ACCUMULATION OF VACUOLAR H-PYROPHOSPHATASE AND H+-ATPASE DURING REFORMATION OF THE CENTRAL VACUOLE IN GERMINATING PUMPKIN SEEDS()

Protein storage vacuoles were examined for the induction of H+-pyropho sphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyroph osphate-dependent H+-transport activities. H+-ATPase activity was sens itive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activ ities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits , 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar me mbranes of cotyledons, and the levels of the subunits of enzymes incre ased parallel to those of enzyme activities. Small amounts of the subu nits of the enzymes were detected in dry cotyledons. Immunocytochemica l analysis of the cotyledonous cells with anti-H+-PPase showed the clo se association of H+-PPase to the membranes of protein storage vacuole s. In endosperms of castor bean (Ricinus communis), both enzymes and t heir subunits increased after germination. Furthermore, the vacuolar m embranes from etiolated cotyledons of pumpkin had a polypeptide that c ross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The result s from this study suggest that H+-ATPase, H+-PPase, and VM23 are expre ssed and accumulated in the membranes of protein storage vacuoles afte r seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacu oles during the growth of seedlings.