Apoptosis in plants: a cytological analysis

Abstract

This thesis embodies a flow cytometric study on apoptosis in plant cells. A large decrease in fluorescence intensity of propidium iodide-stained nuclei was observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (l) DNA accessibility to DNAse I, (2) histone disassociation induced by HCl, (3) saturation of binding sites by the PI fluorochrome, (4) and (5) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed the identification of different stages in the apoptosis pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed the identification of different stages in the apoptosis pathway in plants. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants. Chromatin condensation and nDNA fragmentation occur in plant cells during senescence and in the presence of cytotoxic agents. Camptothecin, cytochalasin B, okadaic acid, salicylic acid, hydrogen peroxide, the calcium ionophore A23187 and nifedipine induced chromatin condensation. At the same effective concentrations terminal deoxynucleotidyl transferase DNA end-labelling assays (TUNEL) showed that these agents also induced nDNA fragmentation. Exposure of cells to suboptimal concentrations or treatment followed by removal of the cytotoxic agent resulted in an initial phase of chromatin condensation, followed by its reversal. Prolonged exposure to higher concentrations of cytotoxic agents induced loss of membrane integrity, as determined by dual labelling of protoplasts with propidium iodide and fluorescein diacetate. An early indicator of apoptosis in mammalian cells is the loss of the phospholipids membrane asymmetry of the cell resulting in exposure of phosphatidylserine on the outer surface of the plasma membrane. This change in membrane asymmetry can be analysed using annexin V. Annexin V binding was found to be an early indicator of apoptosis, occurring prior to the detection of DNA strand breaks as monitored by the TUNEL assay. Apoptotic plant cells also had changes in their light-scattering properties similar to those of mammalian cells, with both types of cells decreasing in forward scatter while increasing in side scatter. These findings suggest that apoptosis occurs in plants in a manner closely related to that characterised for mammalian cells, with similar cytometric characteristics to those of mammalian cells undergoing apoptosis.