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 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 10h). Na + flux measurements under a wide range of NaCl concentrations (25-150mM) 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 100mM NaCl treatment. With calixarene-based Na ⁺ cocktail 2, a large decreasing Na ⁺ influx (0-15min) followed by small Na ⁺ influx (15-45min) was measured, whereas with ETH-based Na ⁺ cocktail Na ⁺ influx was short-lived (1-3min) and was followed by Na ⁺ efflux (3-45min) that might have been due to K ⁺ and Ca ²⁺ efflux measured together with Na ⁺ influx. In conclusion, Na ⁺⁺ flux measurements in plants.