An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca2+ and H+ reveals new insights into ion signaling in plants.

Summary: Whereas the role of calcium ions (Ca2+) 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 Ca2+ 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, Ca2+‐ and pH‐dynamics revealed interconnections previously not described.In tobacco PTs, we demonstrated Ca2+‐dynamics lag behind pH‐dynamics during oscillatory growth, and pH correlates more with growth than Ca2+. In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ 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 Ca2+‐increases and cytosolic acidification by c. 2 min, suggesting a Ca2+/pH‐independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage‐, Ca2+‐ and pH‐responses.We propose close interrelation in Ca2+‐ and pH‐signaling that is cell type‐ and stimulus‐specific and the pH having crucial roles in regulating PT growth and stomata movement. [ABSTRACT FROM AUTHOR]