RESUMO
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.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Nicotiana/crescimento & desenvolvimento , Tubo Polínico/enzimologia , Tubo Polínico/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Animais , Ânions , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Ativação Enzimática , Ativação do Canal Iônico , Oócitos/metabolismo , Nicotiana/metabolismo , XenopusRESUMO
⢠Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. ⢠With Cl(-) -based intra- and extracellular solutions, three outward-rectifying anion conductances, I(Cl1) , I(Cl2) and I(Cl3) , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+) ](in) ), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1) ), or reaching a maximum value with 8.50 µM (I(Cl2) and I(Cl3) ). ⢠After rundown, the anionic currents measured using NO(3) (-) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3) (-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. ⢠This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+) -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.