Characterization of water channels in wheat root membrane vesicles

The functional significance of water channels in wheat (Triticum aestivum L.) root membranes was assessed using light scattering to measure vesicle shrinking in response to osmotic gradients rapidly imposed in a stopped flow apparatus. Vesicles were obtained from both a plasma membrane fraction and a plasma membrane-depleted endomembrane fraction including tonoplast vesicles. Osmotic water permeability (P(os)) in the endomembrane fraction was high (P(os) = 86.0 micrometers s-1) with a low activation energy (E(A) = 23.32 kJ mol-1 +/- 3.88 SE), and was inhibited by mercurials (K1 = 40 micromolar HgCl2, where K1 is the inhibition constant for half-maximal inhibition), suggesting participation of water channels. A high ratio of osmotic to diffusional permeability (P(d)) (using D2O as a tracer, P(os)/P(d) = 7 +/- 0.5 SE) also supported this view. For the endomembrane fraction there was a marked decrease in P(os) with increasing osmotic gradient that was not observed in the plasma membrane fraction. Osmotic water permeability in the plasma membrane fraction was lower (P(os) = 12.5 micrometer s-1) with a high activation energy (E(A)= 48.07 kJ mol-1 +/- 3.63 SE) and no mercury inhibition. Nevertheless, P(os)/P(d) was found to be substantially higher than one (P(os) = 3 +/- 0.2 SE), indicating that water channels mediated water flow in this fraction, too. Possible distortion of the P(os)/P(d) value by unstirred layer effects was shown to be unlikely