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Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH , Ca 2+ and GABA
Author(s) -
Domingos Patrícia,
Dias Pedro N.,
Tavares Bárbara,
Portes Maria Teresa,
Wudick Michael M.,
Konrad Kai R.,
Gilliham Matthew,
Bicho Ana,
Feijó José A.
Publication year - 2019
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.15863
Subject(s) - extracellular , cotransporter , symporter , biophysics , patch clamp , arabidopsis thaliana , biochemistry , chemistry , ion transporter , pollen tube , biology , membrane potential , microbiology and biotechnology , mutant , pollen , transporter , membrane , botany , receptor , gene , sodium , organic chemistry , pollination
Summary 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 ([Ca 2+ ] cyt ). We investigated the pollen‐expressed proteins At SLAH 3, At ALMT 12, At TMEM 16 and At CCC as the putative anion transporters responsible for these currents. At CCC ‐ GFP was observed at the shank and At SLAH 3‐ 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 PT s 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 At TMEM 16 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 At ALMT 12 transfected COS ‐7 cells, strongly suggesting the direct interaction of GABA with At ALMT12. Our data show that At SLAH 3 and At CCC 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 [Ca 2+ ] cyt through anionic transporters.