Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe : Effects of other anions, membrane potential, and transport inhibitors.

The characteristics of Cl(-) transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K(+) and valinomycin, increased the initial rate of Cl(-) transport, demonstrating that Cl(-) could be electrically driven into the vesicles. Chloride influx was unaffected by SO 4 (2-) , but was competitively blocked by NO 3 (-) , indicating that both Cl(-) and NO 3 (-) may be transported by the same porter. In some preparations, increases in free-Ca(2+) concentration from 10(-8) to 10(-5) mol·dm(-3) caused a significant decrease in Cl(-) influx, which may indicate that cytosolic Ca(2+) concentration has a role in controlling Cl(-) fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl(-) transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.