The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor-induced oxidative burst in Arabidopsis
The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor-induced oxidative burst in Arabidopsis
复制标题
DOI：
10.1111/nph.15550
复制
发表时间：
2019-02-01
期刊：
影响因子：
9.4
通讯作者：
Schwarzlaender, Markus
中科院分区：
文献类型：
--
作者：
Nietzel, Thomas;Elsaesser, Marlene;Schwarzlaender, Markus
过氧化氢（H2 O2）在细胞中无处不在，并且是发育计划和环境反应的中心。H2 O2在细胞中的化学性质使得其难以动态、特异性和高分辨率地检测。基因编码的传感器克服了持续存在的缺点，但pH敏感性，沉默的表达和有限的概念，传感器的行为，在活体植物中阻碍了任何有意义的H2 O2传感。我们建立了H2 O2监测拟南芥细胞质和线粒体与融合蛋白roGFP 2-Orp 1使用共聚焦显微镜和多孔荧光法。我们证实了传感器氧化H2 O2，显示对生理pH变化不敏感，并证明谷胱甘肽在体内主导传感器还原。我们显示了传感器对外源H2 O2的响应，药理学诱导的H2 O2释放，以及对拟南芥活体组织中抗氧化机制的遗传干扰。监测细胞内H2 O2动态响应激发子曝光揭示了后期和长期的影响，在细胞质中的氧化猝发，是在氧化还原mutants.We修改提供了一个定义良好的工具包H2 O2监测在植物，并表明，细胞内H2 O2的测量只携带意义的内源性硫醇氧化还原系统的上下文中。这为剖析植物H2 O2动力学和氧化还原调节（包括细胞内NADPH氧化酶介导的ROS信号传导）开辟了新的可能性。
Hydrogen peroxide (H2O2) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2O2 sensing in living plants.We established H2O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry.We confirmed sensor oxidation by H2O2, show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2O2, pharmacologically-induced H2O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants.We provided a well defined toolkit for H2O2 monitoring in planta and showed that intracellular H2O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.