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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Vacuola chloride transport in the extreme halophyte Messembryanthemum crystallinum

Wissing, Frank January 1999 (has links)
The halophyte Mesembryanthemum crystallinum 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 M.crystallinum. This thesis describes the use of tonoplast vesicles from leaf mesophyll cells of M. crystallinum 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 (N/N'-dimethyl-9,9'-bisacridinium dinitrate). This work was complemented by a patch-clamp study of ionic currents of leaf-mesophyll vacuoles from M.crystallinum. 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 M. crystallinum 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. An inside-acid pH gradient, generated by a K<sup>+</sup>/nigericin clamp, reduced the initial rate of chloride transport into tonoplast vesicles of M.crystallinum. 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. The patch-clamp study of ionic currents of whole vacuoles and excised vacuolar membrane patches from M.crystallinum. 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. 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 M.crystallinum. 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.

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