Transport of Ca2+ across the tonoplast of intact vacuoles from Chenopodium album L suspension cells: ATP-dependent import and inositol-1,4,5-trisphosphate-induced release

The removal of Ca2+ from the medium by intact vacuoles and microsomes of Chenopodium album was investigated by measuring INDO-1 fluorescence emission at 400 and 480 nm and the response of Ca2+ -selective mini-electrodes, The removal of Ca2+ depended on the presence of MgATP, displaying an apparent K-mATP of about 50 mu M, a K-mCa of 400-500 nM, and a nucleotide specificity (%) of ATP (100) > CTP (49) > GTP (28) > UTP (20) > ADP = AMP (0). In the presence of saturating MgATP, the vacuoles reduced the [Ca2+] of the medium below 30 nM. Part of the Ca2+ removed from the medium was released again after adding micromolar concentrations of inositol-1,4,5-trisphosphate. This release of Ca2+ was inhibited by heparin. Since digitonin caused the release of the entire amount of Ca2+ removed from the medium in the presence of MgATP, we argue that the Ca2+ is not bound to membranes or sequestered otherwise, but is transported into the vacuoles (or vesicles) and remains freely mobile there. In accordance with the current literature, we conclude that the plant vacuole is an important store for mobile Ca2+ to be released for purposes of signal transduction. Since changes in the trans-tonoplast Delta pH and inhibition of the H+-translocating pumps had no significant influence on the ATP-dependent removal of Ca2+ from the cytoplasmic side, we argue that in C. album ATP-driven Ca2+ transport is the predominant form of Ca2+ translocation into the vacuole.