Apoptotic Cell Death and Cellular Surface Negative Charge Increase in Maize Roots Exposed to Cytotoxic Stresses

Maize root meristematic tissues were exposed to cytotoxic reagents, the RNA-synthesis inhibitor Actinomycin D (ActD), the protein-synthesis inhibitor cycloheximide (CHX) and the mitosis inhibitor colchicine (COL). Morphological and biochemical evidence of specific apoptotic nuclei and chromosomes in individual treated cells was identified using a simple and highly efficient chromosome spreading-based TUNEL assay, DNA laddering and DNA gel blotting. All of these drugs induced DNA cleavage, dose-dependent oligomeric ladders, and characteristic nuclear and chromosomal condensations. Results from DNA gel blotting showed that DNA ladders could be induced by exposure to 0.1 mg l −1 ActD, 100 mg l −1 CHX and 500 mg l −1 COL for 6 h, 6 h and 12 h respectively. The sequence of changes in single cells was studied in detail. DNA cleavage was found to occur before condensation and disorganization of the nucleus, followed by deformation and condensation of metaphase chromosomes, and marginalization of chromatin. Finally, nucleoli disappeared and fragmentation of the nucleus occurred. Meanwhile, changes in the outer surface charge of apoptotic cells were assessed by electrophoresis. Results indicated quantitatively that the surface negative charge increased during these apoptotic processes. Our results also showed that the apoptotic pathway induced by each of these drugs could be reversed before serious cleavage of DNA into oligonucleosomal fragments and universal chromatin condensation. Copyright 2001 Annals of Botany Company