Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Abstract

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic ("neutral") vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PPi-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PPi-hydrolytic activity and cross reactivity with antibodies including that directed against the Na+/H+-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of "neutral" vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the "neutral" vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na+ sequestration and is accordingly equipped with a high capacity of proton pumping and Na+ uptake via the Na+/H+-antiporter.