pH regulates water transport in Beta vulgaris root cells

GABRIELA AMODEO, MOIRA SUTKA, KARINA ALLEVA RICARDO DORR Y MARIO PA

Buenos Aires, Argentina

Simposio; Satellite Course and Symposia "Volume Regulation in Animal and Plant Cells"; 2002

NOLOSE

We report here the study of water and solute transfers and their putative regulation in parenchymatic cells of the storage root of Beta vulgaris. Experimental techniques, currently used in animal tissue were adapted to study water movements in root sections. Results showed that only in the radial pathway a net water flux increase was observed when an osmotic gradient was applied. Mercurial compounds (1 mM HgCl2) reversibly inhibited this response therefore allowing to conclude that in this pathway water channels are involved in water transfers. Following studies were extended to the cellular level, seeking for putative regulation of the water movements. Two membranes are involved in cell osmoregulation in plants: the vacuolar membrane (the tonoplast) and the plasma membrane. Initially, we achieved the isolation of vacuoles from the root parenchyma and then the volume changes of individual vacuoles in response to aniso-osmotic challenges were measured using videomicroscopy. Hypo-osmotic gradients were either step-applied or progressively imposed in perfusion experiments. Results confirmed what was observed at the tissue level: there is an osmotic response that can be reversibly blocked by mercurial compounds. Additionally, it was observed that low pH affects water and solute transfers (urea) in vacuoles. The osmotic properties of the plasma membrane were also characterized. Plasma membrane vesicles were purified by aqueous two-phase partitioning and its water permeability was studied by means of stopped-flow spectrophotometry. The water permeability values were high showing inhibition by HgCl2, and a reduced activation energy (Ea). These findings suggest the presence of a channel-mediated water transport mechanism in the plasma membrane of Beta vulgaris. Interestingly, the high Pf at pH 8.3 was dramatically reduced by lowering the pH to 5.6. The reduction in Pf at low pH also showed a significative increase in temperature dependence which can be interpreted as water molecules permeating the purified vesicles mostly by the lipid pathway at low pH. Therefore reduced pH appears to shut down the path for facilitated diffusion of water through aquaporins. A pH gradient was established to determine which face of the plasma membrane may be responding to pH changes. With the pH gradient under iso-osmotic conditions no volume change was observed. However, when an osmotic gradient was applied, the Pf was only reduced when pH inside the vesicles was lowered. The evidence suggests that the plasma membrane of Beta vulgaris has aquaporins that are gated by pH on the inside-side face of the vesicle. This normally corresponds to the cytosolic face of the membrane using the isolation technique. It has been suggested that the high water permeability of the tonoplast allows the vacuole to effectively buffer the cytoplasm, minimizing short term volume transients. It has been also reported that a cytoplasmic acidification can be induced by salt stress and also during hypoxia conditions. It is therefore possible to suggest that the inhibition of water permeability as a response to acidification could be a regulation mechanism of water exchange under stress conditions. Supported by FONCYT PICT-99/5145; IFS C3052 and IUPAB Grant Task Force for Capacity Building of Biophysics