The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death

Abstract

SummaryThe key role for mitochondria in mammalian apoptosis, a form of programmed cell death (PCD), is well established, but a similar role for plant mitochondria is just emerging. In order to unravel the molecular mechanisms linking plant mitochondria to the downstream events of PCD, we have developed an Arabidopsis cell-free system that can be used to monitor biochemical and morphological changes in isolated nuclei that are associated with PCD. Using this system, two activities that resulted in nuclear DNA degradation could be distinguished, both of which were facilitated by the addition of mitochondria. One activity mediated the generation of 30 kb DNA fragments within 3 h and chromatin condensation within 6 h, when nuclei were incubated with mitochondria alone. The second activity required cytosolic extract in addition to mitochondria and resulted in oligonucleosome-sized DNA cleavage after >12 h. Submitochondrial fractionation and pharmacological studies suggested the presence of an Mg 2 -dependent nuclease activity in the intermembrane space, which is responsible for the former in vitro activity. The evolutionary conservation of the role of mitochondria in PCD in animals and plants is discussed.

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

“…The samples were centrifuged at 21,000 ϫ g for 10 min to fractionate supernatants and pellets, and the supernatants were used to detect nuclease and cytochrome c, which were released from mitochondria. The nuclease assay was carried out by a modification of the method of Balk et al (29). Aliquots (each 2 l) of the supernatant were added to substrate buffer (pH 8.0 and 18 l) consisting of EcoRI-treated pBR322 (0.5 g) 10 mM HEPES-NaOH, 2 mM NaCl, 2.5 mM KH 2 PO 4 , 2 mM MgCl 2, 0.5 mM dithiothreitol, 0.5 mM EGTA, 220 mM mannitol, and 68 mM sucrose, and the reaction mixtures were incubated at 30°C for 2 h. The DNA in each sample was analyzed by agarose gel electrophoresis (1.5% agarose gel).…”

“…The protoplasts were resuspended in buffer (pH 7.5 and 100 ml) consisting of 10 mM Tris-HCl, 10 mM KCl, 10 mM MgCl 2 , 20% glycerol, 0.02% Triton X-100, 10 mM ␤-mercaptoethanol, incubated at 4°C for 10 min with gentle agitation, and filtered through 40-and then 15-m nylon mesh. All further procedures for isolation of nuclei were performed as described by Balk et al (29).…”

Maltose-binding protein switches programmed cell death in Nicotiana glutinosa leaf cells

“…An Mg 2+ -dependent nuclease was identified in the intermembrane space of the mitochondria that recapitulates chromatin condensation and DNA fragmentation [11]. Certain proteases such as cysteine proteinases have been well characterized as PCD regulatory factors in plants [12].…”

“…Almost, all of the stimuli and molecular signals that activate programmed cell death are similar in all organisms. On the molecular level, activation of certain transcriptional factors [30], induction of enzymes involved in protein metabolism [31,32], DNA degradation [11], lipids metabolism [17], phytohormone synthesis [7,8], ROS production [33] and ion homeostasis alterations [13,14] have been characterized, so far.…”

Wheat Endonuclease WEN1 Dependent on S-Adenosyl-L-Methionine and Sensitive to DNA Methylation Status

“…1 Mg 2+ -dependent endonuclease detected in the intermembrane space of the Arabidopsis thaliana mitochondria is involved in the execution of programmed cell death. 6 Site specificity of plant endonucleases, the nature and role of their various modulators are still poorly investigated compared with that of microbial and animal endonucleases.…”