A workflow for absolute apoplastic pH assessment during live cell imaging in plant roots

Abstract

Abstract Background Apoplastic pH is a central regulator of plant growth, development, and environmental adaptation, influencing cell expansion, nutrient uptake, and extracellular signaling. Many studies have successfully used HPTS to monitor relative changes in apoplastic pH in plants. At the same time, research increasingly targets pH-dependent biochemical and biophysical processes. Many enzymatic activities, ion binding events, and receptor–ligand interactions depend on defined proton concentrations. Accordingly, the development of reliable approaches to measure absolute pH in living tissues is gaining importance. Methods A calibration-based workflow was developed to enable quantitative assessment of absolute apoplastic pH using ratiometric HPTS imaging. The approach integrates a simplified two-point normalization strategy with an in-vitro derived sigmoidal calibration model, thereby minimizing the need for extensive in-vivo calibration curves. Confocal imaging was performed using HPTS excited at two wavelengths followed by ratiometric image processing. Data analysis is supported by a custom Fiji plugin, Ratio2pH, which converts ratiometric images into pixel-resolved maps of absolute pH. Results In vitro characterization revealed a robust, non-linear relationship between normalized HPTS ratios and pH, enabling accurate pH estimation within the physiologically relevant range of pH 5.0–7.0. When applied in-vivo to Arabidopsis thaliana roots, the workflow yielded extracellular pH estimates consistent with the pH of the incubation medium and detected reproducible pH shifts in response to pharmacological treatments. Conclusions This workflow enables reproducible, spatially resolved measurement of absolute apoplastic pH in living plant tissues. By combining a simplified calibration strategy with accessible image analysis tools, it facilitates quantitative extracellular pH measurements and their integration into biochemical and biophysical analyses.

Plant phenotyping relevance

生きた植物組織の絶対アポプラストpHを画像から定量する校正ワークフローを開発・検証し、Fijiプラグインも提供しているため、植物状態の取得法が中心である。

Code and data availability

The paper deposits its authors' analysis code and data publicly: the Ratio2pH Fiji plugin (Zenodo 10.5281/zenodo.15599805), a Python script for sigmoidal calibration curve fitting (Zenodo 10.5281/zenodo.17303477), and source data files and raw confocal images (Freidata 10.60493/t29wb-7my86). The Zenodo 15658668 ratiom�