Medium acidification affects water permeability in isolated root tonoplast vesicles from Beta vulgaris

MOIRA SUTKA, KARINA ALLEVA, RICARDO DORR, MARIO PARISI AND GABRIELA AMODEO

Cuernavaca, México

Congreso; 1st Pan-American Plant Membrane Biology Workshop; 2003

Comite organizador ad hoc

The discovery of water channels (aquaporins) has requestioned the role of the cell to cell pathway versus apoplastic pathway in water transport. Plant plasma membrane and tonoplast express aquaporins abundantly (called PIPs and TIPs, respectively) (Johansson et al.,2000). We particularly focused our interest in water movements across the tonoplast. It has been reported that tonoplast water permeability (Pf) shows a high variability in different species, but higher values than those reported in plasma membrane. It has been suggested that this higher water permeability of the tonoplast allows the vacuole to effectively buffer the cytoplasm, minimizing short term volume transients (Tyerman et al., 1999). In a previous work, water transport in isolated vacuoles of Beta vulgaris storage root was characterized by means of a system that allows following volume changes using videomicroscopy (Amodeo et al., 2002). External medium acidification inhibited water transport across the vacuolar membrane. With the aim of studying water permeability independently of cellular metabolism we isolated Beta vulgaris tonoplast vesicles (TV) from root microsomes by sucrose density gradient centrifugation. The vesicle preparation presented very low contamination with other membranes (enrichment factor 1.7). The mean apparent diameter of membrane vesicles was determined by electron microscopy. The osmotic shrinking kinetics of the vesicles was characterized by means of the stopped-flow technique (light scattering, 90º, 500 nm) and Pf was calculated according to van Heeswijk et al., 1986. These TV presented a Pf of 452.2 ± 65.2 (±SEM; n= 5) showing 76% and 85 % of inhibition, when exposed to 0.1mM and 1mM HgCl2 respectively. The pH effect was also studied in two different conditions: pH 8.3 and pH 5.6. Results obtained showed that with pH 5.6, vesicles water permeability decreased 25%. Our results obtained with Beta vulgaris tonoplast vesicles confirm the inhibition of water transport observed when vacuoles preparation was exposed to an acid external medium. Supported by FONCYT PICT/99 5145 and IFS-Fundación Antorchas Grant C3052/2. References: 1-Amodeo G, Sutka M, Dorr R and Parisi M (2002). Journal of  Membrane Biology 187:175-184. 2-Johansson I, Karlsson M, Johansson U, Larsson C and Kjellbom P. (2000). Biochimica et Biophysica Acta 1465:324-342 3-Tyerman SD, Bohnert HJ, Maurel C, Steudle E and Smith JAC (1999). Journal of Experimental Botany 50:1055-1071 4-van Heeswijk, MPE and van Os, CH (1986). Journal of Membrane Biology 37:157-166.