Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca ) concentration ([Ca ] ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca ] orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca and anion imaging. We triggered Ca -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca ] increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca -permeable channels eliminated the [Ca ] increase, resulting in the abrogation of anion channel activity via Ca -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca ] gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.

(2018) An interconnection between tip-focused Ca 2+ and anion homeostasis controls pollen tube growth, Plant Signaling &