In plants, ions fluxes are involved in several physiological processes such as nutrient uptake, signaling pathways, cell elongation and stomata movements. However, little information is available about the intracellular anionic concentrations and anion fluxes among plant cell compartments. In the last years some promising anion concentration sensors have been developed (ClopHensor, Arosio et al. 2010) but have never been used in plant cells. The ClopHensor sensor (Figure 1) is a genetically encoded non-FRET based sensor developed in mammals cells. It is based on a modified GFP (E²GFP) that is sensitive to chloride and nitrate concentration, and coupled to a DsRed. The DsRed fluorescent protein is used as an internal control for the expression of the construction, because its fluorescence is insensitive to anions concentration and pH, and clearly separated in term of spectral properties. The E²GFP fluorescent protein, excited at 488 nm, is sensitive to pH and anions concentration. But when it excited at 458 nm, the fluorescent signal is only sensitive to anion concentration. Thus, we are able to obtain simultaneously the intracellular pH and anion concentration maps in the cytosol by calculating ratios of fluorescence emission intensity. To do that we used a scanning confocal microscope equipped with an argon laser which provides these laser lines.
In the present collaboration work, Arabidopsis thaliana transgenic lines stably expressing ClopHensor in the cytosol were generated by Elsa Demes (phD student). We are currently performing the in vivo characterization of the ClopHensor sensor in Arabidopsis cells. Indeed, we are establishing the relationship between the fluorescent signal, and pH or anion concentration values, by generating in cellulo calibration curves. This work will lead to the creation of both pH and anions maps to study those parameters at the cell level.
To cite this abstract:Laetitia Besse, Elsa Demes, Alexis De Angeli, Béatrice Satiat-Jeunemaître; In vivo calibration of a ratiometric sensor for pH and anions concentration in Arabidopsis cells. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/in-vivo-calibration-of-a-ratiometric-sensor-for-ph-and-anions-concentration-in-arabidopsis-cells/. Accessed: February 23, 2019
EMC Abstracts - https://emc-proceedings.com/abstract/in-vivo-calibration-of-a-ratiometric-sensor-for-ph-and-anions-concentration-in-arabidopsis-cells/