Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death
Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death
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DOI：
10.1186/1471-2229-5-4
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发表时间：
2005-03-29
影响因子：
5.3
通讯作者：
Somerville, Chris R.
中科院分区：
文献类型：
--
作者：
Cutler, Sean R.;Somerville, Chris R.
背景：我们对植物细胞死亡的形态学终点了解甚多，但对其事件序列和/或其在生化水平上的执行却知之甚少。使用gfp标记的活细胞成像是提供细胞过程动态画像的有力方法，可以反过来为未来的生化和遗传研究提供有价值的描述性基础。结果：在对随机gfp蛋白融合标记进行表征时，我们发现机械损伤诱导GFP-Nitrilase 1融合蛋白在直接靠近伤口部位的拟南芥细胞中快速聚集。这种反应的延时成像显示，这种聚集发生在受伤后30-60分钟死亡的细胞中，表明GFP-Nit1聚集是伤口部位细胞死亡的早期标志。利用GFP-Nit1和细胞核和内质网标记物，采用延时共聚焦成像来表征伤口诱导的细胞死亡。这些分析提供了众所周知的死亡相关反应的动态画像，如核收缩和细胞崩溃，并揭示了新的特征，如核膜分离、内质网囊泡和核腔内容物丢失。作为细胞死亡成像的平行系统，我们开发了一种化学方法来快速触发细胞死亡，使用除草剂溴酰腈或氯酰腈，导致快速GFP-Nit1聚集，细胞核内容物丢失和细胞崩溃，但不是核收缩，将这种反应与植物细胞死亡期间的其他反应分开。结论：我们的观察表明Nitrilase 1的聚集是与伤口和除草剂诱导的细胞死亡相关的最早事件之一，并强调了植物细胞死亡时发生的一些新的细胞事件。我们的数据为植物细胞死亡的未来研究创造了一个详细的描述性框架，并为其细胞和生化分析提供了新的工具。
Background: A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and/or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations.Results: While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30-60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death.Conclusion: Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and biochemical analysis.