ABSTRACT
We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.
Subject(s)
Arabidopsis/metabolism , Calcium/metabolism , Electrophysiological Phenomena , Pollen/metabolism , gamma-Aminobutyric Acid/metabolism , Anions , Arabidopsis/drug effects , Arabidopsis Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Chlorides/pharmacology , Electrophysiological Phenomena/drug effects , Hydrogen-Ion Concentration , Ion Channels/metabolism , Ion Transport/drug effects , Models, Biological , Mutation/genetics , Nitrates/pharmacology , Pollen/drug effects , Pollen Tube/drug effects , Pollen Tube/metabolism , Protoplasts/drug effects , Protoplasts/metabolism , Symporters/metabolismABSTRACT
Mycorrhization is a typical example of a host-pathogen symbiotic interaction where the pathogen cell biology and the host immune response coevolved several functional links. Here, the role played by ion fluxes across the root concerning nutrient uptake, osmoregulation, growth and signaling events is addressed. An ion-selective vibrating probe system was used to determine the net fluxes of protons (H(+)), calcium (Ca(2+)) and anions (A(-)) along nonmycorrhizal and ectomycorrhizal (ECM) roots of Eucalyptus globulus colonized by Pisolithus sp. These data show that, from five root zones analyzed, the main effect of fungal colonization was localized to the elongation zone. Here, strong changes in ion dynamics and rhizosphere acidification capacity were observed. Additionally, ion fluxes exhibited periodic fluctuations. To verify whether these fluctuations corresponded to sustained oscillations, continuous wavelet time spectrum analysis was applied and it was determined that H(+) and A(-) fluxes from ECM roots had longer periods than nonmycorrhizal roots. By contrast, Ca(2+) oscillations were completely abolished following fungal interaction. These results are interpreted in the light of a working model in which nutrient uptake and stimulation of growth are mediated by ECM fungi and may be pH-dependent. Furthermore, the variations detected in ECM roots for H(+) and A(-) fluxes suggest a main contribution from the plant, while the results obtained for Ca(2+) point to a significant involvement of the fungus.