Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin‐treated oats

Abstract

SummaryHistological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been¯oated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H 2 O 2 , suggesting that the victorintreated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

“…Typical apoptotic changes, such as heterochromatin condensation and a TUNEL-positive response, are observed in sensitive cells treated with victorin in vivo [19,20]. To assess the validity of the cell-free system, we examined whether the same apoptotic morphological changes occurred in the isolated nuclei using fluorescence microscopy after DNA staining with DAPI.…”

“…chromatin condensation in oat leaves [19,20]. To determine whether these enzymes directly participate in the executive phase of oat apoptosis, isolated nuclei were incubated for 2 h with the lysates prepared from victorin-treated leaves in the presence of a cysteine protease inhibitor, E-64, a serine protease inhibitor, aprotinin A or a nuclease inhibitor, ATA.…”

“…Although some plant PCD programs induce chromatin condensation and fragmentation of nuclear DNA into a nucleosomal ladder [18][19][20][21], little is known about intracellular components and mechanisms involved in these events in plants. In animal cells, each apoptotic endonuclease such as CAD/DFF40, endoG and DNase II is solely capable of inducing DNA laddering and/or chromatin condensation [10][11][12][13]15].…”

“…Several apoptogenic effectors, such as caspases (cysteine proteases) [9][10][11][12], CAD/DFF40 (caspase-activated DNase/40 kDa DNA fragmentation factor) [9][10][11][12], endoG (mitochondrial endonuclease G) [13], DNase I [14], DNase II [15], AIF (apoptosis-inducing factor) [16] and Acinus (apoptosis chromatin condensation inducer in the nucleus) [17], have been implicated in mediating these nuclear disintegration processes using a cell-free system. In plants, chromatin condensation and DNA laddering are induced during PCD in response to biotic/abiotic stresses and developmental factors [4,6,[18][19][20][21][22]. However, only limited information is available about plant effectors, especially those involved in the executive phase of plant PCD.…”

“…In oat plants, victorin, a host-selective toxin (HST) produced by Cochliobolus victoriae, induces apoptotic cell death, characterized by DNA laddering, chromatin condensation and a TU-NEL-positive response [18][19][20]. In this study, we established a cell-free system comprising a combination of oat nuclei and lysates from cells treated with victorin.…”