The killing of suspension-cultured tobacco cells and stem cells of Japanese black pine (Pinus thunbergii Parl.) by drying, autoclaving, hypertonic treatment or freezing caused the disappearance or deformation of nuclei, as monitored by staining with 4′,6-diamidino-2-phenylindole (DAPI). This observation suggests that disappearance or deformation of the nucleus can be taken as an index of cell death. Using this index, we examined the spreading of the cell death in water-stressed cuttings of Japanese black pine. Various portions of the cuttings were examined over time and DAPI-stained nuclei were seen to disappear more rapidly in the upper than in the lower portions of water-stressed cuttings. In every portion, undeformed, DAPI-stained nuclei disappeared sequentially from the cortex and the pith, from the periderm, from the xylem and finally from the cambium, indicating that cell death caused by water stress spreads from the cortex and the pith to the periderm, then to the xylem, and finally to the cambium. Water conduction was ex amined by immersing the cut ends of cuttings in a red solution of acid fuchsin. Under water stress, water conduction was reduced and the height of the upper limit of the sucked solution above the cut end was reduced. Just below this upper limit, water conduction was inhibited in some regions of the xylem. However, undeformed, DAPI-stained nuclei were distributed homogeneously over both conductive and non-conductive regions of the xylem, indicating that there is no direct relationship between cell death and water conductivity of the xylem. Undeformed, DAPI-stained nuclei disappeared in regions around the upper limit of suction of the dye, and the sequence of disappearance was again from the cortex and the pith, from the periderm, from the xylem and, finally, from the cambium.
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Ishida et al. (1992) studied this question.