Coulter counter cell size determination of protoplasts from Arabidopsis thaliana PIP1b aquaporin antisense lines under iso- and hypo-osmotic conditions

Water uptake of Arabidopsis thaliana protoplasts was measured after transfer into hypo-osmotic conditions. The time-dependent swelling of protoplast populations was monitored by a Coulter counter device. In order to ascertain the contribution of the plasma membrane intrinsic protein 1b (PIP1b) to the membrane's water permeability, protoplasts of five different plant lines that were transformed with a PIP1b antisense construct were compared to controls. The size distribution of 5 independent protoplast preparations provided similar results for control and antisense lines under iso-osmolar conditions. After transfer into hypo-osmotic conditions, a time difference for the swelling of protoplasts from the different sources was observed. The sizes of control protoplasts changed in less than 20 s, which indicates high water influx rates. In contrast, the protoplast populations obtained from 5 different antisense plants look about 75 s to reach a steady-slate cell size distribution. The difference in time by a factor of about 3 confirms the significance of the aquaporin PIP1b for the water permeability of plant plasma membranes and the cellular water transport.