A comparison of Zn, Mn, Cd, and Ca transport mechanisms in oat root tonoplast vesicles

Oat root tonoplast vesicles were used to determine if tonoplast transport of the divalent cations Zn and Mn occurs via an antiport mechanism, like that described for Ca and Cd. Also, inhibitors reported to affect Ca transport were tested for their effects on Cd versus Ca transport and tonoplast ATPase activity. The ability of Ca, Cd, Zn, and Mn to alter the proton gradient was monitored using both the fluorescent probe acridine orange and C-methylamine accumulation. After the proton gradient was established in MgATP-energized vesicles, addition of Ca, Cd, and Zn to the reaction restored the fluorescence of acridine orange, indicating dissipation of the proton gradient. Fluorescence recovery was linearly correlated with metal concentration and followed the order Ca>Cd≧Zn. Addition of Mn did not restore the fluorescence of acridine orange. All four ions released C-methylamine from MgATP-energized vesicles in an ion-concentration-dependent manner, and with relative initial rates in the order of Ca>Cd>Zn>Mn. The observed ion-concentration-dependent release of protons from sealed vesicles suggests that Zn and Mn, like Ca and Cd, can be antiported into the plant vacuole. In an effort to assess whether Ca and Cd use the same carrier, we tested the effects of verapamil, Do-Tea-Br, nifedipine, ruthenium red, and LaCl₂ on Ca versus Cd transport, and also on MgATPase activity. These compounds are shown to alter Ca transport in plants. Although some of the inhibitors had a negative effect on MgMAPase activity, the decrease in this activity did not account for the decrease in Ca or Cd transport observed in any case. Particularly verapamil had a much greater effect on Ca transport than Cd transport activity while not inhibiting ATPase substantially. Data presented provide evidence for Zn and Mn antiport activity in oat root tonoplast and show differences in responses of Ca and Cd antiport activities to several transport inhibitors.