Spatiotemporal Quantification of Cytosolic pH in

Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.