Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings

Partitioning in a biphasic polymer system was used to isolate plasmalemma (PM) from roots and shoots of etiolated pea seedlings. The membrane preparations were used to assess the osmotic water permeability (P-os) with the stopped-flow method. The Western-blot technique was employed to determine the membrane content of the PIP-family of aquaporins, and their activity was estimated by measuring the rate of osmotic vesicle shrinking in the presence of inhibitors, HgCl2 and AgNO3. Monobromobimane fluorescent dye was used to determine the quantity of sulfhydryl groups in cell membranes and follow the effect of SH-oxidizing (diamide) and SH-reducing (dithiothreitol and tributylphosphine) agents on P-os of the root PM and oligomerization of aquaporins. The shoot PM was shown to combine high P-os with low aquaporin content. In the root PM, P-os was lower and the aquaporin content greatly exceeded that in the shoots. HgCl2 and AgNO3 did not decrease the rate of osmotic shrinking in root membrane vesicles, whereas considerably (by 40-50%) inhibited this index in the shoot membranes. Root and shoot PM preparations dramatically differed in their SH-group contents: the former exceeded the latter sixfold. When added to the homogenization medium, diamide and tributylphosphine affected the content of SH-groups and P-os in the root PM. In the roots, diamide decreased the quantity of SH-groups almost twofold and increased P-os fourfold, and the introduction of tributylphosphine produced a twofold increase in the quantity of SH-groups with only slight decrease in the P-os. Immunological analysis of membranes isolated in the presence of diamide showed that the ratio between the monomer and dimer forms of aquaporins in two membrane preparations depended on the presence of dithiothreitol in the denaturing buffer apparently because dithiothreitol exposed and reduced disulfide bonds essential for monomer interactions and inaccessible for interaction with redox modifiers of SH-groups in the membrane. Because of their inaccessibility, these modifiers could not cause the changes of P-os in the root PM produced by oxidation and reduction of SH-groups. This phenomenon is probably related to the change in the status of SH-groups in two cysteine residues at the N-end of the aquaporin loop C oriented outward into the apoplast.