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.