Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes.

Abstract Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na + flux measurements was limited due to poor selectivity for Na + ions of commercially available Na + cocktails. Often, these cocktails cannot discriminate between Na + and other interfering ions such as K + and Ca 2+ , leading to inaccurate measurements of Na + concentration and, consequently, inaccurate Na + flux calculations. To overcome this problem, three Na + -selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na + cocktail for selectivity for Na + ions over other ions (particularly K + and Ca 2+ ). Among the three calixarene-based Na + cocktails tested, cocktail 2 [in % w/w: Na + ionophore (4- tert -butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na + ions over K + and Ca 2+ ions and was highly stable over time (up to 10 h). Na + flux measurements under a wide range of NaCl concentrations (25–150 mM) using Na + cocktail 2 established a clear dose–response relationship between severity of salt stress and magnitude of Na + influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na + cocktail 2 was compared with commercially available ETH 227-based Na + cocktail by measuring Na + fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na + cocktail 2, a large decreasing Na + influx (0–15 min) followed by small Na + influx (15–45 min) was measured, whereas with ETH-based Na + cocktail Na + influx was short-lived (1–3 min) and was followed by Na + efflux (3–45 min) that might have been due to K + and Ca 2+ efflux measured together with Na + influx. In conclusion, Na + -selective calixarene-based microelectrodes have excellent potential to be used in real-time Na + flux measurements in plants.