Induction and Signaling of an Apoptotic Response Typified by DNA Laddering in the Defense Response of Oats to Infection and Elicitors

Abstract

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.

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

“…To detect endonuclease activity and its molecular mass, an in-gel nuclease assay was performed as described previously with some modifications [19]. Samples (100 lg protein per lane) were run on a 12% (w/v) SDS polyacrylamide gel containing 200 lg ml À1 heat-denatured calf thymus DNA (Nacalai Tesque).…”

“…We employed the same system to explore regulators responsible for nuclear degradation and chromatin condensation during apoptotic cell death in oat plants. A cell lysate was prepared from primary leaves of victorin-sensitive oat cultivar Iowa X469 treated with 5 ng ml À1 victorin, by which apoptotic cell death is induced within 2 h [19]. Isolated nuclei were incubated with 2.2 mg protein ml À1 of cell lysate for 0-180 min and then DNA was analyzed by agarose gel electrophoresis.…”

“…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.…”

“…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.…”