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-synthesisinhibitor cycloheximide (CHX) and the mitosis inhibitor colchicine(COL). Morphological and biochemical evidence of specific apoptoticnuclei and chromosomes in individual treated cells was identifiedusing a simple and highly efficient chromosome spreading-basedTUNEL assay, DNA laddering and DNA gel blotting. All of thesedrugs induced DNA cleavage, dose-dependent oligomeric ladders,and characteristic nuclear and chromosomal condensations. Resultsfrom DNA gel blotting showed that DNA ladders could be inducedby exposure to 0.1 mg l^-1ActD, 100 mg l^-1CHX and 500 mg l^-1COLfor 6 h, 6 h and 12 h respectively. The sequence of changesin single cells was studied in detail. DNA cleavage was foundto occur before condensation and disorganization of the nucleus,followed by deformation and condensation of metaphase chromosomes,and marginalization of chromatin. Finally, nucleoli disappearedand fragmentation of the nucleus occurred. Meanwhile, changesin the outer surface charge of apoptotic cells were assessedby electrophoresis. Results indicated quantitatively that thesurface negative charge increased during these apoptotic processes.Our results also showed that the apoptotic pathway induced byeach of these drugs could be reversed before serious cleavageof DNA into oligonucleosomal fragments and universal chromatincondensation. Copyright 2001 Annals of Botany Company Cytotoxin, chromosome spreading, apoptosis, cell electrophoresis