MAJOR CHANGES IN CHROMATIN CONDENSATION SUGGEST THE PRESENCE OF AN APOPTOTIC PATHWAY IN PLANT-CELLS

A large decrease in fluorescence intensity of propidium iodide (PI)-st ained nuclei is observed during senescence of plant cells. The phenome non reflects a decrease in accessibility of DNA to this fluorochrome a nd is a consequence of chromatin condensation. This decrease is substa ntially greater than usually found in animal nuclei whose chromatin un dergoes condensation, e.g., during differentiation or quiescence. Chro matin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturati on of binding sites by the PI fluorochrome (iv), and (v) visual inspec tion by fluorescence and confocal microscopy, The extent of changes re vealed by these assays was used to map progressive changes in chromati n condensation which allowed us to identify different stages in an apo ptosis-like pathway in plants. The initial step of chromatin condensat ion which occurred prior to endonucleolytic DNA degradation was detect ed by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry, 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. (C) 1998 Aca demic Press.