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High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

ABSTRACT: The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+ -ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton-pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.

SUBMITTER: Graus D

PROVIDER: S-EPMC6099232 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

Graus Dorothea D Konrad Kai R KR Bemm Felix F Patir Nebioglu Meliha Görkem MG Lorey Christian C Duscha Kerstin K Güthoff Tilman T Herrmann Johannes J Ferjani Ali A Cuin Tracey Ann TA Roelfsema M Rob G MRG Schumacher Karin K Neuhaus H Ekkehard HE Marten Irene I Hedrich Rainer R

The New phytologist 20180625 4

The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H<sup>+</sup> -ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PP<sub>i</sub> hydrolysis and proton-pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V-PPase-dependent sal ...[more]

PMID: 29938800

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Project description:BackgroundHalophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored.Methodology/principal findingsA halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2-16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45 degrees C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22 degrees C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the alpha-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data.Conclusions/significanceThe solution alpha-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity.

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High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

Publications

High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

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