Exposito-Rodriguez Marino, Laissue Pierre Philippe, Littlejohn George R, Smirnoff Nicholas, Mullineaux Philip M
School of Biological Sciences, University of Essex, Colchester, United Kingdom.
Methods Enzymol. 2013;527:185-201. doi: 10.1016/B978-0-12-405882-8.00010-6.
Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.
将拟南芥叶片组织的光合细胞暴露于高光强度（HL）下，可能会导致叶绿体和与光合代谢相关的亚细胞区室（如过氧化物酶体）中的过氧化氢（H₂O₂）水平迅速升高。据推测，当H₂O₂在其产生部位或附近存在时，它在诱导对高光适应的信号传导中起重要作用。然而，如果这种离散的控制失效且H₂O₂水平超过抗氧化系统清除它们的能力，就会产生氧化应激，进而引发细胞死亡。为了验证这一假设，需要对不同亚细胞区室中H₂O₂的时空积累进行定量。在本章中，介绍了关于使用用基于核编码的位于细胞质的黄色荧光蛋白的H₂O₂传感器（称为HyPer）转化的拟南芥幼苗的初步实验。HyPer允许在两个激发波长下对其荧光进行比率测定，从而使体内H₂O₂的定量不受HyPer可变水平的影响。结果表明，HyPer荧光有潜力通过共聚焦显微镜提供必要的空间、时间和定量分辨率，以研究幼苗对高光的反应。叶绿素荧光成像用于量化显微镜载物台上高光处理诱导的幼苗光合作用光抑制。然而，仍存在几个技术问题，其中最具挑战性的是HyPer在幼苗期之后的表达沉默。这将我们的初步研究限制在子叶表皮细胞，这些细胞虽然不进行光合作用，但对高光的反应是细胞质H₂O₂增加45%。
