Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Plant Methods. 2010 Nov 30;6:27. doi: 10.1186/1746-4811-6-27.
Programmed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD.
The acidification that occurs in the cytosol during plant PCD was monitored by way of the extinction of YFP fluorescence at low pH. This fluorescence was recovered experimentally when bringing the intracellular pH back to 7, demonstrating that there was no protein degradation of YFP. Because it uses YFP, the assay is none-destructive, does not interfere with the PCD process and allows time-lapse studies to be carried out. In addition, changes of sub-cellular localisation can be visualised during PCD using the protein of interest fused to RFP. Coupled to a transient expression system, this pH-based assay can be used to functionally analyse genes involved in PCD, using point mutations or co-expressing PCD regulators. Transfecting mBAX and AtBI-1in onion epidermal cells showed that the pH shift is downstream of PCD suppression by AtBI-1. In addition, this method can be used to score PCD in tissues of stably transformed transgenic lines. As proof of principle, we show the example of YFP extinction during xylogenesis in Arabidopsis. This demonstrates that the assay is applicable to PCD studies in a variety of tissues.
The observation that YFP fluorescence is lost during the plant PCD process provides a new tool to study the genetic regulation and cell biology of the process. In addition, plant cell biologists should make a note of this effect of PCD on YFP fluorescence to avoid misinterpretation of their data and to select a pH insensitive reporter if appropriate. This method represents an efficient and streamlined tool expected to bring insights on the process leading to the pH shift occurring during PCD.
程序性细胞死亡（PCD）是所有多细胞生物生命周期所必需的过程。然而，在高等植物中，人们对负责启动和执行 PCD 的基因和信号分子级联反应知之甚少。为了帮助发现和分析植物 PCD 调节因子，我们设计了一种基于低胞质 pH 作为 PCD 标志物的新型细胞死亡测定法。
通过低 pH 下 YFP 荧光的消光来监测植物 PCD 过程中胞质中的酸化。当将细胞内 pH 恢复到 7 时，实验上恢复了这种荧光，证明 YFP 没有蛋白降解。由于它使用 YFP，该测定是非破坏性的，不干扰 PCD 过程，并允许进行延时研究。此外，使用与 RFP 融合的感兴趣的蛋白质，可以在 PCD 过程中可视化亚细胞定位的变化。与瞬时表达系统相结合，这种基于 pH 的测定法可用于功能分析参与 PCD 的基因，使用点突变或共表达 PCD 调节剂。转染洋葱表皮细胞中的 mBAX 和 AtBI-1 表明，pH 变化发生在 AtBI-1 抑制 PCD 之后。此外，该方法可用于对稳定转化的转基因系组织中的 PCD 进行评分。作为原理证明，我们展示了在拟南芥木质部形成过程中 YFP 消光的例子。这表明该测定法适用于各种组织中的 PCD 研究。
观察到 YFP 荧光在植物 PCD 过程中丢失，为研究该过程的遗传调控和细胞生物学提供了新的工具。此外，如果适当的话，植物细胞生物学家应该注意到 PCD 对 YFP 荧光的这种影响，以避免对其数据的错误解释并选择 pH 不敏感的报告基因。该方法代表了一种高效、精简的工具，有望深入了解导致 PCD 过程中 pH 变化的过程。
