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. 2015 Jan 6;15(1):855-67.
doi: 10.3390/s150100855.

Hyper, a Hydrogen Peroxide Sensor, Indicates the Sensitivity of the Arabidopsis Root Elongation Zone to Aluminum Treatment

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Free PMC article

Hyper, a Hydrogen Peroxide Sensor, Indicates the Sensitivity of the Arabidopsis Root Elongation Zone to Aluminum Treatment

Alejandra Hernández-Barrera et al. Sensors (Basel). .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Hyper expression in transgenic Arabidopsis lines depicts the H2O2 distribution in the growing root. Note the high H2O2 levels in the root tip region where cell division occurs (Dr) and the lower levels in the elongation zone (Er) and root hairs at the root hair emerging zone (Rh).
Figure 2.
Figure 2.
Increased levels of external H2O2 result in increased intracellular changes in ratio values. Arabidopsis roots expressing Hyper were treated with different external concentrations of H2O2 and the intracellular changes were monitored by analyzing Hyper fluorescence ratios. Images were acquired at 10 s intervals. Note that higher concentrations of external H2O2 resulted in increased ratio values.
Figure 3.
Figure 3.
Exposure to Al (800 nM) induces dramatic changes in intracellular H2O2 levels in growing Arabidopsis roots. Note that H2O2 levels are highest in the root tip, but that the elongation zone exhibits the strongest response to Al treatment. Furthermore, the differentiation zone, from where root hairs emerge, is also affected. The arrow indicates when the Al was added and the asterisks where the elongation zone is located. Images were acquired at 1-min intervals.
Figure 4.
Figure 4.
The elongation region depicts dramatic decreases in intracellular H2O2 levels. Intracellular H2O2 levels were measured in several Arabidopsis roots before and after treatment with 1 mM Al. Note that the elongation region exhibits a dramatic decrease in ratio signal after 30 min of Al treatment. n = 25 cells.
Figure 5.
Figure 5.
Intracellular levels of H2O2 in Arabidopsis roots respond to Al treatment in a dose-dependent manner. (A) Roots were treated with different Al concentrations and the intracellular H2O2 levels were evaluated as a ratio change of the Hyper probe. Note that higher concentrations of Al induce lower intracellular H2O2 levels. n = 25 cells; (B) Graph depicting the representative response of Arabidopsis roots to various Al concentrations. Images were acquired at 30 s intervals.
Figure 6.
Figure 6.
Different regions of the roots respond with a similar pattern, but the elongation region exhibits the most dramatic change. Different regions of Arabidopsis roots treated with 1 mM Al were evaluated for changes in intracellular H2O2 levels (see inset). Note that all the regions responded at the same time, but with a different strength. 1 = apical, 2 = meristem, 3 = elongation 1, 4 = elongation 2, 5 = elongation 3, 6 = differentiation, 7 = mature.
Figure 7.
Figure 7.
Arabidopsis roots exhibit increased intracellular H2O2 levels after several days of exposure to Al. After 5 days of Al treatment, roots responded with an increase in lateral root formation. The newly formed lateral roots were shorter than those of control plants of the same age and had higher H2O2 levels in the tip regions. However, the elongation zone remained and exhibited reduced H2O2 levels. Arrows indicate the tip of the root where the division region is located, asterisks the elongation zone, and arrowheads the small lateral root primordia.

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