Abstract
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.
Highlights
In plant cells, reactive oxygen species (ROS) levels affect various processes, including development, the hypersensitive response to pathogen attack, hormone perception, gravitropism, and the stress response [1,2]
We examined the roots of 5-day-old transgenic Arabidopsis seedlings expressing Hyper using ratiometric fluorescence imaging
Hyper depicts a clear H2O2 distribution that is abundant in the root tip region, labeled the division region (Dr), and is present at lower amounts in the elongation region (Er), including the region of root hair cell formation (Rh)
Summary
ROS levels affect various processes, including development, the hypersensitive response to pathogen attack, hormone perception, gravitropism, and the stress response [1,2]. DAB has been used as frequently as Nitro Blue Tetrazolium (NBT) to image the superoxide oxidative burst during the plant-pathogen interaction, and several other biotic and abiotic responses [18] Both of these compounds are toxic and result in cell death. We report the use of Arabidopsis lines expressing a Hyper transgene to detect the intracellular changes in H2O2 levels induced by Al. We found that plants exhibit a rapid decrease in intracellular ROS levels within the first minute of Al exposure. This decrease in ROS level is most pronounced in the elongation zone, where it induces permanent root growth arrest, but stimulates the generation of short secondary roots This approach allows the visualization of ROS changes in living cells and provides an elegant strategy for studying stress responses in plants
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