Temporal and spatial activation of caspase-like enzymes induced by self-incompatibility in Papaver pollen

Abstract

Caspase-like proteases are universal mediators of programmed cell death (PCD). Because plants have no caspase homologs, establishing the nature of their caspase-like activities is of considerable importance to our understanding of PCD in plants. Caspase-3, displaying DEVD specificity, is a key executioner caspase in animal cells. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants by inhibiting incompatible pollen. In Papaver rhoeas, SI activates a caspase-3-like/DEVDase activity in incompatible pollen that plays a pivotal role in regulating PCD. Here we characterize the SI-induced caspase-like activities in detail; our work provides insights into the temporal and spatial activation of plant caspase-like enzymes. We show that SI also activates a VEIDase and a LEVDase and that the VEIDase plays a role in SI-induced PCD. The DEVDase and VEIDase are activated remarkably rapidly: detectable within 1-2 h after SI induction; the LEVDase activity peaks later. Importantly, we show live-cell imaging of a DEVDase activity in a higher plant cell; the SI-activated DEVDase has a cytosolic and nuclear localization. We also demonstrate that SI induces a rapid and substantial cytosolic acidification that matches the in vitro pH optima for the SI-induced caspase activities. Because both cytosolic acidification and nuclear caspase localization are observed during apoptosis in animal cells, our data provide striking parallels between SI-induced PCD and apoptosis in animal cells.

Fig. 1.

Live-cell DEVDase activity in pollen tubes. (a) Specificity of DEVDase in SI-stimulated pollen tubes. Pollen tubes were scored for DEVDase activity by using the NucView probe 5 h after SI induction. Fluorescent nuclei indicated high caspase activity (+, filled bars); low fluorescence indicated no caspase activity (−, open bars); intermediate fluorescence, intermediate activity (+/−, cross-hatched bars). UT, untreated; +DEVD, pretreatment with Ac-DEVD-CHO; +YVAD, pretreatment with Ac-YVAD-CHO. Error bars, ± SEM (n = 3). (b–j) CR(DEVD)₂ imaging of DEVDase activity in SI-induced pollen. (b) CR-(DEVD)₂ live-cell imaging of a normally growing untreated pollen tube (control). No background DEVDase activity was detected. (c) Bright-field image of b. (d) DAPI staining shows nuclear DNA in the GC and VN. (e) Typical CR fluorescence upon SI induction. DEVDase activity is cytosolic and also localizes with the GC and VN. (f) CR fluorescence 2 h after SI induction. (g) Bright-field image of pollen tube in f. Note the highly granular cytoplasm. (h) CR fluorescence 4 h after SI induction. (i) Typical example of CR fluorescence in a pollen grain 4 h after SI induction. (j) No DEVDase activity is detected in an untreated pollen grain at 4 h. Images shown are median plane confocal sections; all were captured using identical detection settings. (Scale bars: 10 μm.)

Fig. 2.

Substrate specificity of SI-induced caspase-like protease activities. (a) SI-induced pollen extracts were tested for substrate specificity by using different caspase substrates: Ac-DEVD-AMC (DEVDase), Ac-VEID-AMC (VEIDase), Ac-LEVD-AMC (LEVDase), Ac-YVAD-AMC (YVADase), Ac-GRR-MCA (GRRase). Relative fluorescence units were measured and are expressed as a percentage of the DEVDase activity. Error bars, ± SEM (n = 3). (b) Initiator caspase activities are not detected in SI-induced samples. The initiator caspase substrates Ac-IETD-AMC and Ac-LEHD-AMC show low or no cleavage activities at 2 h and 5 h after SI induction. Error bars, ± SEM (n = 3).

Fig. 3.

Effect of caspase inhibitors on SI-induced caspase-like activities and DNA fragmentation. (a) Ac-DEVD-AMC (filled bars), Ac-VEID-AMC (open bars), and Ac-LEVD-AMC (cross-hatched bars) substrate cleavage in the presence of caspase-specific tetrapeptide inhibitors. Substrate cleavage 5 h after SI induction is expressed as a percentage of activity in the presence of DMSO (100%). Error bars, ± SEM (n = 3). (b) DNA fragmentation in untreated (UT), SI-induced (SI), and in SI-induced after pretreatment with Ac-DEVD-CHO (SI+DEVD), Ac-VEID-CHO (SI+VEID), or Ac-LEVD-CHO (SI+LEVD) inhibitors before SI induction. Error bars, ± SEM (n = 300).

Fig. 4.

Temporal activation of SI-induced caspase-like activities. (a) SI-induced caspase activities were followed temporally in pollen extracts after SI induction, by using the Ac-DEVD-AMC (♦), Ac-VEID-AMC (○), and Ac-LEVD-AMC (●) substrates. Error bars, ± SEM (n = 3). (b) DEVDase activity in pollen tubes measured with the NucView probe. The number of pollen tubes with highly fluorescent nuclei (+, filled bars), little to no fluorescent nuclei (−, open bars), and nuclei with intermediate fluorescence (+/−, hatched bars) was scored in untreated (UT) and SI-induced (SI) pollen tubes. Fluorescence intensity indicates the level of DEVDase activity. Error bars, ± SEM (n = 3). (c) Early DEVDase activity visualized in a pollen tube with the CR(DEVD)₂ probe 44, 59, and 95 min after SI induction. (Scale bar: 10 μm.) (d) Example of cytosolic and nuclear DEVDase increases detected at 108 and 190 min after SI using CR(DEVD)₂. (Scale bar, 10 μm.)

Fig. 5.

SI-induced caspase-like activities are optimal at an acidic pH, and cytosolic acidification is triggered during SI. (a) Ac-DEVD-AMC (♦), Ac-VEID-AMC (○), and Ac-LEVD-AMC (●) substrate cleavage activities at different pH values, with citrate/phosphate buffers, 5 h after SI induction. Activity is expressed as percentage of maximal activity at pH 5.0. Error bars, ± SEM (n = 3). (b) SI-induced [pH]_i changes measured in individual pollen tubes 1–4 h after SI induction. Error bars, ± SEM (n = 13 and 12).