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 (25-150 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 100mM 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.