pH but not Ca²⁺ Waves Propagate Membrane Potential Oscillations Throughout the Pollen Tube

Polar growth and guidance of pollen tubes (PTs) is regulated by chemical and electrical signaling cues. Central regulators controlling cellular protrusion of PTs tip-focused Ca2+ and H+ cytosolic gradients and ion fluxes depend, but also feedback, on membrane voltage (V-m). However, the functional relationship between V-m, Ca2+, and H+ dynamics is largely unclear. We carried out parallel live-cell Ca2+ and H+-imaging together with V-m recordings followed by cross-correlation analysis to study their interconnection in tobacco PTs. Upon electrode impalement at the tip or shank, PT growth ceased but sustained oscillatory behavior of all three signals continued and remained mostly in phase, although shifted in time. The Ca2+ oscillations were largely restricted to the apex, temporally leading the H+ increases by & SIM;18 s, whereas H+ tip-focused peaks spread into the shank. Voltage recordings in the shank (50-80 & mu;m behind the tip) revealed transient bell-shaped depolarizations that correlated strongly with H+ but not Ca2+ oscillations. In contrast, V-m oscillations readout at the tip correlated equally with tip Ca2+- and H+-signals, having action potential-like appearances that were approximately five times faster compared with those in the shank. Our multiparameter readout approach identified distinct relationships of chemical and electric signals along PTs, where at the tip changes in both Ca2+ and H+ correlated with V-m dynamics, whereas at the shank only H+ correlated with V-m. We propose that spatially confined chemo-electric coupling support the manifestation of propagating voltage and pH waves along nongrowing PTs, which are preceded by short-lasting but high amplitude tip Ca2+ spikes.