Clark, Lawrence J.; (1990) Studies on the mechanism of sodium transport at the tonoplast of Beta vulgaris. Doctoral thesis (Ph.D.), University College London.

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Abstract

The aim of the work described in this thesis was to characterise the mechanism of Na+ transport at the vacuole membrane (tonoplast) of red beet using isolated tonoplast vesicles. It was confirmed that pure tonoplast vesicles could be prepared from red beet storage root. However, it was found that the KI treatment used in published tonoplast vesicle preparation protocols was deleterious to tonoplast, so this was subsequently modified. Attempts to characterise a Na+/H+ antiport reported by Blumwald and Poole (1985a; Plant Physiol. 78, 163-167) by studying the effect of Na+ on pH gradients imposed by a pH jump were not successful. Under the conditions used by these workers the fluorescent pH gradient probe acridine orange misreported the pH gradient and apparently good evidence for a Na+/H+ antiport was shown to be artefactual. With modifications to the media used for these experiments, acridine orange could be used to monitor the pH gradient and these results were confirmed using another pH gradient probe, [14C] methyl amine. However, no evidence was found for a Na+/H+ antiport as it was not possible to distinguish between changes in the pH gradient due to an Na+/H+ antiport and changes caused by electrically-coupled Na+ and H+ fluxes. In an attempt to avoid these problems, +Na uptake by tonoplast vesicles was studied in response to pH gradients generated by the H+-ATPase. No pH gradient-dependent Na+ uptake was found using this approach. It is concluded from these studies that previous evidence for a tonoplast Na+/H+ antiport in red beet is artef actual. It is suggested that Na+ is passively distributed across the tonoplast in red beet.

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