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

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 Ca2+, 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 Ca2+). 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 Ca2+ 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 Ca2+ 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.