| Summary: | <p>The halophyte <em>Mesembryanthemum crystallinum</em> L. accumulates high concentrations of NaCl (up to 1 M) in its leaf cells as a response to soil salinity. While there is evidence that vacuolar sodium transport is mediated by a tonoplast Na<sup>+</sup>/H<sup>+</sup> antiporter (Barkla <sup>et al.</sup>, 1995), little is known about the transport of Cl<sup>-</sup> transport into the vacuole. So far, it has been uncertain whether secondary active transport (e.g. H<sup>+</sup>/C1<sup>-</sup> antiporter) is involved or whether a passive mechanism (Cl<sup>-</sup> channel) is sufficient to mediate Cl<sup>-</sup> accumulation in the vacuole of <em>M</em>.<em>crystallinum</em>.</p> <p>This thesis describes the use of tonoplast vesicles from leaf mesophyll cells of <em>M</em>. <em>crystallinum</em> to study vacuolar Cl<sup>-</sup> transport. Cl<sup>-</sup> uptake into the vesicles was measured using the Cl<sup>-</sup>-sensitive fluorescent dye lucigenin (<em>N</em>/<em>N</em>'-dimethyl-9,9'-bisacridinium dinitrate). This work was complemented by a patch-clamp study of ionic currents of leaf-mesophyll vacuoles from <em>M</em>.<em>crystallinum</em>.</p> <p>Cl<sup>-</sup> transport into tonoplast vesicles showed saturation-type kinetics with an apparent Km between 10 and 36 mM and a maximum initial change of the intravesicular Cl<sup>-</sup> concentration of 4.8 mM min<sup>-</sup>, corresponding to an estimated Cl<sup>-</sup> flux of 31 nmol m<sup>2</sup> s<sup>-</sup><sup>1</sup>. Vacuolar chloride transport was not affected by sulphate, malate, or nitrate, indicating a high specificity of this transport process for chloride over other anions. By imposing insidepositive membrane potentials using a K<sup>+</sup>/valinomycin clamp revealed a sigmoidal voltagedependent relationship with the steepest increase in vacuolar Cl<sup>-</sup> uptake around +30 mV. Only under severe salt treatment with 500 mM NaCl for 3 weeks did 9-week-old <em>M</em>. <em>crystallinum</em> plants show a significant increase (63%) of vacuolar C<sup>-</sup> uptake, along with an increased V-type H<sup>+</sup>-ATPase hydrolytic activity (up to 65%). The apparent K<sub>m</sub> of vacuolar Cl<sup>-</sup> uptake was also increased from 27 mM to 44 mM under these conditions.</p> <p>An inside-acid pH gradient, generated by a K<sup>+</sup>/nigericin clamp, reduced the initial rate of chloride transport into tonoplast vesicles of <em>M</em>.<em>crystallinum</em>. External Cl<sup>-</sup>, in contrast to external Na<sup>+</sup> , did not dissipate an inside-acidic ΔpH generated by various techniques. This is strong evidence against a proton-driven antiport mechanism.</p> <p>The patch-clamp study of ionic currents of whole vacuoles and excised vacuolar membrane patches from <em>M</em>.<em>crystallinum</em>. leaf-mesophyll cells revealed a number of cation channels. At cytosolic free Ca <sup>2</sup><sup>+</sup> concentrations of 1μM and above, ubiquitous slow-vacuolar type cation currents could be observed. In excised patches, eleven different single channel types, with conductances ranging from 2 up to 200 pS, could be described. However, no clear Cl<sup>-</sup>conductance could be identified. The lack of observed vacuolar Cl<sup>-</sup> channel activities is discussed in the light of possible lack of Cl<sup>-</sup>-channel activation due to the loss of a cytosolic factor.</p> <p>The results obtained from the biochemical work on tonoplast vesicles support the hypothesis that a passive transport mechanism (i.e. channel) is sufficient to mediate vacuolar chloride transport in <em>M</em>.<em>crystallinum</em>. The observed upregulation of vacuolar Cl<sup>-</sup> transport under severe salt stress shows that it plays an important role in the salt adaptation of this halophytic plant.</p>
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