Activity of tonoplast proton pumps and Na⁺/H⁺ exchange in potato cell cultures is modulated by salt

The efficient exclusion of excess Na from the cytoplasm and vacuolar Na⁺ accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na⁺ from the cytosol in exchange for H⁺, a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H⁺-PPase and V-H⁺-ATPase and the involvement of Na⁺ compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PPi)-dependent H⁺-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H⁺-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H⁺-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na⁺-dependent dissipation of a pre-established pH gradient was used to measure Na⁺/H⁺ exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the Vmax of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H⁺-coupled exchange was specific for Na⁺ and Li⁺ and not for K⁺. The increase of both the pH gradient across the tonoplast and the Na⁺/H⁺ antiport activity in response to salt strongly suggests that Na⁺ sequestration into the vacuole contributes to salt tolerance in potato.