Protocol for cell-specific ratiometric quantification of apoplastic pH in Arabidopsis seedlings

Apoplastic pH dynamically regulates plant intercellular communication, but its measurement in internal tissues, such as the vasculature, remains technically challenging. Here, we present a protocol for ratiometric quantification of apoplastic pH in Arabidopsis seedlings using genetically encoded sensors. We describe seedling preparation, confocal imaging, and ratiometric image processing. Companion cell-specific expression of the pH sensor enables apoplastic pH readouts in the vasculature and supports in vivo comparative analyses of apoplastic pH across genotypes, treatments, and growth conditions in young seedlings. For complete details on the use and execution of this protocol, please refer to Xiong et al. 1