Oscillatory Chloride Efflux at the Pollen Tube Apex Has a Role in Growth and Cell Volume Regulation and Is Targeted by Inositol 3,4,5,6-Tetrakisphosphate

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations ${\rm Ca}^{2+},\ {\rm H}^{+}$, and K+. Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl- at the apex and steady influx along the tube starting at 12 μm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl- ion traverses the apical region. The Cl- channel blockers 4,4′ -diisothiocyanatostilbene-2,2′ -disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 μM, respectively. Cl- channel blockers also induced increases in apical cell volume. The apical 50 μm of untreated pollen tubes had a mean cell volume of 3905 ± 75 μ m3. DIDS at 80 μM caused a rapid and lethal cell volume increase to 6206 ± 171 μ m3, which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl- efflux from the apex, indicating that Cl- flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate $[{\rm Ins}(3,4,5,6){\rm P}_{4}]$ antagonized pollen tube growth, induced cell volume increases, and disrupted Cl- efflux. ${\rm Ins}(3,4,5,6){\rm P}_{4}$ decreased the mean growth rate by 85%, increased the cell volume to 5997 ± 148 μ m3, and disrupted normal Cl- efflux oscillations. These effects were specific for ${\rm Ins}(3,4,5,6){\rm P}_{4}$ and were not mimicked by either ${\rm Ins}(1,3,4,5){\rm P}_{4}$ or Ins(1,3,4,5,6) P5. Growth correlation analysis demonstrated that cycles of Cl- efflux were coupled to and temporally in phase with cycles of growth. A role for Cl- flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 μm of the apex during the phase of maximally increasing Cl- efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl- ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.