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

Abstract

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 correlating 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 ~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 H₂O₂ 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 pH having crucial roles in regulating PT growth and stomata movement.