Hernández-Barrera Alejandra, Velarde-Buendía Ana, Zepeda Isaac, Sanchez Federico, Quinto Carmen, Sánchez-Lopez Rosana, Cheung Alice Y, Wu Hen-Ming, Cardenas Luis
Department of Plant Molecular Biology, Institute of Biotechnology, National Autonomous University of Mexico, 62210 Mexico.
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
Sensors (Basel). 2015 Jan 6;15(1):855-67. doi: 10.3390/s150100855.
Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.
新出现的证据表明，一些活性氧（ROS），如超氧阴离子自由基和过氧化氢（H₂O₂），是植物对生物和非生物胁迫反应的核心调节因子。因此，ROS的细胞水平被认为受到一个高效且精细的促氧化和抗氧化系统的严格调控，该系统调节ROS的产生和清除。直到最近，植物细胞中ROS的研究还局限于生化分析和荧光探针的使用；然而，这些荧光探针的不可逆氧化使得无法可视化ROS水平的动态变化。在这项工作中，我们描述了使用Hyper（一种最近开发的用于测量活细胞中H₂O₂的活细胞探针）来监测拟南芥根在铝处理下的氧化应激。Hyper由插入大肠杆菌过氧化氢结合蛋白（OxyR）调节结构域的环状排列黄色荧光蛋白（cpYFP）组成，是一种H₂O₂特异性的比率型探针，因此是定量的，可在植物和动物细胞中表达。现在我们证明，在用铝处理的根的伸长区，H₂O₂水平急剧下降。这种反应可能导致根生长停滞，并提供了H₂O₂参与早期铝感知的证据。
