Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

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Abstract
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-Ca2+ concentration from 10−8 to 10−5 mol·dm−3 caused a significant decrease in Cl− influx, which may indicate that cytosolic Ca2+ 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.