An interconnection between tip-focused Ca2+ and anion homeostasis controls pollen tube growth.

Plant reproduction is the basis for economically relevant food production. It relies on pollen tube (PTs) growth into the female flower organs for successful fertilization. The high cytosolic Ca2+ concentration ([Ca2+]cyt) at the PT tip is sensed by Ca2+-dependent protein kinases (CPKs) that in turn activate R- and S-type anion channels to control polar growth. Lanthanum, a blocker for plant Ca2+-permeable channels was used here to demonstrate a strict dependency for anion channel activation through high PT tip [Ca2+]cyt. We visualized this relationship by live-cell anion imaging and concurrent triggering of Ca2+-elevations with the two-electrode voltage-clamp (TEVC) technique. The anion efflux provoked by a TEVC-triggered [Ca2+]cyt increase was abolished by Lanthanum and was followed by an overall rise in the cytosolic anion concentration. An interrelation between Ca2+ and anion homeostasis occurred also on the transcript level of CPKs and anion channels. qRT-PCR analysis demonstrated a co-regulation of anion channels and CPKs in media with different Cl- and NO3- compositions. Our data provides strong evidence for the importance of a Ca2+-dependent anion channel regulation and point to a synchronized adjustment of CPK and anion channel transcript levels to fine-tune anion efflux at the PT tip.

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

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.