An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca and H reveals new insights into ion signaling in plants.

Whereas the role of calcium ions (Ca ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca - and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca -dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca . In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca -increases and cytosolic acidification by c. 2 min, suggesting a Ca /pH-independent early pathogen signaling step. Imaging Ca and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca - and pH-responses. We propose close interrelation in Ca - and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.