Changes in tonoplast protein and density with the development of pear fruit

Protoplasts isolated from pear fruit at the end of the cell-division stage, 30 days after flowering (DAF), had already formed a large central vacuole and the vacuole occupied most of the protoplast. The changes in protein composition and density of the tonoplast (vacuolar membrane) were investigated during fruit development. After a linear sucrose density gradient centrifugation, the distribution of tonoplasts at 30 and 48 DAF was broad and began to narrow with further fruit development. This suggests that the tonoplast of young fruit is heterogeneous and becomes homogeneous with fruit development. The apparent density of the tonoplast at 30 DAF was approximately 1.12 g ml^-1; it decreased with fruit development and was finally 1.09 g ml^-1 in mature fruit. The phospholipid amount on the basis of tonoplast protein was 0.80 mg mg^-1 at 30 DAF. It increased with fruit development, and finally reached 7.49 mg mg^-1. This result indicates that the decrease in the density of the tonoplast was caused by the increase in the ratio of phospholipid to membrane protein. The protein composition of the tonoplast at each stage was quite different. The level of polypeptides of 94, 70, 61, 52, 48 and 41 kDa was low in young fruit and high in the middle or later stages of fruit development. In contrast, the level of a 76-kDa polypeptide was high in young fruit and decreased with fruit development. Although their functions are still unclear, these tonoplast proteins may play important roles in fruit development.