Development of Vacuolar Membranes during Elongation of Cells in Mung Bean Hypocotyls

The development of vacuolar membrane in the elongating hypocotylsof the mung bean was investigated. The hypocotyls from 3-day-oldseedlings were dissected into the dividing, elongating and matureregions. The diameter of protoplasts prepared from the matureregions was about 3-fold greater than the diameter of thosefrom the dividing region. The activity of inorganic pyrophosphatase,an enzyme associated with the vacuolar membrane, was detectableeven in the dividing region. The level of pyrophosphatase wasquantified by slot-blot analysis with the pyrophosphatase-specificantibody. The relative amount of pyrophosphatase per cell, calculatedon the basis of DNA content, increased about 4-fold during cellmaturation. When the densities of vacuolar membranes were comparedby sucrose density gradient centrifugation, there was no markeddifference among the preparations from three regions. Furthermore,most of the major proteins were common to the three purifiedpreparations of vacuolar membranes. From the results, it appearsthat most components of vacuolar membrane may be synthesizedde novo and added to the existing membrane during cell elongation.Furthermore, it is proposed that the H⁺-pyrophosphatase mayactively hydrolyze its substrate to maintain the internal acidityof expanding vacuoles, because pyrophosphate was present ata concentration of more than 70 µM in the dividing andelongating regions. (Received August 7, 1989; Accepted January 11, 1990)