Fluxes Of H～+, K～+ And Na～+ In Salinised Poplar Cells And The Relevance To Salt Tolerance

We used callus of Populus euphratica Oliv., P. popularis, P. davidiana to isolate protoplasts in this study. The concentration of enzymes for protoplasts isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied in this report. Using patch clamp, we examined inward whole-cell K⁺ current in protoplast originated from P. euphratic.Moreover, salt-induced shifts of H⁺, K⁺ and Na⁺ fluxes were measured in callus and protoplast of the three tested poplars by Scanning Ion-selective Electrode Technique (SIET). Main results are listed in below.1 Protoplasts isolation(1) High yield and viability of P. euphratica protoplast was achieved after 6-8 hours incubation of callus in enzyme solution containing 1.5 % (w/v) cellulase R-10, 0.1% (w/v) pectolyase Y-23, 0.2% (w/v) macerozyme R-10, 0.05 % (w/v) hemicellulase and 0.75-0.8 mol L^-1 sorbitol.(2) High yield and viability of P. popularis protoplast was achieved after 6-8 hours incubation of callus in enzyme solution containing 1.5 % (w/v) cellulase R-10,1.5% (w/v)cellulysin , 0.2% (w/v) pectolyase Y-23, 1.0% (w/v) macerozyme R-10, 0.2 % (w/v) hemicellulase and 0.75-0.8 mol L^-1 sorbitol.(3) High yield and viability of P. davidiana protoplast was achieved after 6-8 hours incubation of callus in enzyme solution containing 1.5 % (w/v) cellulase R-10, 1.5% cellulysin, 0.2% (w/v) pectolyase Y-23, 0.2% (w/v) macerozyme R-10, 0.1 % (w/v) hemicellulase and 0.75-0.8 mol L^-1 sorbitol.2 The whole-cell inward currents We used a patch clamp to examine whole cell currents across the plasma membrane, and the application of voltage pulses ranged from -180 to 40 mV. The inward whole-cell current were activated when the membrane voltages were more negative than - 80 mV. Moreover, the inward currents increased with the more negative membrane voltage. 3 Cellular H⁺, K⁺, Na⁺fluxes(1) Initial salinity-induced the shift of H⁺ from influx to efflux were found in both callus and protoplasts of P. euphratica, P. popularis and P. davidiana, implying the enhanced activity of PM H⁺-pumps caused by a salt-shock.(2) We found that the higher external Na⁺ mediated high affinity K⁺ absorption in protoplasts of the three genotypes. But a reverse trend was observed in callus of the three poplars. Salt-shock induced K⁺ effluxes, especially in P. popularis and P.davidiana, which is presumably accounted for that the K⁺. inward rectifier channels were inhibited and K⁺ outward rectifier channels opened.(3) After subjected to a 30-min salt-shock, significant Na⁺ effluxes were observed in callus and protoplasts of P. euphratica. This may be the result of NaCl-increased activity of PM H⁺-ATPase and Na⁺/H⁺ antiporter. In contrast, Na⁺ influxes appeared in protoplasts of P. popularis and P. davidiana after cells subjected exposure to a salt-shock. The same trend was observed in the callus. Accordingly, we conclude that the sensitivity of salt-sensitive poplars to NaCl is resulted from the lower capacity for salt exclusion in cells.