Plasma Membrane Vesicles of Beta vulgaris Storage Root Show Very High Water Permeability that is Shut Down by pH and Calcium

KARINA ALLEVA, CHRISTA M. NIEMIETZ, MOIRA SUTKA, RICARDO DORR, CHRISTOPHE MAUREL, MARIO PARISI, STEPHEN D. TYERMAN AND GABRIELA AMODEO

Montpellier, Francia

Workshop; 13th International Workshop on Plant Membrane Biology; 2004

Comité organizador ad hoc

Plasma membrane vesicles isolated by two-phase partitioning from Beta vulgaris storage root show atypically very high water permeability that is equivalent to those reported only for active aquaporins in tonoplast or animal red cells (Pf= 542 x 10-6m s-1). The values were determined from shrinking kinetics measured by light cattering. This high Pf was only partially inhibited by mercury (HgCl2), but was strongly inhibited by AgNO3. The Pf showed low activation energy (Ea) consistent will water permeation through aquaporins. In order to study regulation of water transport in this preparation, we tested pH and pH gradients, divalent cations, and protection against dephosphorylation. The observed high Pf at pH 8.3 was dramatically reduced by lowering the pH to 5.6. This was independent of the buffer employed to change the pH. The reduction in Pf at low pH corresponded to a large increase in activation energy. This suggests that at low pH water molecules permeate the purified vesicles mostly by the lipid pathway. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) and independent of pH gradient, shut down the path for facilitated diffusion of water through aquaporins. Dephosphorylation was discounted as a regulatory mechanism in this preparation, but calcium has a clear inhibitory effect on aquaporin activity. This inhibition occurred on the cytoplasmic face of the membrane. These findings establish cytoplasmic pH, and Ca2+ as important regulatory mechanisms involved in aquaporin gating. Furthermore we demonstrate that plasma membrane vesicles from Beta vulgaris storage root are an excellent system for the study of aquaporin regulation in plant plasma membranes