Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel.
Physiol Plant. 2010 Apr;138(4):493-502. doi: 10.1111/j.1399-3054.2009.01334.x. Epub 2009 Nov 23.
Redox-sensitive GFP (roGFP) localized to different compartments has been shown to be suitable for determination of redox potentials in plants via imaging. Long-term measurements bring out the need for analyzing a large number of samples which are averaged over a large population of cells. Because this goal is too tedious to be achieved by confocal imaging, we have examined the possibility of using a fluorometer to monitor changes in roGFP localized to different subcellular compartments during development and dark-induced senescence. The degree of oxidations determined by a fluorometer for different probes was similar to values obtained by confocal image analysis. Comparison of young and old leaves indicated that in younger cells higher levels of H(2)O(2) were required to achieve full roGFP oxidation, a parameter which is necessary for calculation of the degree of oxidation of the probe and the actual redox potential. Therefore, it is necessary to carefully determine the H(2)O(2) concentration required to achieve full oxidation of the probe. In addition, there is an increase in autofluorescence during development and extended dark stress, which might interfere with the ability to detect changes in oxidation-reduction dependent fluorescence of roGFP. Nevertheless, it was possible to determine the full dynamic range between the oxidized and the reduced forms of the different probes in the various organelles until the third day of darkness and during plant development, thereby enabling further analysis of probe oxidation. Hence, fluorometer measurements of roGFP can be used for extended measurements enabling the processing of multiple samples. It is envisaged that this technology may be applicable to the analysis of redox changes in response to other stresses or to various mutants.
已证实，定位于不同隔室的氧化还原敏感 GFP（roGFP）可通过成像适用于测定植物中的氧化还原电势。长期测量需要分析大量样本，并对大量细胞进行平均处理。由于通过共聚焦成像实现此目标过于繁琐，因此我们研究了使用荧光计监测 roGFP 在不同亚细胞隔室中定位的变化以用于发育和暗诱导衰老的可能性。荧光计确定的不同探针的氧化程度与共聚焦图像分析获得的值相似。比较幼叶和老叶表明，在较年轻的细胞中，需要更高水平的 H ₂ O₂才能实现 roGFP 的完全氧化，这是计算探针的氧化程度和实际氧化还原电势的必要参数。因此，有必要仔细确定实现探针完全氧化所需的 H ₂ O₂浓度。此外，在发育和延长的暗应激期间，会出现自发荧光增加的情况，这可能会干扰检测 roGFP 氧化还原依赖性荧光变化的能力。尽管如此，仍然有可能在第三天的黑暗和植物发育期间确定不同细胞器中不同探针的氧化和还原形式之间的全动态范围，从而能够进一步分析探针的氧化情况。因此，roGFP 的荧光计测量可用于扩展测量，从而能够处理多个样本。预计这项技术可能适用于分析其他应激或各种突变体的氧化还原变化。
