Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspens ion cells and seedlings from spruce [Picea abies (L.) Karst. Proton transpo rt activity and uncoupler(1 mu M nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the prep aration of spruce tonoplast vesicles From roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) t onoplast membranes exhibiting vacuolar PPase as well as ATPase activity wer e used as a probe and added to the homogenization medium prior to the prepa ration of spruce vesicles. The preparation of spruce vesicles was more inhi bitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzym atic properties. After isopycnic centrifugation on continuous sucrose gradi ents the vacuolar PPase from spruce suspension cells co-purified with the v acuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonopl ast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spru ce are differentially energized by the vacuolar pyrophosphatase that may in dicate a difference in pyrophosphate metabolism between embryogenic and dif ferentiated spruce cells, and (4) tonoplast vesicles From spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATP ase in the absence of vacuolar PPase activity.