An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca<sup>2+</sup> and H<sup>+</sup> reveals new insights into ion signaling in plants
Kunkun Li, Juan Prada, Daniel Santa Cruz Damineli, Anja Liese, Tina Romeis, Thomas Dandekar, José A. Feijó, Rainer Hedrich, Kai R. Konrad
Abstract
Summary 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.