Yamada Tetsuya, Takatsu Yasumasa, Kasumi Masakazu, Ichimura Kazuo, van Doorn Wouter G
Plant Biotechnology Institute, Ibaraki Agricultural Center, 319-0292 Ibaraki, Japan.
Planta. 2006 Nov;224(6):1279-90. doi: 10.1007/s00425-006-0307-z.
We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.
我们研究了裂叶牵牛花朵花瓣在程序性细胞死亡(PCD)过程中的DNA降解和核碎片化情况。在琼脂糖凝胶上观察到的DNA降解显著增加。使用能对DNA进行染色的DAPI以及针对DAPI荧光的流式细胞术,我们发现每片花瓣中DNA团块的数量至少增加了一倍。这表明染色质在细胞核内或细胞核外发生了碎片化。用阳离子亲脂性荧光探针DiOC6染色表明每个DNA团块都有一层外膜。对细胞核和DNA团块进行荧光显微镜观察发现,其直径最初减小,同时染色质发生凝聚。这些凝聚细胞核的直径约为原来的70%。在核碎片化的初始阶段观察到了两种核直径群体,一种的平均直径约为另一种的一半。随后DNA团块的直径进一步逐渐减小。观察到的最小DNA团块直径小于原始细胞核直径的10%。环己酰亚胺处理阻止了流式细胞术测定的DNA荧光变化,表明该过程需要蛋白质合成。乙烯抑制剂(氨基乙氧基乙烯基甘氨酸和1-甲基环丙烯)对流式细胞术测定的DNA变化没有影响,这表明这些过程不受内源性乙烯的控制。