Direct Evidence of Active and Rapid Nuclear Degradation Triggered by Vacuole Rupture during Programmed Cell Death in Zinnia

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.

导管分子（tracheary element, TE）的分化是维管植物发育进程中程序性细胞死亡（programmed cell death, PCD）的典型实例。在该程序性细胞死亡过程中，导管分子会降解自身细胞内含物，最终形成中空的死细胞以执行水分运输功能。本研究利用百日草（Zinnia elegans）细胞培养体系，通过激光共聚焦扫描显微镜对经历TE PCD的单个活体细胞进行了连续观测。通过活体染色并测定相对荧光强度，研究发现导管分子中肿胀液泡的液泡膜（tonoplast）在发生物理破裂前，便丧失了对荧光素（fluorescein）的选择透过性。液泡破裂后，细胞核会在10至20分钟内快速降解，该过程中未出现明显的染色质固缩或细胞核碎裂现象。叶绿体类核（nucleoids）的降解时序与细胞核基本一致。经丙磺舒（probenecid）处理诱导液泡破裂的非导管分子细胞，则未发生此类降解过程。上述结果表明，导管分子分化属于一种独特的程序性细胞死亡类型，其主动且快速的细胞核降解由液泡破裂所触发。