Tip-localized Ca2+-permeable channels control pollen tube growth via kinase-dependent R- and S-type anion channel regulation

Abstract

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca2+) concentration ([Ca2+]cyt) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca2+]cyt orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca2+ and anion imaging. We triggered Ca2+-channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca2+]cyt 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 Ca2+-permeable channels eliminated the [Ca2+]cyt increase, resulting in the abrogation of anion channel activity via Ca2+-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 Ca2+-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 [Ca2+]cyt gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.