期刊
NEW PHYTOLOGIST
卷 229, 期 5, 页码 3026-3036出版社
WILEY
DOI: 10.1111/nph.17021
关键词
BCECF; fluorescence; NHX antiporters; patch‐ clamp; PI(3; 5)P-2; plant vacuole; proton
资金
- Italian 'Progetti di Ricerca di Interesse Nazionale' [2015795S5W_003]
- Spanish 'Agencia Estatal de Investigacion' (AEI) [RTI2018-094027-B-I00]
- 'Ministerio de Ciencia, Innovacion y Universidades' (MCIU)
- European Regional Development Fund
This study combined patch-clamp technique with ratiometric fluorescence imaging to detect NHX antiport activity-induced acidification in Arabidopsis mesophyll vacuoles. NHX activity was specifically impaired by the low-abundance signaling lipid PI(3,5)P-2, which may regulate salt accumulation in plants by coordinately shutting down secondary active carriers responsible for cation and anion uptake inside the vacuole.
We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K+) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P-2, which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.
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