title: Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20.
abstract: Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.
authors.affiliation: Gulbenkian Institute of Science, P-2780-156 Oeiras, Portugal.
authors.forename: Timo
authors.initials: T
authors.lastname: Gutermuth
authors.forename: Roman
authors.initials: R
authors.lastname: Lassig
authors.forename: Maria-Teresa
authors.initials: MT
authors.lastname: Portes
authors.forename: Tobias
authors.initials: T
authors.lastname: Maierhofer
authors.forename: Tina
authors.initials: T
authors.lastname: Romeis
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: Borst
authors.forename: Rainer
authors.initials: R
authors.lastname: Hedrich
authors.forename: José A
authors.initials: JA
authors.lastname: Feijó
authors.forename: Kai R
authors.initials: KR
authors.lastname: Konrad
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D058503:Mesophyll Cells / Q000378:metabolism; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D030821:Plants, Genetically Modified; D053205:Pollen Tube / Q000254:growth & development* / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D000838:Anions; D029681:Arabidopsis Proteins; D002118:Calcium; D007473:Ion Channels; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C582432:SLAH3 protein, Arabidopsis; C000592511:calcium-dependent protein kinase 20, Arabidopsis thaliana; C490307:ATCDPK2 protein, Arabidopsis