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

Abstract

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 (PP(i))-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 V(max) 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.

Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+exchange

“…A control step enhancing transcription or protein translation rates and/or diminishing the turnover of the protein is most likely involved in the S. tuberosum cells in response to salt. 35 Similarly, an increased accumulation of the 68 kDa V-H + -PPase catalytic subunit in vacuolar membrane vesicles isolated from Salicornia bigelovii grown in 200 mM NaCl was observed. 46 In tonoplast vesicles from wheat (Triticum aestivum) roots exposed to severe NaCI stress (200 mmol/L) for 3 days the strong reduction in V-H + -PPase substrate hydrolysis activity correlated with lower amounts of V-H + -PPase protein.…”

“…Evidence for a decreased activity of V-H + -PPase with exposure to NaCl has been described several times, [37][38][39][40][41][42] but it has been shown that the activity of V-H + -PPase increases in several plants grown within saline environments. 35,[43][44][45][46] In salt adapted cell line of Solanum tuberosum, the activity of V-H + -PPase increased about threefold over cells cultivated in the absence of salt. 35 In the halophyte Suaeda salsa only in the case of 0.1 M NaCl treatment was V-H + -PPase markedly increased over the entire duration of the experiment, all other treatments only led to a small transient increase of V-H + -PPase activity or to a decrease of activity compared to controls; thus, under salt stress and osmotic stress conditions in S. salsa, V-H + -PPase activity seems to be less important physiologically than V-H + -ATPase activity.…”

“…35,[43][44][45][46] In salt adapted cell line of Solanum tuberosum, the activity of V-H + -PPase increased about threefold over cells cultivated in the absence of salt. 35 In the halophyte Suaeda salsa only in the case of 0.1 M NaCl treatment was V-H + -PPase markedly increased over the entire duration of the experiment, all other treatments only led to a small transient increase of V-H + -PPase activity or to a decrease of activity compared to controls; thus, under salt stress and osmotic stress conditions in S. salsa, V-H + -PPase activity seems to be less important physiologically than V-H + -ATPase activity. 47 As discussed by these authors, NaCl responses of the V-H + -PPase depend on plant species and type of treatment and cannot be generalized.…”

“…31 In several plant models the V-H + -PPase seems to be able to generate and maintain across the vacuolar membrane a higher pH gradient than the V-H + -ATPase, at PP i concentrations in the micromolar range. [32][33][34][35] Generally, V-H + -PPase activity is high in young tissues, whereas V-H + -ATPase activity is relatively constant during growth and maturation. 26 In pear fruit the ratio of V-H + -PPase activity to V-H + -ATPase activity indicated that V-H + -PPase is a major H + -pump of the vacuolar membranes of young fruit and that the contribution of V-H + -ATPase increases with fruit development, finally, V-H + -ATPase becomes the major H + -pump during the later stages of fruit development.…”

“…Such correlation between enzyme activity and protein content was also found for the tonoplast V-H + -ATPase in potato cell lines when western blotting analysis revealed that the relative amount of A subunit of the V-H + -ATPase remained constant in NaCl-tolerant calli despite the observed increase in both hydrolytic and H + -pumping activity in the salt-tolerant cell line. 35 Therefore, since the amount of the subunit A is likely to represent the protein level of V-H + -ATPase, and post-translational modifications such as phosphorylation/ dephosphorylation, the assembly of other subunits or the action of regulatory proteins might be involved. Phosphorylation and dephosphorylation of proteins is a common example of a posttranslational modification that has the potential to alter protein activity.…”