title: 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.
abstract: Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), 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 microm 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 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), 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 [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). 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 microm 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.
authors.affiliation: Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Pernikarce 15, 160 00 Prague 6, Czech Republic. zonia@ueb.cas.cz
authors.forename: Laura
authors.initials: L
authors.lastname: Zonia
authors.forename: Sofia
authors.initials: S
authors.lastname: Cordeiro
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Tupý
authors.forename: José A
authors.initials: JA
authors.lastname: Feijó
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D007295:Inositol Phosphates / Q000494:pharmacology*; D027762:Lilium / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D009544:Niflumic Acid / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D011058:Pollen / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000254:growth & development
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D007295:Inositol Phosphates; D009579:Nitrobenzoates; C451920:inositol-1,3,4,5,6-pentakisphosphate; C081655:inositol-1,3,4,5-tetrakisphosphate; C081652:inositol-3,4,5,6-tetrakisphosphate; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid