An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca(2+) and H(+) reveals new insights into ion signaling in plants

Whereas the role of calcium ions (Ca(2+)) 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(2+) 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(2+)- and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca(2+)-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca(2+). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca(2+) 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(2+)-increases and cytosolic acidification by c. 2 min, suggesting a Ca(2+)/pH-independent early pathogen signaling step. Imaging Ca(2+) and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca(2+)- and pH-responses. We propose close interrelation in Ca(2+)- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.