Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants

Abstract

Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H2O2-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H2O2, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H2O2 production by plants using spectrofluorometry and microscopy.

Keywords: Arabidopsis; ContPY1; cells suspension; fluorescence; hydrogen peroxide; microscopy; protoplasts.

Figure 1. (A) Relative fluorescence of the ContPY1 probe (5 µM in HEPES pH 7), the control in light gray and in presence of H₂O₂ (250 µM) in dark gray. (B) Relative fluorescence of the ContPY1 probe (5 µM in HEPES pH7) in presence of H₂O₂ (1.5 mM) (dark gray) and with other reactive oxygen species (1.5 mM) (light gray).

Figure 2. (A) and (B): Relative fluorescence of ContPY1 (A) and DCFDA (B) added to elicited suspension-cultured cells (COS-OGA) and non-elicited cells (Control). (C) and (D): measure of relative fluorescence of ContPY1 and DCFDA on protoplast of cell culture for elicited (COS-OGA) and non-elicited protoplast (Control). Notes: Results and standard deviation were based on 8 biological replicates.

Figure 3. Time lapse acquisition of a wounded leaf of Arabidopsis thaliana. The leaves were incubated during 1 h in 30 µM ContPY1. (A), (B), and (C) represent wounding after 1, 20, and 40 min, respectively. Scale bars = 100 µm.