Activity, amount and subunit composition of vacuolar-type H⁺-ATPase and H⁺-PPase in wheat roots under severe NaCl stress

Tonoplast-enriched membrane vesicles from wheat (Triticum aestivum) roots exposed to severe NaCl stress (200 mmol/L) for 3 days were isolated using the Dextran T₇₀ step gradient method. High purity of the tonoplast vesicle preparations was indicated by a high degree of Bafilomycin A₁ and nitrate inhibition of ATP-hydrolysis activity at pH 7.5. Severe NaCl stress strongly reduced the V-ATPase and V-PPase substrate hydrolysis activity compared with controls. Immunoprecipitation analysis showed that the relative amount of V-ATPase protein related to total tonoplast proteins was reduced by a factor of 2.4 due to NaCl stress. Antisera directed against the Kalanchoe daigremontiana V-ATPase holoenzyme cross-reacted with polypeptides present in the T. aestivum root tonoplast vesicle fraction exhibiting apparent molecular masses of 67, 58, 44, 41, 34, 32 and 17 kDa, which are likely to be subunits of the wheat V-ATPase. In preparations from salt-treated seedlings an additional 33 kDa polypeptide was immunodecorated by an antiserum against V-ATPase subunit A, which is discussed to represent a proteolytic fragment of subunit A. By cross-reaction with a specific antiserum a 70 kDa polypeptide was identified as V-PPase. Similar to V-ATPase subunits the V-PPase protein amount was lower in preparations from NaCl-treated plants.