Vacuolar Na+/H+ antiporter from barley: identification and response to salt stress.

One of the protective mechanisms used by plants to survive under conditions of salt stress caused by high NaCl concentration is the removal of Na+ from the cytoplasm. This mechanism involves a number of Na+/H+-antiporter proteins that are localized in plant plasma and vacuolar membranes. Due to the driving force of the electrochemical H+ gradient created by membrane H+-pumps (H+-ATPases and vacuolar H+-pyrophosphatases), Na+/H+-antiporters extrude sodium ions from the cytoplasm in exchange for protons. In this study, we have identified the gene for the barley vacuolar Na+/H+-antiporter HvNHX2 using the RACE (rapid amplification of cDNA ends)-PCR (polymerase chain reaction) technique. It is shown that the identified gene is expressed in roots, stems, and leaves of barley seedlings and that it presumably encodes a 59.6 kD protein composed of 546 amino acid residues. Antibodies against the C-terminal fragment of HvNHX2 were generated. It is shown that the quantity of HvNHX2 in tonoplast vesicles isolated from roots of barley seedlings remains the same, whereas the rate of Na+/H+ exchange across these membranes increases in response to salt stress. The 14-3-3-binding motif Lys-Lys-Glu-Ser-His-Pro (371-376) was detected in the HvNHX2 amino acid sequence, which is suggestive of possible involvement of the 14-3-3 proteins in the regulation of HvNHX2 function.

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

The synthesis of HSPs in sugar beet suspension culture cells under hyperthermia exhibits differential sensitivity to calcium.

Experiments aimed at testing the effect of Ca2+ on heat shock-induced changes in protein synthesis of cultured sugar beet cells were performed. Heat shock inhibits the synthesis of non-heat shock proteins (non-HSPs) and promotes the synthesis of a set of HSPs. Extracellular Ca2+ appeared to be strictly required for the synthesis of non-HSPs. Calcium was found to differentially exert its effect on the HSP synthesis: calcium induced (96 and 76 kDa), stimulated (94, 67, 58, 52, 32, 30, 26 and 22 kDa) or did not influence (82, 17 kDa) the de novo production of various HSPs. Cell injury increased if the cells were exposed to high temperature in a Ca(2+)-deficient medium. Calcium supplement to Ca(2+)-depleted cells resulted in the recovery of HSP synthesis and reduced cell injury by heat shock.

Plant cell pH-static circuit mediated by fusicoccin-binding proteins.

On sugar beet protoplasts that carry two types of fusicoccin-binding sites, a pH downshift in a physiological range (7.0-6.6) markedly enhanced the efficiency of fusicoccin (FC) binding, mainly owing to increased avidity of low-affinity FC-binding sites. This may allow the FC-binding proteins to act as pH-sensitive modulators of cell activity, for instance, via plasma membrane H+-ATPase or potassium channels.