposed to 15 kJ m⁻² UV-C and kept in light for 20 min, and then excited by a 458-nm laser. Chloroplasts, ECFP, and EYFP were recorded, and the fluorescence intensity of ECFP, EYFP, and chlorophyll as well as the EYFP/ECFP ratio were measured during a time lapse. Representative images and data from one experiment are presented. Repetition of experiments led to results very similar to those shown. a.u., Arbitrary units. Bars = 5 μm.

Figure 8.

Western-blot evidence that the ECFP-DEVD-EYFP fusion protein was cleaved in response to UV-C treatment. Protoplasts transfected with ECFP-DEVD-EYFP (A) or ECFP-DEVG-EYFP (B) were irradiated with (+) or without (−) 15 kJ m⁻² UV-C and then kept in the light for different times. The extent of cleavage of ECFP-DEVD-ECFP or ECFP-DEVG-EYFP was detected by western blotting with anti-GFP antibody.

Figure 9.

Caspase-3-like activity tested in an in vitro assay using the caspase-3 substrate. The extracts from protoplasts irradiated with UV-C and kept in the light for 0.5, 1, 1.5, 2, 3, or 4 h were used to test caspase-3-like activity. The samples were incubated with the caspase-3 substrate Ac-DEVD-pNA (200 μm) in assay buffer containing phenylmethylsulfonyl fluoride and EDTA. Error bars indicate sd values for five replicates.