The presence of both calcium (Ca 2+ ) and proton (H + ) apical gradients is necessary for polarized cell elongation to occur in pollen tubes. So far, most of these studies have been carried out in lily pollen tubes, using chemical probes. Yet, lily is a refractory model for molecular genetics, with no easy protocol available for the construction of stable transgenic lines. Tobacco, however, is well suited for both transformation and cell biology, with sexual organs that are accessible, easy to handle and visualize. Pollen tubes are in an ideal size range for subcellular imaging analyses using modern microscopy techniques. Ion homeostasis in tobacco pollen tubes has not been precisely characterized so far. Here, we characterize the H + and Ca 2+ spatial and temporal patterns in tobacco pollen tubes by the use of two fluorescent genetic probes, pHluorin and the YC3.1 yellow CaMeleon, and direct measurement of extracellular flux by ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to stretch from the tip up to 40 lm into the tube shank. This gradient intensity displayed 1-4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. Furthermore, sub-membrane and sub-apical alkaline domains were detected. Extracellular H + fluxes oscillated between 10 and 40 pmol cm -2 s -1 . Fourier and continuous wavelet analyses showed tubes with one or two major oscillatory components in both extra and intracellular H + oscillations. Cytosolic Ca 2+ was imaged by confocal microscopy, showing a V-shaped 40 lm gradient extending from the tip, from 0.2 to 1.0 lM, which oscillates with a 1-4 min period, but with only one major oscillatory component. Extracellular Ca 2+ fluxes oscillate in most pollen tubes, between 2 and 50 pmol cm -2 min -1 and, like in H + , with one or two major oscillatory peaks. A combination of confocal and widefield microscopy showed that H + and Ca 2+ displayed different patterns and shapes inside the cell, sometimes suggesting a structurally complementary role for these 2 second messengers in the growth process. These data suggest that fluxes at the apex of the pollen tube are directly responsible for establishment and maintenance of the gradient.

Summary• 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 lM (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.